SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

KeY'Nord Index 

Abdomen pain, C II 

Absinthe, B9 

Acetaminophen, C17, P58 

6-Acetylmorphine, C2, F8 

ADH,A45 

Adulteration, F2, F4 

ACE inhibitors, A 13 

Acetaldehyde, A47 

Acotinine, A59, ClO, P4 

Acute narcotism, P38 

Addiction, M13, M32 

Adducts, C23 

AEME,M26 

Alcohol, B8, B13, B17, P37 

Alcohol intoxication, B6 

Alcohol markers, A36, A 76, Mil 

Alcohol testing, A18, A24, A60, M37 

Alcoholic beverages, P63 

Alcoholic ketoacidosis, P64 

Alfentanil, P49 

Alpha-methy ltryptamine, P2l 

Alternative matrices, M28 

Aluminum, C3 

Amitriptyline, PI7, P23 

Amphetamine precursor drugs, B4 

Amphetamine type stimulants, F15 

Amphetamines, AI, A2, A3, A4, A7, 

A50, A63, F12, MIO, M16, M41, 

Amphetamines in hair, P36 

Anabolic steroids, M30 

Anaesthetist, M 13 

Analysis, B9 

Anti-impotence drugs, A43 

Anti-depressants, A25, PI 

Antihistamines, P59 

Arsenic, C34, P45 

. AT II antagonists, A 13 

Atomoxetine, A46, P2 

Atractyloside, PI2 

Automated method, A48, A56 

Automation, Pl6 

Azide, A66 

Barbiturates, M44 

Basic drugs, A37 

Benzodiazepines, A8, AIO, A40, F2l, 

M20, M25, P27 

Benzoylecgonine, A44 

Beta-blockers, A38 

Beta-hydroxybutyrate, P64 

Biological matrices, A52, A54 

Biomarker, A60, A 77 

Blood alcohol, A21, A58, P65 

Blood analysis, A31, PIO, P13,P15, P22, 

P47 

Blood sample preparation, P32 

Body sway, BI0 

Bone, P27 

Brain, Bl 

Brain death, C21 

Brazil, A5 

Breath, A 18, A24 

Breath testing, B 15 

Brown mixture, FI6 

Buprenorphine, A33, A49, A84, C33, 

F23 

Bulla, CI5 

Caffeine,P57 

Calendar year 2003, FI9, F20 

Callilepis laureola, PI2 

Cannabidiol, B2 

Cannabinoids, A44, B2, P41 

Cannabis, A 75, B 1, B2, M48, M49 

Capillary column, A21 

Capillary Electrophoresis, All, A 74 

Carbmazepine, A48 

Carbon Monoxide, P32, P35 

Carboxy-THC, A31 

Carisoprodol, M39, PI8 

Cause of death, P33 

CEDIA, A33, C2 

Page 402

Channelopathy, C34 

Chemical bums, C 15 

Chemical warfare agents, C23 

Children, C28 

Chloramphenicol, A39 

Chloramphenicol glucuronide, A39 

Chlorinated MDMA, F25 

Chloroformates, Al 

Chloropyrifos, C24 

Chromium, F2 

Chronic abuse, P49 

Chronic administration, C 1 0 

Citalopram, P3 

Clandestine, C28 

CLND,A69 

Clomipramine, P6 

Clozapine, M14 

Cluster analysis, P64 

Cocaine, A29, C31, Fl1, M26, M28, 

M51,P27,P29,P33 

Cocaethylene, M43 

Codeine, F16, M29 

Collection, C3 

Comparative analysis, A 17 

Consolidated workstation, A 19 

Contamination, A 75, M8 

Controlled cocaine administration, M51 

Conversion, A81 

Corticosteroids, A37 

Cotinine, M45 

Crack, M26 

Criminal poisoning, C 19 

Criteria, A6 

Cryogenic Grinding, M2 

Cytochrome P450, B4 

Cytotoxicity, M43 

Cyanide, P44 

Dark Agouti rats, B4 

Death, P25, P61 

Death investigation, P53 

Demethylation, A 16 

Depot, M49 

Derivatisation, An 

Designer drugs, Al 

Detection window, C32 

Detoxification programme, C 1 

Dextromethorphan, M39 

Diagnosis, C23 

2,4-dichlorophenol (2,4-DCP), C20 

2,4-dichlorophenoxyacetic acid (2,4-D), 

C20 

Dietary supplements, A43 

Diethylene Glycol, C26 

Differential display, C17 

2,5-dimethoxy-4-bromoamphetamine, 

C29 

2,4-Dinitrophenol, P62 

Direct and affordable analysis, A60 

Disinfectant, C 14 

Distribution, P 11 

Diuretics, C30 

DNA,A77 

DOB overdose, C29 

Doping, A65, M30 

DRE, M34 

Driving, B 12, B 13 

Dronabinol, B3 

Drug, P28, P37 

Drug analysis, A69 

Drug diversion, C27 

Drug-facilitated crime, M9 

Drug-facilitated sexual assault, B8, B 17, 

C5, C32, P23 

Drug identification, A 70 

Drugs of Abuse, AI9, A28, An, B7, 

B12, B13, Cl, Fl, M2, M15, M24, M33, 

M35,M37,M42,M46,M50,P31 

Drug testing, F13 

Drug Use History, M6 

Drunken driving, B 15 

Dual-column, A25 

DUI, A73, B3 

Ecstasy, AI2, AI7, P25, P43 

EIA, M34, A50 

ELISA, A57, A65, F23, M16, M24, 

M40 

Emblica officinalis, C 18 

EMIT II Plus immunoassay, A44 

Enantiomer, A63, P42 

Enantiomer separation, A4 

Enantioselective Separation, M 10 

Entomology, M44 

Page 403

Environmental toxicology, C4 

Enzyme immunoassay, A32, C 13 

Epidemiology, P28 

Ephedrine, A8I, P42 

Ephedrone, C7 

Epidemiology, C6 

Error, P65 

Estazolam, B5 

Ethanol, A27, A35, A45, A55, BIO, C25, 

MI8, P35, P47 

Ethanol metabolism, A36 

Ethyl-glucuronide, A35, A53, MIl 

Ethyl sulfate, A36, A76 

Exhaled breath, M45 

Exposure, M46 

External contamination, P45 

Extraction, A30 

FAS, A77 

Fatal Intoxication, C29, P8, Pl4 

Fatality, P46, P48, P52, P62 

Femoral, P65 

Fentanyl, P22, P48 

Fire, P35 

Flunitrazepam, A23, B8 

Food products, P63 

Forced ingestion, C31 

Forensic, A6, A52 

Forensic Laboratories, A5 

Forensic Toxicology, P36 

Formalin-induced, P39 

FOXY,C9 

Free-morphine, FlO 

Friction coefficient, B5 

Fungi, A75 

Furosemide, A 14 

GHB, A73, P46, P63 

GC-FID, A27, C24 

GC-HRMS, M41 

GC-MS, AI, AI5, A29, A30, A32, A4I, 

A47, A63, A70, An, A73, A80, A8I, 

B7, B9, CI5, C2I, C24, C30, FI7, F24, 

F25, F27, MI5, M2I, M23, M29, M45, 

PI, P57 

GC-MS/MS, A3I, A53, A56, C8, M35, 

P49 

GCIMS/SIM, CIO, M7 

Glucuronide metabolites, A84 

Goat, C12 

Go-pills, FI2 

Hair, M3, M4, M8, M9, M13, MI6, 

M28,M35,M41,P9,P49 

Hair analysis, M2, M5, M7, MIO, MIl, 

MI2,M20, M39,M40, P30 

Hallucinogens, P21 

Haulage drivers, F22 

Headspace GC, A2I, A58 

Headspace-SPME, A54 

Heroin, F8, FlO, P24 

Heroin maintenance program, MI2 

Heroin related death, P29 

HHS-certified laboratories,FI9, F20 

HPLC, AIO, AI7, A20, A62, C20, P5 

HPLC-MS, Pl2 

Human, BI1 

Hydrocodone, P20 

Hydroxychloroquine, P52 

Hydroxy radical, A55 

ICP-MS, C3 

IDA, A83 

Illicit drug, C7 

Illicit substances, F22 

1midacloprid, P 14 

ImmunoassaY,A3, F5, F24, PI8 

Immunoassay comparison to GC/MS, F6 

Immunoassay confirmation rates, F19, 

F20 

IMS, C5 

Inductively Coupled Plasma, A22 

Infant, P40 

Information, A9 

Information dependent acquisition, A68 

Inhalation, Cll 

Insecticide poisoning, P34 

Intect, F4 

Interaction, P29 

Internal standard, P32 

Internet, P44 

Interpretation, PI9, P59 

Intoxication, P7, P40, P44, P57 

Intoxilyzer 8000, A 18, A24 

In Utero Exposure, M47 

Iodine, F4 

Page 404

Ion chromatography, A66 

Ion-paired extraction, F3 

Ion trap, C8 

Ketamine, C32, M7, M40 

Labetalol, P8 

L-Ascorbyl-2-phosphate, A55 

LA-ICP-MS, M4 

Laser spray, A59 

Laxatives, C30 

LCIAPCIIMS, P9 

LC-MS, A42, A43, A79, FI8, P14, P50 

LC-ESI-MS, AlO, A25, A28, A51, A67, 

A83,M25,M27,M3l,M42 

LC-MS/APCI, A34, A38 

LC-MSIMS, A8, A13, A37, A39, A40, 

A49, A52, A76, A78, A83, F7, F2l, F26, 

M9, 

MI4, M20, P48 

LC-MSIMS library, A68 

Lead, BI6 

Legal and judicial issues, A9, M32 

Lethal dose, C26 

Levetiracetam, P 15 

LiquidlLiquid, FI7 

Liver, B16 

Liver fibrosis, C 18 

LOD,A6 

Lorazepam, A15, P53 

Low dose, P58 

LSD, A78 

Marijuana, Fil 

Mass detector, A22 

Mass spectrometry, A42, A59, A64, A69, 

C31 

Matrix, A7l 

MBTFA,AI6 

MDA,A4,A7 

MDMA, A3, A4, All, A20, Fll, M5, 

M36, P43 

Measurement uncertainty, F3 

Meconium, M47 

Medical professional, F26 

Melanin, M3, M14 

Mellanby, B17 

Meprobamate, C6 

Mercury analysis, C 19 

Mercury poisoning, C 19 

Metabolic acidosis, C25 

Metabolism, C9, C33 

Metabolites, A29, C3l 

Metalloids, A22 

Metals analysis, C4 

Metaxalone, P7 

Methadone, C22, F17, Ml, P19, P20, 

P40,P60,P6l 

Methamphetamine, A4, B14, CIl, C28, 

M4l,M47,P39,P4l 

Methamphetamine chronic toxicity, P36 

Methcathinone, C7 

Method development, A42, A 79 

Method Validation, A57, FI5, M5 

5-Methoxy-alpha-methyltryptamine, P2I 

5-Methoxy-N,N-diisopropyltryptamine, 

C9 

Methylation, P39 

Methylhippuric acid, A5I 

Microdiffusion method, A66 

Microvascular Endothelium, M43 

Microwave-assisted Extraction, M 19 

Mirtazapine, P 13 

6-mono-acetyl Morphine, FlO 

Monoliths, A 7 

Morphine, F8, F13, F16, P24, P25 

Multicenter evaluation, A48 

Multiple Analyte Enzyme Immunoassay, 

FI 

Multi target screening, A68 

Multisite sampling, MI8 

Nail analysis, M30 

Nails, M26 

Naloxone, A83 

Napoleon, C34, P45 

Narcotics, C27 

NAS, C22 

Neonate, P54 

Nerve gas, C8 

Nicotine, C22, M22 

Nicotine metabolites, M22 

Nitrite, F2 

N,N-dimethylamphetamine, F18 

NNSL, F14 

Page 405

Non-controlled psychotropic substances, 

A61 

Non-negative, Fl4 

Non-regulated testing, F20 

11-nor-delta-9-carboxytetrahydrocannabinol, 

F5 

Norephedrine, P42 

Norketamine, C32 

Nornaloxone, A83 

Noscapine metabolites, F9 

On-Site testing, F6, M33, M36 

Opiates, A44, A64, C2, F7, M12, P30 

Opioids, A16, BI4 

Oral administration ofTHC, B2, M17, 

M23 

Oral Cannabinoids, M38 

Oral fluid, MI5, M22, M24, M27, M31, 

M33,M34,M36,M37,M48,M49,M50 

OraSure, M17 

Organophosphorous pesticides, A54 

Outcomes, C22 

Overdose, C6, PI, P8, P51, P55 

Oxcarbazepine, P9 

Oxycodone,A32,CI3,F24,P20,P26 

Oxymorphone, CI3 

P450, C33 

Paclitaxel, A 79 

Paint thinner abuser, A51 

Papaverine metabolites, F9 

Paramethoxyamphetamine, P41 

Pediatric, P52 

Perfonnance toxicology, B 11 

Pennethrin, P34 

Phannaceutical testing, C27 

Phannacogenomics, CI7, P60, P61 

Pharmacokinetics, A84 

Phenazepam, A80 

Phenethylamines, A61, A67 

Piperazine, A 7 4 

Plasma, A38, M23, M24 

PMA,A26 

Poisoning, C24, C26, P22 

Portugal, B 12 

Positive chemical ionization, A 15 

Positive rates, F26 

Postmortem, PII, P13, PI5, PI8, P24, 

P26,P31,P34,P37,P38,P47,P5I,P56, 

P58,P62 

Postmortem blood concentrations, P33, 

P59 

Postmortem diffusion, P4 

Postmortem ethanol, P50 

Postmortem fluids and tissues, PI9 

Postmortem redistribution, P3, P6, P 17 

Postmortem specimens, M 18, P2, P5 

Postmortem toxicology, P23, P46, P52, 

P53,P55 

Post-rotational nystagmus, B6 

Posturography, B5 

Pregnancy, M46 

Prenatal, C22 

Profiling, A26 

Profiling MDMA, AI2 

Propylene glycol, C25 

Protease, F5 

Psilocin, A34, FII 

Psychomotor performance, B 10 

Puffer fish, C 16 

Putrefaction, P4 

Pyrolysis products, A29 

Pyrosequencing, P60 

Quantitation, A45, A49, A71, A80 

Quetiapine, PII, P55, P56 

Rat, C18, P3 

Redistribution, P43 

Residue cytotoxicity ACTP, C12 

Retention time locking, A 70 

rhuEPO, A65 

RIA, A57 

Roadside survey, B 15 

Saliva, All, M21, M24, M42 

Salivette, M17 

SAMHSA, F14 

Sampling, A20 

San Francisco, P28 

Scatchard Analysis, M3 

Science and law, A9 

Screening, A28, A40, A62, F23 

Sebum, MI9 

Segmental Hair Analysis, M6, M32 

Self-injurious behavior, B14 

Page 406

Serotonin metabolites, PSO 

Sertraline, PS4 

Serum, B16 

Sexual assault, B7 

Sildenafil, PS 

S ilymarin, B 16 

Skelaxin, P7 

SNRI, A46 

Sodium chloride, A27 

Soils, C4 

Solid Phase Extraction, A14, A23, A41, 

AS3, A62, MI, Pl6 

Solid Phase Microextraction, A2, Ml 

Specimen validity testing, F2 

STA, A83 

Stability, A47, PIO 

Standard addition, A 71 

Statistical evaluation, P38 

Street heroin markers, F9 

Strychnine, A41 

Substance abuse, P30 

Substitution therapy, C 1 

Sulfonylurea, PIO 

Supercritical Fluid Extraction, M 19 

Surfactant, C 14 

Survey, AS 

Sweat, M29, MSI 

Sweat patch, M38 

Tetrodotoxin, C16 

THC, A30, AS6, M27, M31, M38, M48, 

M49 

THC metabolites, F27 

Therapeutic drug monitoring, A 19 

Tissue Distribution, P2 

Tissue Samples, P16 

Toxicity, C14, PS6, PS8 

Toxicokinetic, Cl2 

Toxicology, A9, A46, CS, M44, P26 

Trace analysis, M4 

Traffic safety, BI 

Tramadol, PI7, PSI 

Tricyclic antidepressants, P6 

Tryptamines, A6I, A67 

Turkey, BII 

Turnaround time, FI 

Underground water, C4 

Urinalysis, F6, FlS, F18 

Urinary sulfate metabolites, F3 

Urine, A23, A33, F7, F12, F21, F27 

Urine Drug Screen, M34 

Urine Drug Testing, MSO 

Vagina, C16 

Validation, A8, C21 

Vegetables analysis, C4 

Vigilance, F22 

Vitamin C, Bl6 

Vitreous fluid, P47 

Vitreous humor, P6S 

Washing, M8 

Whole blood, A14, A23, A34, ASO, A64, 

A78 

Withdrawal, PS4 

Workplace, F13 

Page 407

Presenting Author Index 

. 

· A' Zary, Edward 

F17 

I Chen Meng 

A47 

i Adamowicz Piotr 

C32 Cheng. W C 

Fl8 

I Akgur 5 A Bll · Chien Beckie F4 

AI-Ahmadi Tareq A72 Choo Robin E C22 

Anderson Dan P2 I Chung, Hee-5un MS I 

· Andrenyak, David M A1S • Chung, Heesun PS 

~r,MikeK A46 i Chung, H 5 P44 I 

Anne Lakshmi C13 ! Clarke Joe M1S 

Augsburger, Marc B13 • Clarkson Jayne E PS3 i 

Avella, Joseph P6 i Clelland Brandi L All 

Avila,S B12 I Coffinq, Mary J A48 i 

• Aydin Handan C20 Colangelo C H C4 

Baker, Daniel D P20 Collison Ines B P63 

LBaker, Ginger PSl Cone Edward J M49 

! Balikova, Marie C29 Cooper, Gail Ml6 

Balikova, Marie P36 • Coopman, Vera P48 

• Barroso M A4l I Couper, Fiona J P40 

Battah A H PlO i Coyer Michael J Fl 

Beck Olof A33 ! Croal Bernard P3l 

I 

• Behonick George 5 P22 • Cruickshank Yvonne M2 

Belhadj-Tahar H C7 : Darwin t William D Ml7 

Bellissima Brandi L AlO Davis Paul J A56 ! 

• Bernhard, W P24 De Jesus Rosa E A70 

Beyer, J C30 • De MartiniS, Bruno 5 Ml8 

· Bishop, Sandra C A74 I Diaz-Vazquez liz M Ml9 

~m LeeM C3 ! Dresen, Sebastian A76 

Boettcher, Michael C2 I Druid Henrik P30 

! 

Bogusz, Maciej J A39 Drummer Olaf H PS8 

Boland Diane M P2l ! Duffort, G M20 

· Bosman I J P33 I Dumestre-Toulet, V P49 

I Braithwaite R A Pl3 I Edinboro Leslie E F7 

I Brewer William E A30 Elliott Simon P43 

I Burrows David L F5 · Fang, WenfCl~ A83 

! Burt, Martha J P55 FeeneYI Michael A58 

: Burton Fiona C B8 Feldman, Michael A3S 

I Bush, Donna M F19 Ferrara Santo D B10 

~I§!r, Jessie C 

C8 

C26 

~ii LA 

I Bynum Nichole D 

P17 Ferrari l A 

P3S 

CalieryL Patrick 5 P39 ~erald Robert l C25 

Cardona Patrick 5 A29 est, A RW P64 

! Cawley, Adam F3 Fortner Neil F8 

Caylor Curtis P47 Frison G A2 

: Cazenave, S.O.S. A7S Fritz, Kristina P23 

Cervenak Juraj P7 Fucci Nadia P38 

Chang, Yan C33 Fuller Dwain C A16 

Chasin, A M AS Gautam l 

Lata M3 

Page 409

Gelhaus, Stacy L 

A77 • Laloup, Marleen 

A50 

! Gergov Merja 

A49 Lambert Willy 

A79 

Gerostamoulos Jim P25 Larson Scott F27 

Giroud C B3 · Lebrun B A12 

Gordon Ann M C28 i Lechowicz, Wojciech A34 

• 

i Goulle, J P A22 i Lemos, N P P28 i 

• Hackett Jeffery A23 Leong, H S M7 

Hamzah Asimah F23 I Lewis Russell J P42 

• Harden Wat L M4 ! Lim, Mi-ae F1S 

Hayakawa, Hideyuki P4 Linden Rafael A62 

Hegstad, S F21 

! 

Liu, HSiu-Chuan F16 

Helander A A36 Liu, Ray H A63 

i Hieda Yoko C14 Lood Yvonne A1 

Hirata Yukari A66 Lyle Charles R M21 

Hodda A E P6S Maralikova Barbora A13 

Horak Erica All • Maresova Vera F2S I 

Huang, Wei A84 Margalho C A14 

Huestis Marilyn A M47 Marinetti Laureen J I PS4 

Humbert, L C31 Martinez, Maria A I C24 

Itoh, Shinobu ASS Martins, Liliane M10 

i Jannetto Paul J AS7 Marti-Ortiz, A A6 

• Jayashanker G CS Maurer, Hans H A38 

i Johansen Sys S A78 Mayer J P26 

Johnson, R D PSO • Mazarr-Proo, Susan P46 

Jordan S A19 McGrath Kelly K P27 

Juhascik Matthew B7 McIntyre lain M P62 

Jurado C Mll l\1cKaque Kathleen A40 

Kacinko Sherri L MS1 McMullin Matthew F26 

Kala Maria BS Meatherall Robert A64 

• Kaneko, Rina A59 Meenan Gerard A32 

! Karch Steven B C34 Merves, Michele L M4S 

Kardos Keith M48 Mireault Pascal AS2 . 

~ovsek Majda Z B6 Mobley, P L F12 

shik Romina A60 ! Monforte Joseph R F24 

Kerrigan Sarah PS7 Moody, David E F6 

Kiat Wenceslo Cll Morl, Tomohisa 814 

Kikura-Hanajiri R A61 Moringa Masatoshi AS1 

Kim Insook M22 Mozayani Ashraf S 

• Kim Jin Y M41 Muller 18 A20 

• Kinkennon Amy A31 \\1ura, P B1 

! Kintz P M13 Musshoff F M12 

· Klys Malgorzata P9 Mykkanen, Sirpa A80 

i Koski A P37 Namera A A7 

Kraemer Thomas B4 Namiki, Mizuho C17 

Kristoffersen Lena A4S i Nebro Wesenyalsh M23 

Kronstrand, Robert M14 Nippert H P52 

! 

Kucmanic, John J A24 Noort D C23 

Kuqelberq, F C P3 • Nunes-Daniel F P18 

Kuntz, David J F2 O'Hanlon, L M1 

• Kupiec, Thomas M24 Ojanpera I1kka A69 

! Labat, L F22 Papa, Vincent M Fll 

Page 410

Parker Dawn R 

PS6 

Paterson Sue 

F9 

I Paul Buddha D 

A4 

~e MichelieR 

M44 

Pehourcq, Fabienne 

C6 

Peters, Frank T 

C21 

Petrova, V 

P41 

Pichini S 

M46 

Pirnay,S 

P45 

i Poklis l Alphonse 

C19 

Poklis Justin L 

P34 

· Polettini A P29 

: Portman M B1S 

i Pragst, Fritz 

B2 

Proenca P 

P14 

I Quintela, 0 

M2S 

i Ragoucy-Sengler, Catherine 

M26 

Raikos, Nikolaos 

A54 

: Rasanen, llpo A21 

Razatos Gerasimos 

A18 

Rivadulla, Manuel 

M27 

· Ropero-Miller Jeri D P19 

ISahu C R 

C12 

I Saisho, K 

A43 

· Samkova, H P32 

Sandberg, Michelle 

PS9 

Sasaki, Tania A 

A42 

Schaffer Michael 

M8 

Scheidweiler 1 Karl B 

M28 

• Schur B C 

P60 

Schwilke Eugene W 

M29 

I Scott Karen S 

M6 

Shalan M G 

B16 

· Simonsen K W A44 

! Sklerov, Jason H A2S 

• Smith AC 

B9 

• Snyder Ann 

MSO 

i Soper John W 

flO 

• Stankova, Marie 

P1 

Steenkamp, Paul A 

P12 

Steentoft, A 

A8 

Stimpfl T 

P16 

Stripp, Richard 

A6S 

Sutheimer Craig A 

• Swienton Anjali R 

Tacker, Danyel H 

Takaichi Kenichi 

Tasduqi SA 

Teixeira, H M 

Tsatsakis Aristidis 

Tsujino Yoshio 

Tyler Mark 

i Vallejo! Yli R 

I Valouch, Tara J 

! Van Bocxlaer, Jan F 

• Verstraete Alain 

Villain M 

i Vorce Shawn P 

· Vorisek V 

Wada Kentaro 

! WagnerL Michael A 

Wan Terence SM 

Wang, M 

• Waumans D 

Weinmann Wolfgang 

Westenbrink, William 

Wicks John 

• Wille, Sarah 

Wilson J M 

Wilson Lisa 

~ 

Wolf Carl E 

Wonq, Raphael C 

i Wong L Steve 

i Woodall, Karen 

I Wtsadik Abraham T 

i Wu Anthon'i 

· Yang, Wonkyung 

Yashiki, Mikio 

• Yazzie Janice 

~nglCY 

~g, Michael S 

Zaney, M E 

Zuba, Dariusz 

Zweigenbaum, Jerry 

F20 

A9 

M43 

M30 

C18 

M31 

M32 

C1S 

617 

M33 

Pll 

A82 

A3 

M9 

A67 

C1 

C10 

M34 

A37 

AS3 

A26 

A68 

F13 

M35 

A81 

C9 

M36 

C27 

M37 

P61 

P8 

M38 

Fl4 

M39 

C16 

A27 

M40 

M42 

A73 

A17 

A28 

Page 411

Scientific Session 

Abstracts: 

Analytical Methods 

Page 113

At 

DETERMINATION OF PHENETHYLAMINE· AND PIPERAZINE·DERIVED DESIGNER 

DRUGS IN URINE BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Yvonne Lood* and Arne Eklund, National Board of Forensic Medicine, Department ofForensic Chemistry, 

University Hospital, SE-5S1 S5 Linkoping, Sweden 

New designer drugs have appeared on the illicit drug market during the last years. Many of these drugs are 

easily available on the Internet and there are several web sites that give a lot of information about these 

substances. Forensic laboratories are continuously in need of complementary addition of existing methods 

or developing new methods for these substances. Screening of urine samples for amphetamines in our 

laboratory is routinely done by enzyme immunoassay and those giving positiv results are confirmed by 

another analyze. The urine is analyzed by gas chromatography-mass spectrometry (GC-MS) in the selected 

ion monitoring mode (81M) after liquid-liquid extration with isooctane followed by derivatization with 

tritluoroaceticanhydride. This method is quantitative for amphetamine, methamphetamine, MDMA, MDA 

and qualitative for MDEA, MBDB, phentermine, phenylpropanolamine, ephedrine, pseUdoephedrine and 

phenmetrazine. If the result of this method is negative in spite of positive immunological testing or if the 

police or the pathologists have a request about other central stimulating designer drugs we use our method 

for phenetylamines and piperazines. It is a rapid and simple method because we use the sample extract 

from the former analysis and just reinject it on the GClMS using the SIM. Verification of a positive result 

is done by full scan analysis and searching in our own reference library. The method was developed for 

identification of the phenetylamines p-methoxyamphetamine (PMA), p-methoxymethamphetamine 

(PMMA), 4-methylthioamphetamine (4-MTA), 3,4,5-trimethoxyamphetamine (TMA), 2,5-dimethoxy-4 

methylamphetamine (DOM), 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-chloro-2,5 

dimethoxyamphetamine (DOC), 4-ethyl-2,5-dimethoxyamphetamine (DOET), 4-bromo-2,5 

dimethoxyphenetylamine (2C-B), 4-iodo-2,5-dimethoxyphenetylamine (2C-I), 4-ethyl-2,5 

dimethoxyphenetylamine (2C-E), 3,4,5-trimethoxyphenetylamine (mescaline) and the piperazines N 

benzylpiperazine (A2), N-(3-tritluoromethylphenyl)piperazine (TFMPP), N-( 4-methoxyphenyl)piperazine 

(MeOPP) and N-(3-chlorophenyl)piperazine (CPP). The selection ofsubstances included in the method was 

dependent on the seizures done by the police and the availability ofthe substances. Seven ofthe substances 

were kindly received from the National Laboratory ofForensic Science, Linkoping, Sweden. Immunoassay 

screening methodology is of limited value in detecting designer drugs in urine because of low cross 

reactivity for most of these substances. When a case, with clearly symptoms of being under the influence of 

central stimulant, needs to be investigated the described method is used even if the urine has been tested 

negative with immunological methods. In November 2003 one 17-year- and two IS-year-old males were 

found positive for 2C-E. They were caught by the police in the street showing symptoms that indicated 

ingestion of some narcotics. The urine samples were negative for screening but we analyzed further with 

the method for phenetylamines and piperazines and could by that identify 2C-E. The concentrations were 

2,1 IlglmL, O,S Ilg/mL and 0,06 IlglmL respectively. Until today we have in 54 cases identified and verified 

seven ofthese substances in urine samples, 2C-E, MeOPP, A2, DOC, PMA, PMMA and 4-MT A. 

Keywords: Designer drugs, Amphetamines, GC-MS 

Page 115

A2 

DETERMINATION OF AMPHET AMINE-RELA TED DRUGS IN BIOFLUIDS BY GCIMS 

AFTER AQUEOUS-PHASE DERIV A TIZA TION WITH 2,2,2 TRICHLOROETHYL 

CHLOROFORMATE AND SOLID-PHASE MICRO EXTRACTION 

G. Frison"', L. Tedeschi, D. Favretto, S. Maietti, F. Castagna, A. Nalesso, S.D. Ferrara, Forensic 

Toxicology and Antidoping, University Hospital of Pad ova, Via Falloppio 50,1-35121 Padova, Italy 

The potential of 2,2,2-trichloroethyl chloroformate derivatization for the GCIMS analysis of a large range 

of amphetamine-related drugs (ARDs) and ephedrines in plasma, urine and hair samples has recently been 

demonstrated. 

A new and simpler analytical approach has been developed for ARDs bioanalysis, based on aqueous-phase 

2,2,2 trichloroethyl chloroformate derivatization, subsequent direct solid-phase micro extraction (SPME) of 

derivatives, and their GCIMS detection. 

Preliminary studies on amphetamine, MDMA, MBDB, TMA and DOB have been carried out to check 

feasibility of derivatization in aqueous conditions and to optimize reaction conditions. Comparative studies 

were undertaken to optimize SPME conditions: choice of SPME fiber, working pH, effects of salts, 

adsorption temperature, and adsorption and desorption times. 

Results demonstrate that: ARDs can be derivatized in aqueous phase with 2,2,2 trichloroethyl 

chloroformate; carbamate derivatives can be extracted by direct immersion SPME; a simple and fast 

analytical procedure can beappUed for sample preparation and GCIMS analysis (1.2 ml sample total 

volume, 5 III derivatizing agent, reaction time 5 min, use of a PDMS 100 SPME fiber, buffered working 

conditions at pH 9, absence of salts, SPME adsorption temperature 30°C and time 20 min). 

The procedure has been successfully applied to the analysis of blank, spiked and true positive plasma, urine 

(0.5 ml) and hair (50 mg) samples. 

Key words: Amphetamines, Chloroformates, SPME. 

Page 116

A3 

COMPARISON OF THE SENSITIVITY AND SPECIFICITY OF SIX IMMUNOASSAYS FOR THE 

DETECTION OF AMPHETAMINES 

Alain Verstraete' and Fien Vander Heyden, Laboratory of Clinical Chemistry, Section Toxicology, Ghent University 

Hospital, De Pintelaan 185, B·9000 Gent 

Introduction: In drug of abuse screening, the ideal amphetamine immunoassay should detect amphetamine and the 

different illicit amphetamine analogues (e.g. MDMA, MDEA, MDA) without false positive results from anorectics, 

other stimulants or other drugs like ranitidine. We compared the sensitivity and specificity of 6 commercial urine 

amphetamine immunoassays for the analysis of the urine samples that were sent to our laboratory during a 2.5·month 

period. 

Methods: Two hundred twenty five urine samples that had been sent to our laboratory for screening or confirmation 

of amphetamines were analyzed for amphetamines with the FPIA amphetamine/methamphetamine II assay (on 

Abbott ADx and AxSYM instruments), EMIT (Emit II Plus Monoclonal AmphetaminelMetamphetamine Assay and 

the new EMIT II Plus Amphetamines Assay) and KIMS (standard protocol and MDMA protocol, KIMS and KIMS 

X respectively). All assays were calibrated and used semi·quantitatively. All samples that screened positive by any 

amphetamine screening method and 15% of the negative samples were confirmed by LC-MS/MS. Briefly, 10 ilL of 

urine was mixed with 90 ilL of a mixture of deuterated internal standards and 20 ilL was injected in the LC-MSIMS. 

The assay LOQ is less than 15 ng/mL of amphetamine, methamphetamine, MDMA, MDEA, MDA, 4·MTA and 

PMA. A sample was considered positive for amphetamines if any of these substances was present at > 200 ng/mL. 

Results and discussion.·_Ninety·one (40%) of the samples were positive by LC-MS/MS. The number of positive 

samples, lowest, median and highest concentration (in ng/mL) are 74, 71, 2560 and 155000 for amphetamine, 1,33, 

33 and 33 for methamphetamine, 27, 46,5975 and 108000 for MDMA, 23, 15,516, 12400 for MDA and 4,27, 1530 

and 24800 for MDMA. MBDB, 4-MTA and PMA were not found. 

Some c haractenstlcs 0fthe d'ff

ADx AxSYM EMIT EMITN KIMS KIMSX 

Area under the ROC curve 0.999 0.988 0.977 0.984 0.975 0.972 

95% confidence interval 0.982 

1.000 

0.963· 

0.998 

0.948 

0.993 

0.958 

0.996 

0.944· 

0.991 

0.941 

0.990 

Optimal cut-off (ng/mL) 350 677 565 271 404 493 

Sensitivity at the cut-off (%) 98.9% 95.6% 96.6% 90.9% 94.4% 93.3% I 

Specificity at that cut-off(%) 98.5% 97.8% 90.2% 100% 88.5% 89.3% 

# false negatives at that cut-off 1 4 3 8 5 6 

# false positives at that cut-off 2 3 13 0 15 14 

i # false negatives at 500 ng/mL 2 4 3 19 11 7 

, # false positives at 500 ng/mL 2 15 18 0 11 14 

Discussion and conclusion: The best results were seen with the Abbott ADx assay that is not available anymore in 

Europe. If the cut-off is increased to 677 ng/mL, the AxSYM gives a low number of false positives and negatives. 

The new EMIT assay has excellent specificity, but misses more true positive samples: 2 samples containing 

amphetamine (225 and 253 ng/mL), 1 sample containing MDA (231 ng/mL), 4 samples containing MDMA (319 

2760 ng/mL and MDA (113-516 ng/mL) and one sample containing amphetamine and MDMA). For the older EMIT 

assays and both KIMS methods, there was more overlap between negative and positive samples, reSUlting in a high 

number of false positives. The optimal cut-offs, calculated by analysis ofthe receiver operating characteristic curves, 

varied between 271 and 677 ng/mL. Use of 500 ng/mL cut-off doesn't change much for the ADx and KIMS X 

assays, increases the number of false positives for AxSYM and EMIT, and increases the number of false negatives 

for the new EMIT method and the KIMS method. 

Keywords: Amphetamine, MDMA, Immunoassay 

Page 117

A4 

(R)-(-)-a-METHOXY-a-(TRIFLUOROMETHy)PHENYLACETYL CHLORIDE (MTPA) AS AN 

USEFUL CHIRAL REAGENT FORENANTIOMERIC SEPARATION AND QUANTITATION OF 

AMPHETAMINE, METHAMPHETAMINE, MDA, MDMA, AND MDEA IN URINE SPECIMENS 

Buddha D. Paui', John Jemionek, David Lesser, Aaron Jacobs, and Douglas A. Searles, Division of 

Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces Institute of Pathology, 

Rockville, Maryland20850 and Navy Drug Screening Laboratory, San Diego, California 92134 

In drug testing, the presence of methamphetamine in urine is generally confirmed by a GC-MS method. 

Prior to confirmation, the basic drug is derivatized to a suitable pertluoroalkylamide for better 

chromatographic separation. Once methamphetamine is detected, a second GC-MS test is necessary to 

distinguish positive results from medical use of Vick's inhaler or selegiline (Deprenyl). R-( -) 

Methamphetamine is the urinary product from use of these medications. The second GC-MS test is to 

confirm the illicit use of (S)-(+)-methamphetamine. In the procedure, the two methamphetamine isomers 

are changed to the chromatographically separable diastereomers by a chiral derivatizing agent, (S)-(-) 

trifluoroacetylprolyl chloride (TPC). But the method has inherent limitations. Racemization of the reagent 

produces mixed diastereomers even from pure (S)-(+)-methamphetamine. Instead of using TPC, we utilized 

(RH-)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (MTPA) to prepare the diastereomers of 

methamphetamine amide. The reagent showed no recemization because it contained no a-proton next to the 

carbonyl function. The method was extended to resolve GC peaks of (R)-(-)- and (S)-(+)-isomers of 

amphetamine, 3,4-methylenedioxyamphetamine (MDA), N-methyl-MDA (MDMA) and N-ethyl-MDA 

(MDEA). Three ions from the drug and two ions from the deuterated internal standard were monitored to 

characterize and quantitate the drugs. For MDEA, only one ion was used. The compounds in urine after 

acid-base separation and solvent extraction were derivatized by (R)-(-)-MTPA and tested by the GC-MS. 

The quantitation was linear over 25 to 5,000 nglmL for MDEA and 25 to 10,000 nglmL for all other drugs. 

Correlation coefficients were >0.996. Precision calculated as the coefficient of variation at the calibrator 

concentration of 500 nglmL was within ±ll% for all drugs. The method was applied to test forty-three 

urine specimens. In 95% of the methamphetamine-positive specimens, only the (S)-(+)-isomer was 

detected. In all MDMA-positive specimens, the concentrations of (RH-)-isomer were greater than that of 

the (S)-(+)-isomer indicating longer retention of (R)-(-)-MDMA in the human body. In all specimens the 

total enantiomer concentrations (R + S) detected by the MTPA method compared well with that of a nonchiral 

method that used 4-carboethoxyhexafluorobutyryl chloride as derivatizing agent (I ~ 0.650, tail 

2.069, N=24, 95% confidence). But the MTPA method has some advantage. It alone can replace the two 

GC-MS methods needed to confirm the presence of (S)-(+)-isomers of amphetamine and 

methamphetamine. 

Keywords: Amphetamine!Methamphetamine, MDAlMDMA, Enantiomer separation 

Page 118

AS 

SURVEY ON FORENSIC TOXICOLOGICAL ANALYTICAL LABORATORIES IN BRAZIL 

E.F. Mendes, V.F. Barbosa, N.F.G. Pereira, and A.M. Chasin*, REBLAS/ANVISA I Brazil; Forensic 

Toxicology Lab, Medical Legal Institute- SP and College ofPhannacy Oswaldo Cruz, Sao Paulo- Brazil 

Forensic Toxicology in Brazil is carried out, principally, by public laboratories, except for Doping and 

Workplace Drug Testing that are done in private laboratories. Compared to other countries, there are neither 

established guidelines to orient these analyses nor a system of certification to orient Toxicological Analyses, 

in general, and specifically for forensic purposes. 

This paper had as objectives the following: to identify the laboratories that work with or have the potential to 

carry out tests pertaining to Forensic Toxicology and the respective assays, as required by the current public 

health legislation, as well as the methodologies used and the Quality Systems followed by this labs; to 

evaluate, beforehand, the technical capacity of these laboratories and what they must do in order to be 

qualified by the Brazilian Network of Laboratories in Public Health REBLAS/ANVISA (the Brazilian 

Agency for Health and Sanitary Safety Vigilance and Management). 

A survey was prepared of several assays carried out by the labs and were divided in three major groups: 

Group I - Analysis of Controlled Substances and Abusive Drugs in materials seized by the Police (in natura), 

with 120 assays; Group II - Presumptive Tests and Confinnation of Controlled Substances and Abusive 

Drugs in Biological Matrices in vivo, with 1000 assays; Group III Presumptive Tests and Confirmation of 

Controlled Substances and Abusive Drugs in Biological Matrices post mortem, with 906 assays. Also 

included were a "Survey on Quality Systems" and a "Registration Card". 

The surveys were prepared with the aim of dividing substances up into categories and respective subcategories, 

with the most representative substance tests being included. The presumptive and the confinnation 

tests were included for all groups. Fifteen labpratories, representing all Brazilian geographic regions, took part 

in the research. The regions of South and Southeast Brazil were the most represented, each one with six 

participating laboratories, although the regions of North, Northeast and Middle-East Brazil also participated, 

with only one laboratory, however, presenting analyses of Group I. Of the 15 laboratories taking part in the 

research, only 9 of them answered to the Group I survey; 3 answered to both Group I and Group II; 2 

laboratories answered only to Group II and only 1 laboratory, located in Southeast Brazil, declared that they 

had completely carried out the Group III survey. 

Most of the laboratories taking part (80%) carried out the Group I assays- Analysis of Control led Substances 

and Abusive Drugs. Group II Presumptive Tests and Confirmation of Controlled Substances and Abusive 

. Drugs in Biological Matrices in vivo was carried out by 33% of the institutions and Group III - Presumptive 

Test and Confirmation of Controlled Substances and Abusive Drugs in Biological Matrices post mortem was 

carried out by only one laboratory (6.6% of the laboratories studied). It was concluded that 26% of the 

laboratories taking part in the research still do not follow any of the quality parameters. The infonnation from 

the Survey will serve as a basis for preparing policies, establishing and managing Forensic Toxicological 

Laboratories in Brazil. 

Keywords: Brazil, Forensic Laboratories, Survey 

Page 119

A6 

APPLICABLE CRITERIA IN DETERMINING LIMITS OF DETECTION (LOD) 

A. Marti-Ortiz·, V.R. Hernandez, B. Mauricio, and N. Santiago 

University of Puerto Rico School of Pharmacy, Chemical Forensic Laboratories, San Juan, Puerto Rico 

00936 

The proposed HHS SAMHSA guidelines for the analysis of drugs of abuse will affect directly or indirectly 

a very large number of laboratories involved in workplace drug testing. The acceptance of the analysis of 

alternative matrices and the varied instrumentation used poses regulatory and forensic challenges. The 

criteria that a laboratory employs to attest as to the presence of drug are of the utmost importance. 

Chromatographic and mass spectral behavior of chemical species at the instrumental limit of detection is 

subject to variability as the method is being challenged at its limit of performance. A number of recent 

publications have suggested a "relaxation" of the chromatographic parameters and propose stressing more 

the mass spectral behavior ofchemical species this could be problematic for forensic defensibility. 

As the criteria used to determine the limit of detection will affect the limit of quantification and the lower 

limit of linearity of calibration curves and fundamentally the performance of the method it is of the utmost 

importance that a laboratory clearly and objectively define the criteria to apply. A comprehensive study 

using GCIMS was undertaken to revisit generally accepted chromatographic and mass spectral criteria at 

the limit of detection. Benzolyecgonine, ll-nor-~ 9 - tetrahydrocannabinol-9-carboxylic acid (THC) and 

amphetamines were used to assess the following contributors on limits of detection determinations: 

matrix background , chromatographic profiles, mass spectral ion variability and statistical acceptance 

criteria. For example, we observed with benzolyecgonine chromatographic variations of the individual ion 

fragments and ion ratios that would be acceptable or not for the determination of LOD depending on the 

laboratory criteria. 

Advances in analytical instrumentation and techniques are increasingly lowering the limits of detection. 

Acceptable criteria must comply with existing scientific principles and provide sound forensic evidence as 

to be useful in the detection of prohibited substances not only in workplace drug testing but also in 

laboratories where only the presence ofthe drug is required. 

Keywords: LOD, Criteria, Forensic 

Page 120

A7 

QUANTITATIVE DETERMINATION OF AMPHETAMINES AND METHYLENEDIOXY 

AMPHETAMINES IN URINE BY MONOLITHIC COLUMN AND HPLC-DAD 

A Namera*", M Nishida", M Yashiki", A Nakamoto b , Y Takei", Y Shintani", M FurunoC, H Minakuchid, K 

Nakanishi", K Kimura" 

a Department of Legal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, 

Hiroshima, b Scientific Investigation Laboratory, Hiroshima Prefectural Police Headquarters, Hiroshima, C 

GL Sciences Inc., Tokyo, d Kyoto Monotech, Kyoto, e Department of Material Chemistry, Graduate School 

ofEngineering, Kyoto University, Kyoto, JAPAN 

Aims: A monolithic silica was developed for a new material of a micro or capillary scale HPLC. The 

material consists of continuous porous silica having a double-pore structure (through-pore and meso-pore). 

In this study, we developed a simple extraction of amphetamines and methylenedioxyamphetamines from a 

small volume of urine by using a capillary column packed with a Cis-bonded monolithic silica. 

Methods: The mixture of sample urine (0.1 ml), buffer and an internal standard was introduced into the 

monolithic capillary column using a gas tight syringe. Analytes were adsorbed on. the column and 

endogenous interferences were washed with an acidified buffer. Analytes were then eluted with 20-micro-L 

of a mobile phase and direct injected into a HPLC. 

Results: The recoveries of amphetamines and methylenedioxyamphetamines from urine were over 90%. 

The calibration curves showed linearity in the range of 100 - 50,000 ng/g in urine. The coefficients of 

variation of intraday and interday were below 10%. 

Conclusion: These results demonstrated that the C1s-bonded column was useful for the extraction of 

amphetamines in urine. This column has a potential as a new tool for the extraction of chemicals in 

biological materials. 

Keywords: Amphetamines, Methylenedioxyamphetamines, Monoliths 

Page 121

AS 

QUANTITATIVE DETERMINATION OF DIAZEPAM AND NORDIAZEPAM IN WHOLE 

BLOOD BY 

LC-MS/MS 

A. Steentoft* and K. Wiese Simonsen, Department of Forensic Chemistry; University of Copenhagen; 

Denmark 

Aims: To present a full validated LC-MSIMS method for quantification of diazepam and nordiazepam in 

whole blood for routine use. 

Methods: Blood samples were extracted with ethyl acetate at pH 9. After evaporation of the organic 

solvent, the residue was dissolved in solvent. Spiked blood samples (0.0025-2.5 mg/kg diazepam and 

nordiazepam) were used for the calibration curve. The analysis was performed on a Quatro micro MSIMS 

(Waters) coupled to an HPLC (Agilent). The separation column was a Zorbax SB-C8. The solvent consist 

of formic acid: ammonium acetate buffer pH 5.0: methanol (0.16:400:400). The masses: mlz 140 for 

nordiazepam and mlz 154 for diazepam were used for quantification. 

Results: Detection limit was 0,001 mglkg and the quantification limit was 0.002 mg/kg for both 

compounds. The calibration curves were linear in the measuring interval. The linearity was evaluated with 

polynomial regression. Within day precisions for blood controls spiked at 0.05 mg/kg, 0.5 mglkg and 1.5 

mglkg (diazepam and nordiazepam) were

A9 

. RESOURCE GUIDE FOR USERS OF SCIENCE AND TECHNOLOGY AND THE NATIONAL 

CLEARINGHOUSE FOR SCIENCE, TECHNOLOGY AND THE LAW 

Anjali R. Swienton*, Interim Deputy Director, Director of Outreach, National Clearinghouse for Science, 

Technology and the Law, Stetson University College of Law; SciLawForensics, Ltd., 25 Wahlutwood Ct., 

Germantown, MD; Carol Henderson, Director, National Clearinghouse for Science, Technology and the 

Law, Stetson University College of Law, 1401 61 51 Street South, Gulfport, FL 

New challenges for expert witnesses and the legal community have arisen due to recent developments in 

science and technology. New technologies and methodologies, as well as fields long considered 

established, such as latent print identification and tool marks are facing increased scrutiny. Given this 

explosion of scientific evidence litigation, scientists, law enforcement, laboratory personnel, judges and 

lawyers are overwhelmed by the amount of information required to educate them to meet these legal 

challenges. Until now, judges, lawyers, scientists and law enforcement personnel did not have one source 

that allowed them to navigate all the existing case law, journals, reports, proceedings and other resources 

necessary to conduct effective investigations and litigation. The Resource Guide for Users ofScience and 

Technology was created to fill an information need specifically relating to legal issues implicated by the use 

of new technology in criminal and civil justice. Supported by a joint cooperative agreement between the 

NFSTC and NIJ (#2000-RC-CX-K001), the project developed a comprehensive searchable database from a 

variety of sources covering a wide range of topics. The database provides information on topics such as 

bloodstain pattern analysis, body scans/retinal scans, digital image enhancement, entomology, expert 

witness malpractice, fingerprints, questioned documents, smart cards, toxicology, trace evidence and tool 

marks. The Resource Guide covers existing court rulings, pending court cases, scientific and legal articles 

from applicable sources, relevant information from books, current and pending legislation, conference 

proceedings, university and continuing education courses, and pronouncements from professional 

organizations. The NFSTCINIJ project produced a searchable CD. The information contained in the 

Resource Guide will be included in and expanded upon in the online resource being developed by the 

National Clearinghouse on Science, Technology and the Law at Stetson University College of Law. The 

Clearinghouse is supported by a grant from the National Institute of Justice (#2003-I1-CX-K024). In 

addition to the development of the online resource, the Clearinghouse Program is building partnerships 

with law schools, professional associations and federal agencies, sponsoring a forensic science/science and 

technology seminar series, convening Community Acceptance Panels at the request of NIJ, developing 

training modules qn a variety of forensic disciplines with an emphasis on distance education, and building a 

reference collection of law, science and technology literature available through interlibrary loan to other 

institutions. 

Keywords: Science and law, Information, Toxicology 

Page 123

AIO 

IDENTIFICATION OF BENZODIAZEPINES USING HPLC-ESI-MS-MS 

Brandi L. Bellissima·, Diane M. Boland, and W. Lee Hearn 

Miami-Dade Medical Examiner Department, Toxicology Laboratory, 1851 NW 10 th Avenue, Miami, 

Florida 33136 

A confirmatory method for twenty-three commonly detected benzodiazepines in postmortem biological 

matrices has been developed using solid phase extraction (SPE) coupled with high performance liquid 

chromatography positive ion electro spray ionization tandem mass spectrometry (HPLC-ESI-MS-MS). 

Isolation and purification of the drugs was performed using mixed-mode SPE columns installed on a 

Zymark RapidTrace system. After addition of internal standard, samples were diluted with sodium 

phosphate buffer and extracted using UCT Clean Screen® SPE columns. The SPE columns were 

sequentially rinsed with 2% ammonium hydroxide (NH 40H) in ethyl acetate, methanol, de-ionized water, 

and phosphate buffer. Samples were loaded onto the column at a flow rate of 2.0mLlmin. The cartridges 

were then washed with de-ionized water, followed by a wash with 20% acetonitrile in de-ionized water, 

and dried under vacuum prior to elution with 2% NH 4 0H in ethyl acetate mixture. Residues were 

reconstituted with the HPLC mobile phase and injected onto a Restek reversed phase C8 HPLC column. 

The analytes were eluted at a flow rate of 3751ll/min with a solvent mixture composed of methanol:water 

containing 0.1% formic acid, and detected using an Advantage LCQTM ion trap mass spectrometer. 

Positive ion ESI-MS resulted in ion spectra for each of the benzodiazepines at their expected retention time 

on the LC column. Unique MS-MS spectra for each of the benzodiazepines were obtained and matched the 

direct infused data acquired with neat standards (See Table I). The limit of detection for each 

benzodiazepine was estimated from extracted samples with decreasing concentrations. This method 

provides a rapid, sensitive approach to isolate, purify and confirm a broad spectrum ofbenzodiazepines. 

Table 1 Benzodiazepine Spectral Data 

Benzodiazepine Molecular Product Benzodiazepine Parent Product 

Ion Ion Ion Ion 

IM+Hr (amu) [M+Hr (amu) 

(amu) 

-(amu) 

7 -aminoclonazepam 286 250,222 Estazolam 295 267,192 

7 -aminoflunitrazepam 284 264,256 

H!! 

304 276,290 

7 -aminonitrazepam 252 121,114 raze pam 388 315,288 

Alphahydroxymidazolam 342 203,324 321 303,275 

Alphahydroxytriazolam 259 331,279 Midazolam 326 291,244 

i Alphydroxyalprazolam 325 297,279 Nltrazepam 282 236,254 

: Alprazolam 309 281.274 Nordiazepam 271 243,208 

Bromazepam 316 288,261 Norflunitrazepam 300 254,243 

Chlordiazepoxide 300 283,241 Oxazepam 287 269,259 

Clonazepam 316 270,251 Temazepam 301 283,255 

Desalkylfurazepam 289 261,226 Triazolam 343 226,308 

I Diazepam 285 257,228 

Keywords: Benzodiazepines, HPLC, ESI-MS-MS 

Page 124

All 

DIRECT ANALYSIS OF MDMA AND METABOLITES IN SALIVA USING CAPILLARY 

ELECTROPHORESIS 

Brandi L. Clelland-' I, Stephen L. Morgan l , Demi Garvin2, William E. Brewer 3 

1 University of South Carolina, Department of Chemistry and Biochemistry, 631 Sumter St, Columbia, SC 

29208; 2Richland County Sheriffs Department, 5623 Two Notch Rd, Columbia, SC 29223; 3Clemson 

University Veterinary Diagnostic Center, 500 Clemson Rd, Columbia, SC 29223 

Ecstasy, or 3,4-methylenedioxymethamphetamine (MDMA), has gained popularity among young adults in 

the past few decades mainly for its euphoric and stimulating effects. Though this drug has a "street" 

reputation of being safe, case studies have shown its toxic effects. Drug testing in recent years has focused 

on. analysis of alternative biological specimens because of the invasiveness and ease of adulteration of the 

more commonly used blood and urine. Testing of saliva as an alternative biological matrix has advantages 

over conventional matrices: saliva sampling is noninvasive and the presence ofMDMA in saliva correlates 

with individual being under the pharmacological effects of the drug. MDMA is a basic drug with an 

approximate pK.= 9.9. At two pH units below the pK., MDMA is fully charged and amenable for 

separation by capillary electrophoresis. Analysis of MDMA in saliva by CE also does not require 

derivatization or extraction; consequently minimal sample pretreatment is required. 

Capillary electrophoresis separates analytes by electrophoretic mobility, and allows a simplified rapid 

method for drug analysis. On-capillary detection in CE is generally performed by a diode array detector 

(DAD). Sample stacking is used to increase the sensitivity of the CE-DAD system. This paper demonstrates 

methods for the direct analysis of MDMA in saliva employing both CE-DAD as well as capillary 

electrophoresis/diode array/quadrupole time-of-flight mass spectrometry (CE/DAD/Q-Tof-MS). The DAD 

and Q-Tof-MS provide extremely sensitive on-line detection for CE as well as rapid component 

identification by UV-Vis or mass spectra. Limits of detection and reproducibility for the detection of 

MDMA and its primary metabolites will be discussed. 

Key words: MDMA, Saliva, Capillary electrophoresis 

Page 125

A12 

ANALYSIS OF XTC SEIZURES IN THE COUNTY OF TOURNAI (BELGIUM) 

B. Lebrun*, E. Noel; IPHB, Toxicology DepartlIjent, 55 boulevard Sainctelette 7000 Mons (Belgium) 

P. Ludinant; SJA de Tournai, 19 Dreve de Maire 7500 Tournai (Belgium) 

Introduction: By the early 1980s, ecstasy (XTC) was used as a recreational drug. Its consumption became 

particularly prominent in the United Kingdom, Spain, the Netherlands, Belgium and Germany. Recently, the 

dance culture is growing almost everywhere in Europe. Its usage was even described in less-festive events 

like athletic meetings. This led us to collaborate with the "Service Judiciaire d'Arrondissement" of Tournai 

for more than two years. In Belgium the presence of many "megadancing" is on the French - Belgian border. 

Relationship between cross-border consumption and cross-border traffic is not clear. Typological data 

(weight, logo, diameter, thickness, color ...) does not provide any information regarding synthetic procedures 

used by the laboratories nor it provides the location of the clandestine laboratories. In order to control the 

drug trafficking, it is important to know the synthetic procedures used by the laboratories and also the 

distribution networks. In this study we investigated the profiling of impurities in ecstasy and spatial analysis 

of seizures that could provide useful information to the legal authorities in controlling drug trafficking. 

Materials and methods: We analysed 2052 XTC pills received from 857 seizures realised in the County of 

Tournai. Each pill was examined for its colour, diameter, thickness, logo, and weight. The pills were then 

analysed for chemical impurities. Five milligram of the pill were treated by 1 ml of dichloromethane, mixed 

for 15 seconds, and centrifuged. The supernatant were separated and evaporated to dryness under nitrogen. 

The sample was then dissolved in 250 IJ.I of methanol and analysed by GC-MS. Results were encoded in a 

database and the profiling was analysed. Similarities between seizures were recorded in a larger Europol 

database. Analytical data were also introduced into a geographical information system in order to identifY 

clusters of similar profiles. 

Results: The typological analysis showed that sometime logos were not used on the pills. In fact, we 

observed four logo-use patterns: I) period for less than a month, 2) for several months, 3) for a year or 4) 

with a cyclical appearance. The pattem depends not only on the consumer (bad pills identified very fast) but 

also on the trafficker (capacity of production, material available). These observations provide information 

about the behaviour of dealers, traffickers, and tablet makers. The chemical impurities indicated primarily 

two synthetic routes used by the clandestine laboratories: one with reductive ami nation and the other with 

Leukart's method. The information allowed us to identify pills from different geographic locations and 

helped us to monitor and control drug trafficking. 

Conclusion: Spatial, typological and chemical results enabled us to build a database. The information is 

helpful in identifYing clandestine laboratories. It also provides information on the synthetic route used and 

the technical means employed to make the pills. This database permits to unveil links between different legal 

cases. 

Keywords: XTC, Profiling MDMA 

Page 126

A13 

SCREENING FOR ACE INHIBITORS AND AT-II-ANTAGONISTS IN URINE BY LC-MSIMS 

Barbora MaraIikova 1 ,2, Wolfgang Weinmann 1 , Claudia A. Muellerl,' 

lInstitute of Forensic Medicine, University Hospital, Albertstr. 9, D 79104 Freiburg (Germany) 

Phone: ++49 7612036878, Fax: ++49 761 2036858, Email: muel1erc@ukl.uni-freiburg.de 

2University of Par dub ice, FCHT, Department of Analytical Chemistry, nam. Cs.legii 565, 53210 

Pardubice, Czech Republic 

For the detection of Angiotensin-Converting Enzyme inhibitors (ACE inhibitors: enalapril, Iisinopril, 

perindopril, captopril, ramipril, cilazapril as prodrug and active drug) and Angiotensin-II-Receptor 

Antagonists (candesartan, eprosartan, irbesartan, \osartan, telmisartan, valsartan) an LC-MS/MS target 

screening method has been developed to be used in clinical and forensic toxicological analysis. These drugs 

are widely used for lowering blood pressure besides betablockers, diuretics and calcium channel blockers 

which have been the aim of our previous work [I] - and their intake after prescription or even the non 

compliance of patients, not taking the drugs, could have severe effects on traffic safety. Furthermore, 

overdosage ofthese drugs can cause severe lowering of the blood pressure. 

For method development by LC-MSIMS ESI and APCI mass spectra of the compounds have been 

compared. ESI was found to be more suitable in terms of sensitivity for the ACE inhibitors, whereas the 

AT -II-Receptor Antagonists were detectable by both ionization modes. 

Linearity for quantitation was found for captopril, cilazapril, enalapril, Iisinopril, perindopril, ramipril and 

ramiprilat in the range of 10 to 500 nglmL with ESI after extraction from urine. LOD was below 10 ng/ml 

in urine. In patients' urine samples the ACE inhibitors were detectable in their active form (carboxylic 

form) by electrospray ionization after intake of therapeutic doses. However, the active form was not 

available as reference substance for all compounds and had to be generated by hydrolysis of the esters. The 

internal standard still remains a problem for quantitative analysis, since deuterated compounds are not 

available. For patients' urine and serum samples Benazepril was used as internal standard for quantitation, 

since it was not prescribed for these patients. 

Method development for the sartanes included liquid-liquid extraction (LLE). LC-ESI-MRM was found to 

be suitable for target screening for these drugs after spiking to urine; irbesartan was detected in a forensic 

case. 

Results of the method development and applications to patients' urine and also some serum samples are 

presented. 

[1] Mueller CA, Baranda AB, Weinmann W. Screening for l,4-dihydropyridine calcium channel blockers 

in plasma and serum by solid-phase extraction and LC-MS-MS. J Mass Spectrom (in Press, 2004). 

Keywords: ACE inhibitors, AT II antagonists, LC-MS/MS 

Page 127

A14 

DETERMINATION OF FUROSEMIDE IN WHOLE BLOOD USING SPE AND GCIEI-MS 

C. Margalho',!, E. Gallardo!, M. Barroso!, S. Avila!, E. Marques\ D. de Boer, D. N. Vieira! 

!Instituto Nacional de Medicina Legal - Portugal 

2Instituto Nacional do Desporto Laboratorio de Analises de Dopagem e Bioquimica - Portugal 

Introduction: Furosemide (4-chloro-N-furfuryl-5-sulfamoylanthranilic acid) is an anthranilic acid derivative 

with strong diuretic potential. It exerts its action on the luminal side ofthe thick ascending limb ofthe Henle's 

loop by inhibiting chloride transport or sodium chloride co-transport. This loop diuretic is employed for the 

treatment of renal diseases, congestive heart failure and hypertension. Although furosemide is included on the 

Iist of prohibited doping substances, indicated by the World Anti-Doping Agency (W ADA), it is widely used 

in sports, mainly in competitions classified by weight or to avoid detection ofother drugs. 

The aim of this work was the development and validation of a methodology for application in forensic 

toxicology. A simple, rapid and validated gas chromatography-electron ionization-mass spectrometry (GeIEI 

MS) method is described to evaluate the use of whole blood in the quantitation of furosemide. A solid-phase 

extraction (SPE) technique was used to extract this compound from blood samples. 

Materials and Methods: Stock solutions of furosemide and ketoprofen (internal standard) were prepared in 

methanol, protected from light and stored in the freezer at 4°C until use. Extraction - To 1 ml of whole blood 

were successively added 50 /ll of internal standard solution (10 /lglml) followed by furosemide at different 

concentrations (0.10,0.25,0.50, 1.00,2.50 and 5.00 /lglml). The mixture was vortex-mixed, sonicated for 15 

min and centrifuged for 15 min at 3000 rpm. The sample was extracted using BondElut® -LRC Certify 300 mg 

SPE columns and the organic solvent evaporated to dryness in a vacuum rotary evaporator. Selective 

derivati=ation - The dry residue was dissolved with 25 /ll oftetramethylammonium hydroxide 0.2 M (TMAH) 

and injected directly into the chromatographic system. ChromatographiC conditions - Initial oven temperature 

was 160°C for 1 min, increased by 20°C/min to 270°C and held for 8.50 min. The temperatures of the injector 

and detector were set at 2500C and 280°C, respectively. The mass spectrometer was operated at 70 eV in the 

electron ionization (El) mode using selected ion monitoring (SIM). The ions were monitored at mI::, 81, 372 

and 96 for furosemide and 209, 205 and 268 for ketoprofen. 

Results: Calibration curves were measured daily, based in one-day assay protocol between 0.10 and 5.00 

/lglml, and correlation coefficients were above 0.9910. Control samples at low, middle and high 

concentrations of furosemide were measured in the same day. The calculated LOD and LOQ were 10.05 and 

45.44 nglml respectively. Intraday precision (coefficient of variation - CV) was inferior to 7.9 % for all 

concentration levels. For the interday precision, the calculated CV was inferior to 13.9 % for all control 

samples. The relative recovery calculated for the three levels of the control samples (0.30, 0.75 and 3.00 

/lglml) were respectively 104%, 89% and 91 %. 

Conclusion: A sensitive, specific and reliable procedure has been developed for the determination of 

furosemide in whole blood samples. The experimental work was performed so that all validation parameters 

are considered simultaneously in one day of assay. We may conclude that the validated methodology is 

suitable for the application in forensic toxicology routine analysis. 

Keywords: Furosemide, Whole blood, Solid-phase extraction 

Page 128

A15 

ANALYSIS FOR LORAZEPAM IN BLOOD AND URINE BY GAS CHROMATOGRAPHY 

MASS SPECTROMETRY - POSITIVE CHEMICAL IONIZATION 

David M. Andrenyak·. Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84112, 

USA. 

Lorazepam is a benzodiazepine drug that is used to treat anxiety and seizures. It is also prescribed as a pre 

operative sedative. Because lorazepam is a powerful CNS depressant and can enhance the effect of other 

CNS depressants, effective procedures for the analysis of lorazepam in biological samples are essential. 

This study reports on the analysis for \orazepam by gas chromatography - mass spectrometry (GC-MS) in 

biological samples. Liquid - liquid extraction was performed on 1 mL sample volumes using clean, 

separate 16 x 100 mm silanized culture tubes with PTFE lined screw caps. Prior to extraction, the urine 

samples were hydrolyzed by adding 1 mL of 5000 units I mL ~-glucuronidase in 0.1 M sodium Acetate, pH 

5.0. The urine samples were incubated 3 hours at 40' C in an incubation oven. Following incubation, 

internal standard (100 ilL of 1 ng/!lL lorazepam-d 4 ) was added to the urine and unhydrolyzed blood 

samples. A 1 mL volume of saturated sodium borate buffer (pH 9) and 5 mL oftoluene: dichloromethane 

(70:30) was added to each urine and blood sample. After mixing 20 minutes on a reciprocal shaker, the 

tubes were centrifuged at 2000 rpm for 10 minutes. The organic layer was collected into clean, separate 13 

x 100 mm culture tubes. The extracts were evaporated to dryness under a stream at air at 40' C using the 

Turboevap evaporator. The extracts were derivatized by adding 100 ilL of N,O-bis(trimethylsilyl) 

trifluoroacetamide +1% trimethylchlorosilane) to each tube. The extract tubes were heated at 70' C for 20 

minutes by using a dry block heater. After heating, the extracts were cooled to room temperature and 

transferred to clean, separate autosampler vials. The derivative reaction incorporated 2 trimethylsilyl 

(TMS) groups on the lorazepam molecule. The derivatized extracts were analyzed by GC-MS using an 

Agilent 5973 GC-MSD system. Chromatographic separation was achieved using an HP-1 MS 30 M x 0.25 

mm, 0.33 11m film capillary column (AgiJent) and UHP helium (1 mL/min) as the carrier gas. The column 

oven temperature program was 125' C, hold O. 2 minutes, 20· C/ minute to 300', hold 7.5 minutes. 

Splitless injection with injection port temperature at 250· C was used. The transfer line temperature was 

set at 300' C. Positive chemical ionization was utilized with ammonia as the reagent gas and an ion source 

temperature at 200' C. Selected ion monitoring was employed to analyze (mlz) 465 for 10razepam-diTMS 

and (m/z) 471 for lorazepam-d 4 -diTMS. The 10razepam-diTMS mass spectrum had a base peak at (m/z) 

465 and the 10razepam-~-diTMS mass spectrum had a base peak at (mlz) 469. Since 10razepam-diTMS 

had a significant (m/z) 469 ion fragment, the (m/z) 471 ion was used to monitor for the lorazepam-~diTMS. 

Calibration standards were prepared with blank bovine blood and ranged from 2.5 nglmL to 1000 

ngimL. In house controls were prepared with blank blood at 3.5 nglmL, 100 nglmL, 650 nglmL and with 

blank human urine at 100 ngimL. In the intra-assay accuracy evaluation, the lorazepam concentration was 

within 13% of target. In the intra-assay precision evaluation, the coefficients of variation were within 8%. 

This method was used to analyze blood and urine samples from a post mortem case. 

Keywords: Lorazepam , GC-MS, Positive Chemical Ionization 

Page 129

A16 

MATRIX DEPENDENT O-DEMETHYLATION OF 3-METHOXY OPIOIDS DURING 

DERIVA TIZA TION 

Dwain C. Fuller* and Paul J. Orsulak. Department of Psychiatry, University ofTexas Southwestern 

Medical Center at Dallas, Dallas, TX and VA North Texas Health Care System, Toxicology 113, 4500 

South Lancaster Road, Dallas, TX 75216 

Several in-vitro conversions of compounds to their metabolites during the extraction process have been 

observed by various investigators. Among these are the conversion of methamphetamine to amphetamine, 

methadone to EDDP, cocaine to benzoylecgonine, and ethanol to acetaldehyde. These authors have 

observed the O-demethylation of the 3-methoxy opioids, codeine, hydrocodone and oxycodone to their 

respective metabolites, morphine, hydromorphone and oxymorphone, during derivatization with N 

methylbistritluoroacetamide (MBTFA). The transformation was observed to be dependent on specimen 

matrix, incubation time and incubation temperature. It is important for the toxicologist to be aware of this 

phenomenon when developing analytical assays and interpreting results. 

Key words: Opioids, Demethylation, MBTF A 

Page 130

A17 

HIGH.PERFORMANCE LIQUID CHROMATOGRAPHY AS AN ALTERNATIVE METHOD 

FOR COMPARATIVE ANALYSIS OF SEIZED DRUG SAMPLES 

2 

Bogumila Byrska\ Dariusz Zuba*\ Malgorzata Swisf, Andrzej Parczewski l • 

I Institute of Forensic Research, ul. Westerplatte 9,31-033 Cracow, Poland; 2 Faculty of Chemistry, 

Jagiellonian University, ul. Ingardena 3, 30-060 Cracow, Poland 

The comprehensive testing of seized drug samples performed in forensic laboratories allows identification 

and determination of the controlled psychoactive substances, identification of additives (e.g. caffeine or 

paracetamol) and diluents (e.g. palmitylic acid or glucose), as well as chemical impurities. The results of 

analysis deliver information about synthesis methods applied by illegal producers. It is also possible to 

establish if the seizure drug samples originate from the same production batch or the same illegal 

laboratory and even to link the dealer with drug users. Hitherto, mainly gas chromatographic methods 

(GClFlO, GCIMS) are used for this purpose. 

The aim of the present study was to develop an analytical procedure that makes possible separation of the 

substances occurring in the seized ecstasy tablets by means of the high-performance liquid chromatography 

(HPLC). The studies have been carried out on the samples of 3,4-methylenedioxymethamphetamine 

(MDMA, ecstasy) prepared by different routes: reductive amination, Leuckart reaction and safrole 

bromonation. The reductive amination was performed using various reducing agents: sodium 

cyanoborohydride, aluminium amalgam and sodium borohydride. In order to estimate the repeatability, 

each synthesis route was repeated three times. HPLC analyses were carried out on LaChrom D·7000 

System (Merck-Hitachi) liquid chromatograph. 

The solid-phase extraction (SPE) was used as the sample preparation method. FIrst, 200 mg of ecstasy 

sample was dissolved in mixture of 1.5 ml of the ammonium buffer (pH 8.5) and 0.5 ml of 30 mg/l 

diphenylamine solution (internal standard). Than the suspension was vigorously shaken for 25 min at 2000 

rpm. The optimisation ofSPE conditions was focused on selection of the appropriate column filling, which 

enables to bind the main compound and to thicken the impurities that are important in view of comparative 

analysis. Bakerbond spe Octadecyl (3 ml, 500 mg) turned out to be the most effective. SPE was 

performed as follows: 1.5 ml of sample was rinsed with 6.0 ml of water and 0.3 ml of methanol and the 

impurities were extracted with 0.8 ml of methanol. Than, 0.3 ml of collected fraction was diluted with the 

phosphoric acid solution up to pH of 6. 

Chromolith Performance RP-18 e (100 - 4.6 mm) monolithic column was used in HPLC analysis. Taking 

into account the variety of physicochemical properties of the impurities, the separation was done in 

gradient conditions. In order to optimise the resolution, the simplex method was applied. Four factors were 

taken into account which describe gradient conditions. After eight steps of optimisation the following 

conditions were selected: 0 min - 100% of phase A (water + 0.1 mIll phosphoric acid), 21.3 min - 59% of 

phase A + 41 % of phase B (acetonitryle), 32.7 min 100% of phase B, 34 min 100% of phase A. The 

flow was 1.0 mUmin. Diode array detector (DAD) was used. 

The applied conditions of SPE extraction and HPLC analysis allow good separation of the characteristic 

impurities occurring in ecstasy samples. Because most of the impurities give similar UVIVIS spectra, it is 

impossible to identifY them using diode-array detector. It is highly probable that application of LCIMS 

system will solve this problem. The obtained HPLC chromatograms could be treated equivalently to FlO 

chromatograms where the impurities pattern is a subject to further chemometric analysis. The preliminary 

results show that the worked-out method allows to distinguish the samples prepared by different synthesis 

route as well as it could be useful in comparative analysis of seized drug samples. 

The study was supported by the grant of the State Committee for Scientific Research, Warsaw, Poland, 

number OT OOC 01024. 

Keywords: Comparative analysis, Ecstasy, HPLC 

Page 131

Al8 

EV ALUA nON OF A PORTABLE EVIDENTIAL BREA TH ALCOHOL ANALYZER 

Gerasimos Razatos*, Ruth Luthi, Sarah Kerrigan 

New Mexico Department of Health, Scientific Laboratory Division, Toxicology Bureau. PO Box 4700, 

Albuquerque, NM 87196-4700. 

The Scientific Laboratory Division of the Department of Health acts by mandate as the regulatory agency 

for the Implied Consent Program for the State ofNew Mexico. The Laboratory is responsible for all blood 

and breath alcohol testing activities for law enforcement statewide. The geographical size and the nature of 

the state, characterized by a highly rural population, demands portable breath alcohol testing equipment. 

Moreover, future expansion and success of the breath testing program has focused on instrument portability 

and data management as critical issues amongst law enforcement agencies and the courts. Thus, the Implied 

Consent Section of the SLD evaluated the performance of the Intoxilyzer® 8000, a portable instrument, 

against the Intoxilyzer® 5000, a stationary instrument which is currently approved for use. 

Instrument performance was evaluated at various alcohol concentrations, ranging from 0.04 to 0.55 g/dL in 

blood or gl210L breath. Special attention was placed on instrument performance at the per se and 

aggravated DWI levels of 0.08 gldL and 0.16 gldL, respectively, due to their legal significance. Precision 

and accuracy were evaluated using in-house ethanol controls in a wet bath simulator. Coefficients of 

variation using the Intoxilyzer® 8000 ranged from 0.30 - 1.3% (n=102), while CV ranges for the 

Intoxilyzer~ 5000 were 0.7 2.1 % (n=102). Calibration stability was assessed in addition to the 

distribution of data at concentrations between 0.04 and 0.55 g/210L. Accuracy was 100 102% for the 

Intoxilyzer® 5000 and 99-101 % using the Intoxilyzer® 8000. Linear regression analysis of more than 700 

comparative measurements revealed an R2 of 1.000 (y = I.005x 0.001), where the lntoxilyzer® 5000 and 

the Intoxilyzer® 8000 were plotted on the y and x-axis respectively. Instrument response to mouth alcohol 

and volatile interferences was also investigated. Potential interferences were evaluated alone or in 

combination with ethanol using a wet bath simulator at 34.0"C. 

The effects of extreme temperature and altitude were also examined using wet bath simulators and dry gas 

cali brant. Accuracy and precision were evaluated at high and low temperatures. High altitude performance 

was evaluated at 10,600 feet above sea level at a local ski resort. In addition to the scientific study, field 

evaluations were also conducted by law enforcement personnel. Based upon the results of the study, the 

Intoxilyzer® 8000 was approved as an evidential breath alcohol analyzer in the State of New Mexico. 

Keywords: Intoxilyzer 8000, Breath, Alcohol 

Page 132

A19 

PERFORMANCE OF ASSAYS FOR THERAPEUTIC DRUG MONITORING AND URINE 

DRUGS-OF-ABUSE SCREENING ON A CONSOLIDATED WORKSTATION 

H. Luthet, I. Domke 2 , S. 10rdan·· J, M. Oellerich I, W. Stockmann 2 

IGeorg-August-Universitaet, Goettingen, Germany, 2Roche Diagnostics GmbH, Mannheim, Germany, 

JRoche Diagnotics Corp., Indianapolis, USA 

Introduction: The study objective was the assessment of performance of nine Therapeutic Drug Monitoring 

(TOM) assays and nine Drugs-of-Abuse assays (OAT) when processed in combination with simulated 

routine workload on Modular Analytics P (Roche Diagnostics). 

Methods: All TOM and OAT assays involve the kinetic interaction of microparticles in solution (KIMS) 

except one enzymatic method. The study design comprises imprecision in simulated routine runs. The 

experiment tests for potential systematic or random errors by comparing the imprecision of reference results 

(standard batch) with results from samples run in a pattern simulating routine sampling. The study was 

supported by a program for computer aided evaluation (CAEv). 

Results: Within-run CVs of the reference runs ranged from 2 to 5 % for the majority of TOM 

(Acetaminophen, Amikacin, Carbamazepine, Digitoxin, Gentamicin, Lidocaine, Phenobarbital, Phenytoin) 

and OAT assays (Barbiturates, Cannabinoids, Cocaine Metabolite, Methadone, Opiates, Phencyclidine, 

Propoxyphene). CVs up to 7 % were obtained for Digoxin, Amphetamines, and Benzodiazepines. Routine 

simulation resulted in slightly increased CVs of maximal 2 %. This is in agreement with experience from 

previous routine simulation studies. 

Conclusion: With these results we conclude that no relevant reagent interactions occur on Modular Anaiytics 

P when using it as a consolidated workstation for TOM, OAT, serum proteins and general chemistry assays. 

Keywords: Therapeutic Drug Monitoring, Drugs-of-Abuse-Screening, Consolidated Workstation 

Page 133

A20 

VALIDA TION OF A HPLC METHOD FOR QUANTIT A TION OF MDMA IN TABLETS AND 

REPRESENTA TIVE DRUG SAMPLING 

LB. MUlier', B.B. Klinke; Department of Forensic Chemistry, Institute of Forensic Medicine, University of 

Copenhagen, Frederik V's Vej 11, DK-2100 Copenhagen, Denmark 

A method for quantification of MDMA (ecstasy) in powder/tablets using HPLC-DAD will be described. 

The powder was extracted with HPLC solvent (KHzP0 4 -buffer, pH 3.2:Acetonitrile (9:1» in 5-10 minutes, 

and analyzed using HPLC-DAD. The column used was a Chromspher B5 column at 30 °C and 0,4 ml/min 

solvent flow. The identity was confirmed using UV-detection with a UV-curve fit of900 or more and peak 

purity of 990 or more. The specificity of the method has been examined for other phenyl amines, and 

showed that only PMMA co-elutes with MDMA. The robustness of the method was good, and minor 

changes in solvent composition and solvent flow was possible. The method uncertainty was found to be 

10%. The limit of detection was 0.001 mg/ml and the limit of quantification was 0.003 mg/mt. 

Representative sampling of the ecstasy tablets was applied prior to MDMA analysis. Visual inspection was 

done to establish if a seizure was heterogeneous or homogenous. Heterogeneous seizures were divided in 

populations of similar appearance and were treated separately in further analysis. The validated method of 

MDMA analysis and sampling of tablets will be presented in the poster. 

Keywords: MDMA, HPLC-DAD, Sampling. 

Page 134

A21 

ALCOHOL DETERMINATION BY HEAD SPACE DUAL COLUMN CAPILLARY GAS 

CHROMATOGRAPHY 

Jlpo Rasanen', Ilkka Ojanpera, Kaisa Kurkinen, Juhani Vartiovaara, Erkki Vuori 

Department of Forensic Medicine, PO Box 40, FIN-00014 University of Helsinki, FINLAND 

A head space dual column capillary gas chromatographic method was developed for the simultaneous 

determination of ethanol, methanol, acetone, isopropyl alcohol, ethyl methyl ketone and n-propanol in 

blood and urine. The method consisted of two identical instruments and conditions, but using different 

internal standards, I-propanol and 2-butanol. 

The gas chromatographs were 6890 Series GCs equipped with two flame ionisation detectors and 

ChemStation software (Agilent Technologies, USA). The autosamplers were Combi Pal Head space 

samplers (CTC Analytics, Switzerland). The parallel capillary columns were a Rtx-BACI, 30m x 0.53mm, 

3 !lm film and a Rtx-BAC2, 30m x 0.53mm, 2 !lm film (Restek, USA). The columns were connected to 

single injector with a Graphpack Divider (Gerstel, Germany). The head space conditions were as follows: 

incubation temperature 60 °C, incubation time 5 min (agitation speed 500 rpm), syringe temperature 65 °C, 

injection volume 500 !ll. The GC conditions were as follows: split ratio I :20, column flow rate 10.7 mllmin 

(He), injection port temperature 200 °C, oven temperature 45°C (4 min), detector temperature 250°C, H2 

flow 40 ml/min, air flow 450 mllmin, make-up flow (N2) 45 mllmin. The total analysis time was 5 min 50 

s. 

A baseline separation was obtained for all six compounds on both columns. Thirty-eight compounds were 

tested for interference. Methyl acetate co-eluted with I-propanol on Rtx-BACI and with acetone on Rtx 

BAC2. No other compound was found to co-elute with an analyte on both columns. However, ten 

compounds co-eluted with an analyte on one column. The reporting limit was set to 0.2 mg/g for ethanol 

and 0.1 mglg for the other analytes. The method was accredited for the determination of ethanol in blood 

and urine according to the standard SFS-EN ISO/IEC 17025. The uncertainty of measurement was 4 % for 

both blood and urine samples. 

The present method takes advantage of the facility of the head space sampling by gas-tight syringe over 

loop or pneumatic systems. Two dual-column instruments with different internal standards comprise a 

reliable but still compact set up for forensic alcohol determination. 

Key words: Head space GC, Blood alcohol, Capillary column 

Page 135

A22 

METAL AND METALLOID MULTIELEMENTARY ICP-MS VALIDATION IN WHOLE 

BLOOD, PLASMA, URINE, AND HAIR. REFERENCE VALUES 

J.P. Goulle", L. Mahieu, J. Castermant, N. Neveu, L. Bonneau, G. Laine, D. Bouige, C. Lacroix 

Groupe Hospitalier du Havre, B.P. 24, F-76083 Le Havre, France 

Four multi-elementary metal and metalloid quantification methods using inductively coupled plasma mass 

spectrometry (ICP-MS) have been developed and validated in human whole blood, plasma, urine and hair 

using a single preparation procedure for each sample. 

The ICP-MS measurements were performed using a Thermo Elemental X7CCT series and PlasmaLab 

software without a dynamic reaction cell. Twenty-four to thirty-two elements are simultaneously quantified 

in biological matrices: Li, Be, B, AI, Y, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Pd, Ag, Cd, 

Sn, Sb, Te, Ba, W, Pt, Hg, TI, Pb, Bi, U. 0.4 ml whole blood, plasma and urine sample are acid diluted with 

purified water prepared extemporaneously, triton X100 and butanol. In and Rh are used as the internal 

standards. For the urine samples, the results were corrected after enzymatic creatinine determination. 25 mg 

hairs are acid mineralized after a decontamination procedure and diluted as previously described for the 

biological fluids. To be validated, each element has to force: linearity with a correlation coefficient higher 

than 0.99. The intra-assay and inter-assay imprecision measured as the CY should be below 5% and 10% 

respectively. Global performance was assessed by quality control program. Our laboratory is a registered 

participant of the Institut National de Sante Publique du Quebec (Sainte-Foy, Canada) inter-laboratory 

comparison program for whole blood, urine, and beard hair of non-occupationally exposed individuals 

spiked with selected elements. 

Multi-element metal and metalloid analysis has been validated for 24 elements in the whole blood, 27 

elements in the plasma, 29 elements in the urine and 32 elements in the hair, from 0 to 25, 250 or 1000 

ng/ml owing to the element. Quantification limits range from 0.002 ng/ml (U) to 4.4 ng/ml (B) for the 

whole blood, from 0.002 nglml (U) to 7.7 ng/ml (AI) for the plasma, from 0.001 nglml (U) to 2.2 ng/ml 

(Se) for the urine, and from 0.2 pglmg (Tl) to 0.5 nglmg (B) for the hair. Normal values have been 

determined in the whole blood, the plasma, the urine (n=100), and the hair (n=45) based on healthy 

volunteers, leading to approximately 10,000 analyses. AI results are presented and discussed. Some clinical 

toxicology and forensic toxicology applications are reported. 

ICP-MS has made significant advances in the field of clinical biology, particularly important in 

toxicological analysis. This is due the use of extremely effective equipment that permits better clinical and 

forensic toxicological analysis of metals and metalloids status of each individual. 

Keyswords: Inductively coupled plasma, Mass detector, Metalloids 

Page 136

A23 

THE EXTRACTION AND ANALYSIS OF FLUNITRAZEPAM FROM WHOLKBLOODIURINE 

USING A NOVEL SOLID PHASE EXTRACTION PROCEDURE AND HIGH PERFORMANCE 

LIQUID CHROMATOGRAPHY-PHOTODIODE ARRA Y DETECTION 

Jeffery Hackett*, Forensic Toxicology Laboratory, Center for Forensic Sciences, Syracuse NY 

Jeanna M. Marraffa" Fellow in Clinical Toxicology, Poison Control Center, Syracuse NY 

Flunitrazepam, has been well recognized as a drug of concern within the forensic toxicology community 

for many years. Several methods have been published for the analysis and detection of the drug 

previously.I-3 The method described in this poster takes a novel yet simplified approach to the extraction! 

detection of flunitrazepam in biofluids. Solid phase extraction followed by high performance liquid 

chromatography is employed without the need for a mass spectrometry unit for detection of low levels of 

the drug. Data is presented to exhibit the efficiency of the method in terms of limit of detection and 

quantitation_ 

Blood and urine were spiked with both the drug (flunitrazepam) and an internal standard (Prazepam) over a 

range of concentrations (O-IOng mL- 1 ). The samples were buffered with a small volume ofO.IM acetic acid 

and applied to commercially available mixed mode (Cs/SCX) columns that had been previously 

conditioned. The columns were washed, dried and eluted twice, once using ethyl acetate (3 mL) and again 

using an elution solvent containing ethyl acetate/ acetonitrile! ammonia (3 mL). Both portions ofthe eluates 

were collected and combined. Evaporation was performed at room temperature. The residue was dissolved 

in 100 JlI of0.1 % aqueous trifluoroacetic acid. 

LC Conditions: 

Column: 

Mobile phase: 

Flow Rate: 

Column Temperature: 

Detection: 

Injection Volume: 

CIS, ISO x 2.lmm, 3.SJlm 

0.1 % Trifluoroacetic acidl Acetonitrile (67: 33) 

0.3 mL min-I 

. 40°C 

220, 2S0 nm 

SOJlL 

References: 

1. LeBeau M.A., Montgomery M_A., Wagner J.R., and Miller M.L., J Forensic Sci. 2000 

Sep;4S(S): 1133-41. 

2. Ahrens B., Schwandt H.J., and Schutz H., Ar:neimitteiforschung. 2000 Dec;SO(12):10S7-62. 

3. Elian A.A., Forensic Sci Int. 1999 Apr 26;101(2):107-11. 

Keywords: Flunitrazepam, Solid-phase extraction, Blood and urine 

Page 137

A24 

OHIO DEPARTMENT OF HEALTH EV ALVA TION OF THE INTOXILYZER 8000 

John J. Kucmanic*, Dean M. Ward, James E. Dykes, Ohio Department of Healthl Bureau of Alcohol and 

Drug Testing. 35 E. Chestnut Avenue, Columbus, Ohio 43215, USA 

Recently, a new Breath Alcohol testing instrument was evaluated by the Ohio Department of Health. The 

Intoxilyzer 8000 is a smaller shielded plastic body breath alcohol analyzer that utilizes a pulsed infra-red 

energy source.as well as makes use of a primary filter in the nine micron range to quantifY alcohol. The 

instrument is engineered with the latest generation of power converters. This technology significantly 

reduces the weight of the instrument so that it can easily be carried using the attached handle. Furthermore, 

the instrument can operate in a limited fashion with an optional battery pack or continuously with a 12 

VDC source such as a cigarette lighter plug. No electrical inverter is necessary to operate the device in 

mobile situations. 

We examined several properties ofthis unit. Specifically, we looked at the in-vitro performance oftwo like 

instruments. Nine solutions were prepared in eight liter carboys to contain a range of concentration of 

ethanol and or acetone while purified water was used to evaluate a negative sample. The prepared solutions 

were analyzed by the Bureau on a Hewlett Packard 6890/7693 gas chromatograph using a dual capillary 

column headspace method. Standards were prepared from SRM 1828a and reagent grade acetone. All 

simulators used throughout the study were certified by the manufacturer prior to the evaluation. 

Furthermore, simulators were dried and replenished with a new bottle of solution after every twenty tests. 

As a consequence of rapid acetone depletion, acetone solutions were replaced after every ten tests. Transfer 

hoses which were connected to the calibration inlet during the evaluation were purged with dry air after 

every five tests. This procedure minimized the build up of excess condensate that would accumulate over 

the course oftesting. 

Mean concentrations of ethanol reported and the % coefficient of variance did not deviate by more than 5% 

in both instruments. A regression analysis between the mean alcohol concentration of twenty tests and the 

target concentration indicated a correlation of greater than 0.999 for both instruments over the respective 

concentrations. Prepared solutions containing acetone at a clinically significant concentration with or 

without ethanol were detected as an interferent for both instruments. Our results correlated well with the 

NHTSA evaluation ofthe Intoxilyzer 8000 which examined ten replicates oftive ethanol concentrations of 

0.020,0.040,0.080,0.160, and O.300g/21OL. 

In conclusion, the results from the in-vitro test performed on the Intoxilyzer 8000 instrument demonstrate 

that it is accurate, precise, and linear at the concentrations of ethanol tested. Furthermore, the results 

demonstrate that the unit is able to detect the presence of acetone vapors or a combination of acetone and 

ethanol vapors at the respective concentration for each volatile. 

Keywords: Intoxilyzer, Alcohol, Breath 

Page 138

A25 

A RAPID METHOD FOR MEASURING ANTI-DEPRESSANTS IN POSTMORTEM BLOOD 

USING DUAL COLUMN LIQUID CHROMATOGRAPHY MASS SPECTROMETRY 

Jason H. Sklerov' 

Office of the Armed Forces Medical Examiner, Division of Forensic Toxicology, Armed Forces Institute of 

Pathology, Rockville, MD, U.S.A. 

A dual column, liquid chromatography mass spectrometry method was developed for the analysis of eleven 

anti-depressants and metabolites in 200uL of postmortem blood. Sample preparation consisted of internal 

standard addition, protein precipitation, and microfiltration. Filtrates were injected onto an extraction 

column (Oasis® HLB, 20 x 2.1mm, dp=25um) and washed with deionized water, 20% acetonitrile, at 

3mLlmin. The trapped analytes were backflushed through a 10-port switching valve onto an analytical 

column (Zorbax® SB-CI8, 30 x 2.1mm, dp=3.5um) using a gradient of acetonitrile (20-80%) and 0.1% 

formic acid at a flow rate of O.5mLlmin. The positive, pseudomolecular ions of imipramine, desipramine, 

amitriptyline, nortriptyline, fluoxetine, paroxetine, fluvoxamine, trimipramine, N-desmethyl-trimipramine, 

clomipramine, and N-desmethyl-clomipramine were monitored following electrospray ionization. Overall 

instrumental analysis time was 11 minutes per sample including extraction and detection. Sample 

carryover, assessed by area% of signals in blank injections, was < 1.4% by the use of both a needle wash 

phase and injector programming. Linear ranges varied within a 0.025-lmg/L range and the online 

extraction recovery was between 75-105%. Additional time savings could be gained by the direct injection 

of diluted plasma, however, due to the inconsistencies in sample collection and the potential for signal 

suppression, a protein precipitation step was adopted for this work. 

Keywords: Anti-depressants, Dual-column, LC/MS 

r-- 

Page 139

A26 

1-(4-METHOXYPHENYL)-1-PROPANAMINE AS CONTAMINANT IN 1-(4-METHOXY 

PHENYL)-2-PROPANAMINE (PMA) PREPARED VIA THE LEUCKART REDUCTIVE 

AMINA TION METHOD 

D Waumans', B Hermans, N Bruneel, J Tytgat 

Laboratory of Toxicology, K.U. Leuven, Eduard van Evenstraat 4, B-3000 Leuven, Belgium 

Introduction: 1-(4-Methoxyphenyl)-2-propanamine (4-methoxyamphetamine, PMA) is an amphetamine 

analogue (co-)responsible for a series of lethal drug accidents the last few years. It is often traded as 

MDMA (N-methyl-I-(3,4-methylenedioxyphenyl)-2-propanamine) and facilely prepared from anethole, the 

main component of (star) anise oil. From a forensic point of view, the profiling of synthesis impurities that 

can be attributed to a specific synthetic pathway is very important. The following presents 1-(4 

methoxyphenyl)-I-propanamine as a new PMA impurity. It is formed via the Leuckart reductive amination 

of 1-(4-methoxyphenyl)-I-propanone, an impurity arising from the peracid oxidation of anethole. 

Methods: Synthesis: The performic oxidation reaction of anethole, the pinacol-pinacolone rearrangement 

and Leuckart reductive ami nation were performed using standard reaction conditions. 1-(4 

methoxyphenyl)-I-propanone was prepared quantitatively via a solventless Friedel-Crafts acylation 

reaction of anisole with lithium perchlorate as catalyst. GCIMS analysis: Agilent 6890 Plus GC coupled to 

Agilent 5973N mass selective detector (EI, 36 - 400 amu, 70 eV). Column: Varian VF5-MS FactorFour 

capillary (30 m x 0.250 mm x 0.25 flm). Injection and oven programming: I flL injection (split I: 10); 50°C 

(hold I min), 5°C/min to 270°C (hold 15 min). Carrier gas: He (I mLlmin). 

Results: The peracid oxidation of anethole yields anethole glycol (and esters), which is converted to 1-(4 

methoxyphenyl)-2-propanone by refluxing in a methanol/sulfuric acid mixture. Also, we have 

demonstrated that 1-( 4-methoxypheny l)-I-propanone is formed as a contaminant during the peracid 

oxidation of anethole. Both substances can serve as substrate in the Leuckart reductive amination reaction, 

where 1-( 4-methoxyphenyl)-I-propanone will yield 1-(4-methoxyphenyl)-I-propanamine and 1-(4 

methoxyphenyl)-2-propanone will yield 1-(4-methoxyphenyl)-2-propanamine. When exammmg 

confiscated PMA preparations, 1-( 4-methoxyphenyl)-I-propanamine is found to be present next to PMA, 

hereby giving an indication for anethole's application as precursor. 

Conclusion: 1-(4-Methoxyphenyl)-I-propanamine is a new impurity formed during the Leuckart reductive 

amination reaction of 1-(4-methoxyphenyl)-I-propanone, a contaminant produced in the peracid oxidation 

reaction of anethole. Its formation is demonstrated by a simulated synthesis, and its applicability is proven 

by its presence in confiscated clandestine PMA samples. There are only few data available on the 

pharmacology and toxicity of this newly discovered impurity. 

Keywords: Profiling, PMA, 1-( 4-Methoxyphenyl)-I-propanamine 

Page 140

A27 

EFFECT OF SODIUM CHLORIDE ON HEADSPACE BLOOD ALCOHOL ANALYSIS BY 

GC-FID 

Janice Yazzie*, Ruth Luthi, Sarah Kerrigan 

New Mexico Department ofHealth, Scientific Laboratory Division, Toxicology Bureau, PO Box 4700, 


Sodium chloride is frequently utilized as an additive for headspace alcohol analysis. The addition of salt to 

the mixture in the headspace vial increases the partial pressure of volatile compounds including alcohol and 

the internal standard. This is advantageous because the concentration of alcohol in the vapor phase is a 

function of both the temperature and the concentration ofalcohol in the liquid phase. 

The effect of salt was investigated in a series of experiments in which sodium chloride was added to the 

internal standard solution containing n-propanol and t-butanol. Ethanol was quantitatively determined using 

an Agilent HP 6890 GC equipped with dual capillary columns and a flame ionization detector (FlO). 

In-house and external whole blood controls were used for comparison purposes. When no salt was added, 

CVs ranged from 1.3 to 4.0% for alcohol concentrations between 0.04 and 0.30 g/dL. By comparison, CVs 

using the salt solution ranged from 1.2 to 4.2%. Accuracy was 100-105% and 99-109%, when either salt or 

no salt was added, respectively. A comparison ofquantitative values obtained by both methods showed that 

the results were not statistically different. A total of 80 antemortem and postmortem casework samples 

were included in the study. These ranged in concentration from 0 to 0.567 gldL ethanol. Linear regression 

analysis showed an R2 value of0.996 and a mean difference of 0.001 g/dL between methods. 

Keywords: Ethanol, Sodium chloride, GC-FID 

Page 141

A28 

HIGH THROUGHPUT SCREENING OF DRUGS OF ABUSE BY LCIMS 

Jerry Zweigenbaum*; Agilent Technologies, Wilmington, DE, USA 

Kolbjorn Zahlsen, Trond Aamo; Department of Clinical Pharmacology, St. Olav Hospital, University 

Hospital ofTrondheim, Trondheim, Norway 

Drugs of abuse are screened in blood and urine throughout the world by immunoassay. This technology 

has the advantages of high-throughput, inexpensive and generally applicable as a screening tool. However, 

there is a reasonable possibility for both false positive and false negative results. In addition, to be reliable 

the methodology must typically utilize a high concentration cutoff. In contrast, screening by a highly 

selective methodology as liquid chromatography/mass spectrometry (LCIMS) is less susceptible to false 

negatives and positives. Because of its sensitivity it has a much lower cutoff than immunoassay. 

One argument for immunoassay lies in its ability to detect classes ofcompounds and therefore would detect 

designer drugs not listed as a sought compound. LC/MS screening for specific drugs of abuse would miss 

this. However, all immunoassay positives must be confirmed, as there is no indication of what the 

substance is. This step would, like LCIMS, miss the non-listed substance. 

I f an immunoassay were to produce a positive screen for amphetamines, there would be no indication of 

what the compound(s) was. However, LCIMS would provide the specific amphetamine and its 

concentration. A clinician reviewing these data in the screen can make assessment about dose and the 

possibility of confounding substances that may also be present. It is this additional data that makes the 

LC/MS screen both reasonable and desirable from a quality of results standpoint. 

We will present how the Department of Clinical Pharmacology performs high-throughput drugs-of-abuse 

screening using LCIMS. The operation of the instrumentation and maintenance procedures will be 

described showing how large numbers of samples are analyzed side-by-side for both drugs-of-abuse and 

therapeutic drug monitoring. Additionally, the quality control processes needed to assure all results are 

accurate will be discussed. These processes encompass the sample handling, preparation, analysis, and 

reporting ofalmost one million determinations (equivalent immunoassays) per year run on 24 instruments 

operating seven days a week. 

Keywords: Screening, Drugs-of-abuse, LC/MS 

Page 142

A29 

SIMULTANEOUS DETERMINATION OF COCAINE, COCAETHYLENE, AND THEIR 

POSSIBLE PENTAFLUOROPROPYLA TED METABOLITES AND PYROLYSIS PRODUCTS BY 

GAS CHROMATOGRAPGY IMASS SPECTROMETRY 

Patrick S. Cardona', Arvind K. Chaturvedi, John W. Soper, and Dennis V. Canfield. Bioaeronautical 

Sciences Research Laboratory (AAM-610), Aerospace Medical Research Division, Civil Aerospace 

Medical Institute, Federal Aviation Administration, U. S. Department of Transportation, P. O. Box 25082, 

Oklahoma City, OK 73125-5066, USA 

Cocaine (COC) is abused by smoking, nasal insufflation, and intravenous injection, and it is also taken with 

ethanol. Therefore, it is important to determine concentrations ofCOC and its metabolites, ethanol analogs, 

and pyrolysis products for establishing the degree of toxicity, the possible ingestion of ethanol, and the 

possible route of administration. In this study, a sensitive and selective procedure is developed for the 

simultaneous analyses of COC, benzoylecgonine, norbenzoylecgonine, norcocaine, ecgonine, ecgonine 

methyl ester, m-hydroxybenzoylecgonine, anhydroecgonine methyl ester (AEME), anhydroecgonine 

(AECG), cocaethylene, norcocaethylene, and ecgonine ethyl ester in blood, urine, and muscle homogenate. 

In the analysis, available deuterated analogs of these analytes were used as internal standards. Proteins from 

blood and muscle homogenate were precipitated with cold acetonitrile. After the removal of acetonitrile by 

evaporation, the supernatants and urine were extracted by solid-phase chromatography. The eluted analytes 

were converted to hydrochloride salts and derivatized with pentafluoropropionic anhydride and 2,2,3,3,3 

pentafluoro-l-propanol. The derivatized products were analyzed on a gas chromatograph (GC)/mass 

spectrometer by selected ion monitoring. 

This method was successfully applied in analyzing 13 case specimens from aviation accident pilot fatalities 

and/or motor vehicle operators. AEME concentrations found in the 13 specimens were consistent with 

those produced solely by the GC inlet pyrolysis of COC controls, suggesting that COC was not abused in 

these cases by smoking. Although AEME remains a potential marker for establishing the abuse of COC by 

smoking, AECG was not a useful marker because of its low recovery and GC inlet production from COC 

metabolites. 

The developed procedure is unique because multiple analytes can be analyzed in urine, blood, and solid 

tissues by a single extraction with increased sensitivity through formation of hydrochloride salts and using 

a one-step derivatization. 

Keywords: Cocaine, Metabolites, Pyrolysis Products, Gas ChromatographylMass Spectrometry 

Page 143

A30 

RAPID ANALYSIS OF THe AND METABOLITES IN WHOLE BLOOD 

William E. Brewer', \ Dwight Flammia 2 , Randall Edwards 2 , and Terri Wood 2 , IClemson Veterinary 

Diagnostic Center, P.O. Box 102406, Columbia, SC 29224, 2Common Wealth ofVirginia, Dept. of 

Criminal Justice Service, 700 N. Fifth St., Richmond, VA 23219 

Extraction of whole blood samples for tetrahydrocannabinol (THC) and metabolites may be timeconsuming, 

especially with liquid-liquid extraction methods. Recently, a novel solid-phase extraction (spe) 

method has been developed for extracting THC and metabolites from whole blood. This spe method uses a 

pipette tip that houses the solid-phase sorbent; the extraction method is referred to as disposable pipette 

extraction (DPX). For this application, a 5 mL DPX tip is utilized to provide the lower detection limits 

required for the analysis. For the DPX extraction, 1 mL blood was spiked with 10 ng of internal standard 

(drTHC and d)-COOH-THC). The blood was vortexed with 2 mL acetonitrile then centrifuged. The 

supernatant was transferred to a clean tube and 4 mL DI water was added. Approximately 3.5 mL of the 

solution and 5 mL of air (for mixing) were drawn into the DPX tip with a syringe attachment. After waiting 

45 seconds, the solution was dispensed to waste. The rest of the sample solution and 5 mL of air were 

drawn into the DPX tip, and after 45 seconds it was dispensed to waste. Subsequently, 300 uL hexane-ethyl 

acetate (50/50) and 5 mL of air was drawn into the DPX tip, and this solution was dispensed into a GC vial. 

Another elution with 300 uL of hexane-ethyl acetate was used and added to the GC vial. The solution was 

dried under nitrogen and heat (90°C). For the derivatization, 25 uL ethyl acetate was added to the dried vial, 

25 uL BSTF A was added, and the solution was capped under nitrogen and vortex mixed. The solution was 

derivatized at 90°C for 20 min. The GC vial was decapped and the solution was transferred to a GC vial 

insert. The insert was returned to the same GC vial, capped, and placed on the GC autosampler for sample 

injection (2 uL split/ess). The parameters for this analysis are crucial because of potential interfering peaks 

from sample matrix components. The GC/MS instrument (HP 6890 with HP 5973 MSD) was equipped 

with a 30 m GC column (RTX-5 ms, 0.25 mm ID, 0.2 urn film). The oven temperature program started at 

80°C for 1 min, then ramped at 40°C/min to 200 C, then ramped at 20°C/min to 240°C with the temperature 

held at 240°C for 5 min, then it was ramped at 20°C/min to 300°C and the temperature held for 8 min. The 

flow rate was set constant at 2 mLlmin. It was found that this higher flow caused separation of the 371 ion 

ofTHC from a prominent interfering ion. For the MS conditions, selected ion monitoring was used and the 

ions mlz 371,374,386,389,303, and 306 were scanned at 50 ms for THC and (int. std.), and mlz == 371, 

374,473,476,488, and 491 for COOH-THC (and int. std.). Using the above parameters, THC and COOH 

THC at levels of 1 ng/mL were detected using 3 ions. A semi-automated extraction device is demonstrated 

to perform 12 extractions simultaneously in just minutes (not including protein precipitation and 

derivatization). Results of extractions from actual case samples are presented, and direct comparisons with 

standard liquid-liquid procedures are presented. It is found that DPX offers a much-improved method for 

the analysis of THC and metabolites from whole blood in terms of sensitivity, selectivity and speed of 

analysis. 

Keywords: THC, GCJMS, Extraction 

Page 144

A31 

ANALYSIS OF TETRAHYDROCANNABINOL (THC) AND CARBOXY· 

TETRAHYDROCANNABINOL (CTHC) IN WHOLE BLOOD 

Amy Kinkennon*, Tim Robert, David Black, Peter Stout 

Aegis Sciences Corporation, Nashville, TN, USA 

THC and CTHC were isolated from I mL of whole blood using SPEWare Cerex PolyChrom THC SPE 

cartridges (35 mg beds). Analytes were eluted, and then derivatized with PFPA. Samples were 

reconstituted in 50 ul of ethyl acetate, and 2 uL injected on an HP 5890 GC interfaced to a Finnigan TSQ 

7000 MSIMS. 

The MSIMS was used in the positive CI mode for THC, and negative CI mode for the CTHC, with methane 

as the reagent gas. The THC molecular ion at 461 mlz was selected in Ql, collided with Ar in Q2, and two 

daughter ions monitored in Q3. The CTHC did not generate a strong molecular ion (622 m/z), so a 602 m/z 

fragment was selected in Q L This fragment was collided with Ar in Q2, and two daughter ions monitored 

in Q3. D3·THC and D3-CTHC were used as internal standards, and appropriate ions were monitored for 

these compounds. Analysis time was -15 min per sample. 

The method was shown to have a linear range of I-50 nglml for THC and 5-50 ng/ml for CTHC. Precision 

and accuracy studies were conducted using known control materials manufactured in drug free blood with 

good accuracy and precision over the linear range. Interference studies indicated that neither ibuprofen 

(110 uglml), acetaminophen (110 ug/ml), salicylate (700 uglml), nor ethanol (63 uglml) affected the 

analysis. Forty authentic blood samples from previous casework were analyzed using this method. Twenty 

had screened positive, and had been confirmed by an outside laboratory. Nine had screened positive, were 

sent for confirmation, and were returned negative. Eleven had screened negative and had not been 

previously confirmed. 

Keywords: Blood analysis, Carboxy-THC, Tandem mass spectrometry 

Page 145

A32 

OXYCODONE ABUSE CONCERN IN METHADONE MAINTENANCE PATIENTS 

Gerard Meenan', Karl Verebey, and Mohan Patel. Ammon Analytical Laboratory, Linden, NJ. 

Detection of oxycodone abuse is a significant problem for drug treatment facilities. A common 

misconception concerns the idea that oxycodone is a synthetic opiate and staff members believe that the 

urine screen for opiates detects oxycodone, also. For example, the amount of oxycodone that yields a 

positive opiates screen on the DRI Opiates Assay is greater than 16,000 ng/mL. The usual level in patients 

taking oxycodone is from 300 to 1,000 nglmL. 

Microgenics Corporation has developed a specific urine oxycodone homogeneous enzyme immunoassay. 

Evaluation of this DRI Oxycodone Assay was added to screening of methadone "intake" patients followed 

by GCIMS confirmation of tests positive for oxycodone. Microgenics has proposed either a 100 or 300 

nglmL cutoff level. The cutoff concentration used for this study was 100 nglmL. The oxycodone 

immunoassay was tested on a Hitachi 717 Analyzer. 224 drug treatment "intake" patients' urine samples 

were screened with the specific oxycodone immunoassay. Using a 100 nglmL cutoff level 45 urine 

samples screened positive for oxycodone yielding a positive rate of 20. I %. All presumptive positive 

samples were extracted through Biochemical Diagnostics GV~65 Detectabuse columns; derivatized by 

BSTFA with 1% TMCS; and analyzed by selective ion monitoring (SIM) on an Agilent Technologies 

5973 GCIMSD system. The GC/MS confirmation cutoff was 25 nglmL. 45 of the 45 samples confirmed 

positive for oxycodone demonstrating a 100% confirmation rate. The amount of oxycodone recovered in 

the urine samples by GCIMS ranged from 25 nglmL to 37,116 ng/mL. 

The DRI Oxycodone Assay identified 20.1 % of the subjects as positive for the synthetic opiate. The 

specificity of the assay was 100% as determined by GCIMS analysis. Thus, in this population 20.1 % of 

the Methadone Maintenance "intake" patients were using oxycodone. With the FDA approval of a 

generic sustained release oxycodone, abuse is likely to increase. The DRI Oxycodone ErA will be very 

useful in differentiating oxycodone from other opiate abuse. 

Keywords: Oxycodone, Enzyme Immunoassay, GC/MS 

Page 146

A33 

EVALUATION OF BUPRENORPHINE CEDIA ASSAY USING SAMPLES FROM PATIENTS IN 

SUBSTITUTION TREATMENT 

Michael Bottcher and Olof Beck*. Arztpraxis f. Medizinische Mikrobiologie, Labordiagnostik & Hygiene, 

Dessau, Germany. Department of Medicine, Division of Clinical Pharmacology, Karolinska University 

Hospital, Stockholm, Sweden. 

Buprenorphine is used as an analgesic drug, and for detoxification and substitution therapy of opioid 

dependence. However, in combination with the increased medical use, buprenorphine also occurs on the 

black market as an illicit drug and fatalities due to poly-drug use has been reported. In addition, pausing 

from buprenorphine intake during opiate relapsing has been observed and can make compliance monitoring 

necessary. As a consequence there is a need for analytical service and toxicological monitoring of patients. 

However, there has been a lack of an immunoassay suitable for automated high-volume screening. The aim 

of this study was to evaluate the prototype buprenorphine CEDIA (Microgenics Inc.) by comparing it with 

existing ELISA (Diagnostix Ltd) and GC-MS methods. Urine samples were collected from patients in 

heroin substitution treatment with methadone, buprenorphine or dihydrocodeine. In total, 1552 samples 

were obtained from approximately 600 patients, consisting of 70% males and having an age range of IS-54 

y. The CEDIA test was performed on Hitachi 911 and 912 (Roche Diagnostics, IN) instruments with a 

semiquantitative test protocol (five calibrators in the range 0-7S IlglL, sample volume 10 ilL) supplied by 

Microgenics Inc. Samples with response >75 Ilg/L were diluted 10- or 100-fold with saline .. 

At the levels exceeding the 5 Ilg/L cutoff level, a variability lower than 10% was observed both within- and 

between-ctays. The correlation of CEDIA and ELISA was studied in 221 samples. There was a 96.S % 

agreement in qualitative results between the methods. In three samples (2.7 % of all positives) CEDIA 

produced a false positive response as the GC-MS confirmation was clearly negative. In one sample the 

CEDIA result was false negative as the sample contained 26 Ilg/L of buprenorphine according to GC-MS. 

The agreement between the CEDIA and GC-MS methods in quantifying buprenorphine was studied in the 

range from 0 to 6000 Ilg/L. The samples were obtained from 72 patients receiving buprenorphine doses 

between 0.5 - 25 mg/day. The slope was 1.09 with an intercept of -12 on the CEDlA axis. The median ratio 

between CEDIA and GC-MS was 0.96 (n=29S). By pooling data for all samples with a buprenorphine 

concentration >5 Ilg/L according to GC-MS or ELISA (n=400), the sensitivity for CEDIA of detecting 

positive samples at the cutoff level of 5 Ilg/L was calculated to be 99.5%. A total of 1011 samples from 

patients who were not receiving prescribed buprenorphine but received other heroin substitution treatments 

(mainly methadone) were used to estimate specificity. Thirty samples (3.0 %) were found to be positive by 

CEDIA at the cutoff level of 5 Ilg/L. One of these had detectable buprenorphine by GC-MS but at a low 

level (0.6 Ilg/L). Fourteen of the 30 samples were from patients prescribed dihydrocodeine and contained 

high amounts of this substance and metabolites. A further 20 samples from patients prescribed 

dihydrocodeine in daily doses of 14 -SO mg were therefore studied and all were positive in the CEDlA 

assay with responses ranging from 5 - 34 IlglL. The 15 false positive samples out of the 1011 (1.5%) all 

had a low response «10) and all expect for one were highly positive in the CEDI A test for opiates, 

indicating that a cross-reactivity with codeine can be suspected. 

In conclusion, the new CEDI A assay for buprenorphine is suitable for use in clinical routine testing at a 

cutoff limit at 5 Ilg/L. 

Key words: Buprenorphine, Urine, CEDI A 

Page 147

A34 

RAPID DETERMINATION OF PSILOCIN IN WHOLE BLOOD BY LC-MS/APCI 

Wojciech Lechowicz', 1, Andrzej Parczewski l ,2, Ewa Chudzikiewiczl, lInstitute of Forensic Reasearch, 

Cracow, Poland, 2Jagiellonian University, Cracow, Poland 

Psilocin and psilocibin are naturally occurring mushroom alkaloids, with their structure being similar to 

serotonin. Hallucinogenic properties of these compounds make them desirable for drug addicts especially 

for those who experiment with so-called "altered state of consciousness". However, there are many species 

of mushrooms similar to psilocybe but not containing psilocibin or psilocin. Ingestion of unknown 

mushrooms may lead to serious intoxication. Rapid method analysis by means of liquid chromatographymass 

spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI) for detection and 

quantification of psilocin in whole blood has been proposed. Optimization of ionization and fragmentation 

parameters was performed using flow injection analysis (FIA). Total ion current (TIC) was recorded. 

Optimization was conducted for most abundant ions of mlz 20S, 160, llS. Seven parameters were chosen 

for optimization: fragmentor voltage (60/120 V), capillary voltage (4000 V), vaporizer temperature 

(320°C), drying gas temperature (300°C), pressure (SO psig) and gas flow (7 Llmin) and corona current (4.5 

IlA); (in parentheses optimal value is given). Liquid-liquid extraction using a mixture of n-propyl 

chloride:dichloromethylene (2:1, v/v) after blood alkalization (pH 11) was applied. Psilocin-D IO was used 

as the internal standard. Separation was carried out using chromatographic column, prepared according to 

customer requirement: LiChroCART (SSx4 mm) filled with Superspher 60 RP-8e stationary phase. The 

stationary phase ensured appropriate retention: Psilocin - TR=2.42 min; Psilocin-D IO - TR=2.41 min. For 

quantification ramped SIM mode was applied. The pseudomolecular ions of mlz 20S, 21S were recorded at 

fragmentor voltage of 60 V and fragmentation ions of m/z 160, 170, lIS, 12S at l20V. The method was 

thoroughly validated. Intra- and inter-day precision (repeatability) for target concentration of 10 IlglL was 

9% and accuracy measured for the concentration range of 4-40 Ilg/L, expressed as a mean recovery, was 

102±lS%. Results of three mushroom intoxication cases have been studied. Only in one tested blood 

sample did the concentration of psilocin exceed S.2 Ilg/L, the level determined to be LOQ. The determined 

concentration was 8 Ilg/L of free psilocin and 12 IlglL of total psilocin. In case of two others person's 

concentration of free psilocin was above 1.7 Ilg/L (LOD) but lower than LOQ. Some important remarks 

concerning ion suppression caused by matrix, adsorption and solubility of psilocin have been shown to 

exert substantial influence on signal intensity. 

Keywords: Psilocin, Blood, LC-MS/APCI 

Page 148

A35 

ETHYLGLUCRONIDE ANALYSIS IN URINE BY LIQUID CHROMATOGRAPHY WITH 

TANDEM MASS SPECTROMETRY 

Michael Feldman*' \ KC Van Home 2 , Zhichun Liu 2 , Patrick Bennete, David J. Kuntz!. lNorthwest 

Toxicology, a division ofLab One, Salt Lake City, UT 84124, USA and 2Tandem Labs, Salt Lake City, UT 

84124, USA 

~-D-ethylglucuronide (EtG) is a metabolite of ethanol which is formed by the conjugation of ethanol with 

activated glucuronic acid in the presence of membrane-bound mitochrondrial UDP glucuronyl transferase. 

EtG is produced at approximately 0.02-0.06% of an ingested dose. EtG is stable, water-soluble and can be 

detected in urine and can be detected for up to three days following ethanol consumption. Urine ethanol 

levels are normally detected only for several hours, thus making EtG a unique metabolite for monitoring 

abstinence of alcoholics in alcohol treatment programs. 

Urine samples for analysis are prepared by the direct addition of 100 IJ,L of urine with 400 IJ,L of internal 

standard (EtG-D5) in water into the liquid sample vial. No extraction is required. The HPLC used in the 

analysis is a Shimadzu SCL-IOA with a Phenomenex Luna 31J,m column with a mobile phase consisting of 

methanol, water and formic acid with a flow rate of 400lJ,Llminute. The mass spectrometer is a Sciex API 

3000 using a Turbo Ion Spray Source in the negative ion mode. Quadrupole 1 was set to pass the 221 and 

226 ions for EtG and EtG-D5, respectively. Transition ions of 75 (quantitation) and 85 (qualifier) were 

monitored for each compound. 

The assay was linear between 40 nglmL to 10,000 ng/mL with a cutoff established at 100 ng/mL with a 

Limit of Detection and Limit of Quantitation of 40 ng/mL. Precision was determined with %CV of less 

than 1 % between the concentrations of 40-150 ngimL. The %CV between runs was determined to be less 

than 7%. Data from dosing studies, including incidental exposure, will be presented. 

Keywords: Ethylglucuronide, Ethanol 

Page 149

A36 

MASS SPECTROMETRIC IDENTIFICATION OF ETHYL SULFATE IN HUMANS - A NEW 

ETHANOL METABOLITE AND A BIOMARKER OF ACUTE ALCOHOL INTAKE 

A. Helander- and O. Beck. 

Karolinska Institute & University Hospital, SE-171 76 Stockholm, Sweden. 

The traditional way to establish whether a person has recently consumed alcohol is by the analysis of 

breath, blood or urine samples for the presence of ethanol. However, because ethanol is rapidly 

metabolized and eliminated from the body, a method to reveal recent drinking even after ethanol has been 

cleared would have considerable importance in clinical and forensic toxicology. A very small fraction 

«0.1%) of the ethanol consumed undergoes a phase II conjugation reaction catalyzed by UDPglucuronosyltransferase 

to produce ethyl glucuronide (EtG), which is excreted in urine. This non-oxidative 

trace metabolite has attracted interest mainly as a specific biochemical marker for recent consumption of 

alcohol. 

Animal studies have indicated that ethanol may also undergo sulfate conjugation to produce ethyl sulfate 

(EtS). In the present study on humans, a direct electrospray LC-MS method was used to confirm ifEtS is 

formed following intake of alcohol and excreted in the urine. Urine samples were collected from healthy 

subjects at timed intervals after drinking a single ethanol dose. Urine samples were also selected at random 

from those sent to the laboratory for routine testing of recent alcohol consumption. Samples were stored at 

-20°C until analysis. 

LC-MS analysis of EtS was performed in the negative ion mode, with selected-ion monitoring at mlz 125 

for EtS (Mw 126.1 g/moJ) and mlz 226 for EtG-ds (used as internal standard). The identity of EtS was 

confirmed by the correct S isotope ratio in authentic specimen, as compared to EtS reference material. The 

EtS concentration of unknown samples was determined from the peak area ratio of EtS to EtG-ds, by 

reference to a calibration curve. The calibration curve was linear (r 2 =0.99, p

..~. 

A37 

HIGH THROUGHPUT SCREENING OF CORTICOSTEROIDS AND BASIC DRUGS IN HORSE 

URINE BY LC-MS-MS 

Gary N. W. Leung, Evonne W. Chung, Emmie N. M. Ho, W. H. Kwok, David K. K. Leung, Francis P. W. 

Tang, Terence S.M. Wan*, and Nola H. Yu 

Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China; 

terence.sm. wan@hkjc.org.hk 

Introduction: Gas chromatography - mass spectrometry (GC-MS) has long been accepted as a powerful 

technique for the screening and confirmation of the presence of prohibited substances in biological samples 

from human and animal athletes. Over the past decade, liquid chromatography - mass spectrometry (LC 

MS) has evolved into a mature technique and is gaining wide acceptance in many doping control 

laboratories. LC-MS or LC-MS' is particularly suited for the analyses of polar, non-volatile and heatlabile 

drugs that cannot be adequately handled by GC-MS. In addition, tedious derivatization steps can 

often be omitted. This paper describes two high throughput LC-MS-MS methods for the screening of two 

important classes of drugs in equine sports, namely the corticosteroids and the basic drugs, at low ppb 

levels in horse urine. The method utilized a high efficiency reversed phase LC column (3 cm L x 2.1 mm 

ID with 2.5 Jlm particles) to provide fast turnaround times as well as achieving significant reduction in the 

consumption of expensive HPLC solvents. The performance of these two methods on real samples was 

demonstrated by analysing drug administration and positive postrace urine samples. 

Method: Corticosteroids and basic drugs were extracted from enzyme-treated urine by solid-phase 

extraction using a Bond Elut CertifY® cartridge and analysed by LC-MS-MS in multiple reaction 

monitoring (MRM) mode using a Thermo Finnigan triple quadrupole TSQ Quantum mass spectrometer. 

Separation of the corticosteroids and basic drugs was achieved using a short reversed-phase CI8 column (3 

cm Lx 2.1 mm ID with 2.5 Jlm particles) on two different LC gradient solvent systems. 

Results: Using the methods developed in this study, the detection of 23 corticosteroids and 42 basic drugs 

could be achieved within a 2.5-min and a 3.5-min LC-MS-MS run respectively. The overall turnaround 

time for the corticosteroid screen was 5 minutes and that for the basic drug screen was 8 minutes, inclusive 

of post-run and equilibration times. The results on the analysis of drug administration and positive 

postrace urine samples also demonstrated that both methods were effective in detecting corticosteroids and 

basic drugs in horse urine at low ppb levels. 

Conclusion: Two high throughput LC-MS-MS methods with the use of a high efficiency LC column have 

been developed for the screening of corticosteroids and basic drugs at low ng/mL levels in equine urine. 

Validation data will also be presented. These methods can be used to control the abuse of these two classes 

of drugs in racehorses. 

Keywords: Corticosteroids, Basic drugs, High throughput LC-MS-MS 

Page 151

A38 

SCREENING FOR, LIBRARY-ASSISTED IDENTIFICATION AND FULLY VALIDATED 

QUANTITATION OF TWENTY-TWO beta-BLOCKERS IN BLOOD PLASMA BY LIQUID 

CHROMATOGRAPHY-MASS SPECTROMETRY WITH ATMOSPHERIC CHEMICAL 

IONIZATION 

Hans H. Maurer*, Oliver Tenberken, Carsten Kratzsch, Annin A. Weber, and Frank T. Peters 

Department of Experimental and Clinical Toxicology, University of Saarland, D-66421 Homburg (Saar), 

Gennany, E-mail: hans.maurer@unikIinik-saarland.de 

Background: Overdose of beta-blockers may lead to life-threatening situations. Although there is no strong 

correlation between plasma concentration of beta-blockers and their pharmacological and toxic effects, 

suitable analytical procedures are necessary for toxicological screening, identification and quantification in 

clinical and forensic toxicology. Therefore, an assay was developed for such purposes in human blood 

plasma using LC-APCI-MS. 

Methods: After mixed-mode (Confirm HCX) solid-phase extraction of 0.5 mL of plasma, the beta-blockers 

were separated on a Superspher 60 RP Select B column (125 x 2 mm J.D., guard column: 10 x 2 mm I.D.) 

using fast gradient elution (ammonium formate buffer/acetonitrile). The compounds were screened for and 

identified using an LC-APCI-MSD (SL version) in the scan mode with fragmentor voltages of 100 and 200 V 

by mass chromatography with selected ions followed by library search of the underlying full APCI mass 

spectra with our new LC-MS reference library. The drugs were quantified in the SIM mode at 100 or 200 V 

using calibration curves. The assay was fully validated according to internationally accepted 

recommendations. 

Results: The assay allowed screening for, library-assisted identification (both in scan mode) and 

quantification (selected-ion mode) ofthe beta-blockers acebutolol, diacetolol, alprenolol, atenolol, betaxolol, 

bisoprolol, bupranolol, carazolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, 

nebivolol, oxprenolol, penbutolol, propranolol, sotalol, talinolol and timolol in blood plasma. The assay was 

found to be selective for all tested compounds. No interfering peaks were observed in the extracts of ten 

different blank plasma samples. Interferences with common drugs typically taken in combination were tested 

and could be excluded due to different retention time and/or mass spectra. The assay was linear from 

sub-therapeutic to overdose concentrations of all compounds. A weighted (lIx 2 ) least squares model was 

used for calculation of calibration curves. Low and high level recoveries ranged from 46.9% to 94.8% for all 

studied analytes. LODs were determined in the full scan mode and they were all lower or equal to the 

corresponding LOQs in the SIM mode. The LOQs corresponded to the lowest calibrator concentrations with 

a signal-to-noise ratio ofat least 10. Within-day, between-day and total precision (combination ofwith in- and 

between-day effects) lay within the required limits of:::;15% RSD (::520% RSD at LOQ). Accuracy data also 

all lay within the acceptance interval of ±15% (±20% at the LOQ) of the nominal values. In processed 

samples, the analytes were stable for a period of more than 24 hours at room temperature. No instability of 

analytes in spiked samples was observed over three freeze/thaw cycles or during storage at -20°C for a one 

month period. The procedure has proven to be applicable in the analysis ofauthentic plasma samples during 

routine work. The presented assay is the first fully validated procedure for the simultaneous detennination of 

a large number ofbeta-blockers in plasma. In emergency toxicology, it should be applicable to confine to one 

point calibration because the y-intercepts of the calibration curves were either not significant (p < 0.05) or 

small compared to the response at high therapeutic concentrations. 

Conclusions: The LC-MS assay has proven to be appropriate for screening, identification and quantification 

of beta-blockers in plasma after intake oftherapeutic as well as of toxic dosages. It was successfully applied 

to authentic plasma samples allowing confirmation ofdiagnosis ofoverdose situations as well as monitoring 

of patients' compliance. 

Keywords: liquid chromatography-mass spectrometry; beta-blockers; plasma 

Page 152

A39 

RAPID DETERMINA nON OF CHLORAMPHENICOL AND ITS GLUCURONIDE IN FOOD 

PRODUCTS BY LIQUID CHROMATOGRAPHY- ELECTROSPRA Y NEGA nVE IONIZAnON 


Madej J.Bogusz*, Huda Hassan, Eid Al-Enazi, Mohammed AI-Tufail 

King Faisal Specialist Hospital and Research Centre, P.O.Box 3354, 1211 Riyadh, Kingdom of Saudi 

Arabia 

Chloramphenicol (CAP) is subjected to monitoring in food products, with a mllllmum required 

performance level set at 0.3 ng/g. CAP was isolated from chicken meat and seafood by very simple solvent 

extraction procedure. For honey, a fast SPE procedure was applied. CAP-D5 was used as internal standard. 

HPLC separation was done on RP18 123 x 3mm column in acetonitrile-ammonium formate 10 mM, pH 3.0 

(40:60) at flow rate of 0.3 m/min. A TSQ Quantum instrument with ESI source has been used in negative 

ionization mode. A MRM procedure has been applied and following transitions were monitored: rn/z 

321>152 (quantifier), 321>194, 321>257 (qualifiers), 326>157 (IS). CAP peak was eluted at around 5 min; 

the total run time was 7 min. LOD was around 0.1 nglg meat or 0.05 nglg honey. For confirmation, ion 

intensity ratios were used. Matrix effects were studies using various approaches. The method allows 

analyzing up to 30 duplicate samples/day, including all calibration standards. Additionally, the method for 

determination of CAP glucuronide (CAP-G) was established. As a source ofCAP-G urine specimens taken 

from rats which were given CAP were used. For the isolation of CAP and CAP-G a SPE procedure, used 

also for honey, was applied. 

Keywords: Chloramphenicol, Chloramphenicol glucuronide, LC-MS-MS 

Page 153

A40 

BROAn..SPECTRUM BENZODIAZEPINE SCREENING BY LC-MSIMS 

Kathleen McKague* and Michael Whiteside 

Centre of Forensic Sciences, Toronto, Ontario, Canada 

Screening biological samples for benzodiazepines is often carried out using immunoassay (IA) techniques. 

However, due to the chemical diversity of this class of drugs, specific identification, confirmation and 

quantitation of the benzodiazepines present in forensic toxicology generally requires multiple analytical 

systems (LC, GC & GCIMS). Prior to validation of this work, confirmation of a putative positive result by 

IA in this laboratory necessitated the use of 11 separate analytical methods with the potential to consume 

19 mL of sample. This led us to develop a single analytical technique for the screening and identification 

of benzodiazepines. A solid phase extraction (SPE) method coupled with LC-MSIMS analysis, was 

developed to simultaneously detect 14 benzodiazepines and 9 benzodiazepine metabolites in 1 mL of blood 

or urine. The compounds and their respective limits of detection (LOD) in blood included in this assay are 

alprazolam (1.5 nglmL), a-hydroxyalprazolam (1.5 nglmL), bromazepam (1.5 nglmL), chlordiazepoxide (5 

nglmL), demoxepam (5 ng/mL), clobazam (3 nglmL), clonazepam (1.5 nglmL), 7-aminoclonazepam 

(3nglmL), diazepam (20 ng/mL), nordiazepam (5 ng/mL), flunitrazepam (1.5 ng/mL), 7 

aminoflunitrazepam (1.5 ng/mL), n-desmethylflunitrazepam (1.5 nglmL), flurazepam (1.5 ng/mL), n 

desalkylflurazepam (1.5 nglmL), lorazepam (3 ng/mL), midazolam (1.5 nglmL), nitrazepam (3 nglmL), 7 

aminonitrazepam (1.5 nglmL), oxazepam (3 ngfmL), temazepam (20 ng/mL), triazolam (1.5 nglmL) and a 

hydroxytriazolam (1.5 ngfmL). 

The extraction method utilizes Waters Oasis HLB extraction cartridges on a Zymark Rapidtrace SPE 

workstation. Samples are precipitated with acetonitrile and then diluted with pH 6 phosphate buffer prior 

to loading onto the extraction columns. After washing with water, phosphate buffer, and 5% methanol, the 

compounds are eluted with 95% methanol. The samples are subsequently derivatized with acetic 

anhydride. Analysis ofthe extracted samples is performed on a Thermo-Finnigan LC-MSIMS system. The 

HPLC is a ThermO-Finnigan Surveyor equipped with a Waters Symmetry® CIS (2.1 mm x 20 mm, 5 Ilm 

particle) guard column followed by an Agilent Zorbax SB-CIS (2.1 mm x 150 mm, 3.5 Ilm particle) 

analytical column. Separation is achieved isocratically using a 54:46 methanol:ammonium formate pH 3 

buffer at a flow rate of 225 ilL/min and a temperature of 35°C. The MSIMS system is a Thermo-Finnigan 

TSQ Quantum operated in APCI/SRM (atmospheric pressure chemical ionization/selected reaction 

monitoring) mode. With the exception of alprazolam, a-hydroxyalprazolam, fl unitrazepam , 7 

aminoflunitrazepam a-hydroxyalprazolam and lorazepam, the limit of detection for this method is 

significantly lower than any of the previously used target analyses utilized by our laboratory. 

From July 2003 to April 2004, we have analyzed 382 case samples. 52% of these samples were positive 

for at least one benzodiazepine or benzodiazepine metabolite. The most commonly found benzodiazepines 

were clonazepam, diazepam, lorazepam, and temazepam. No case samples were positive for either 

flunitrazepam or its metabolites. Of the positive findings, 81 % were below the limit of detection for the 

original target analysis used in our laboratory. While the detection of low concentrations of these 

compounds is not usually an essential requirement in death investigations, they can be critical in drugfacilitated 

sexual assault investigations. In the latter instance, sexual assaults are often associated with the 

ingestion of low drug doses and a long time elapse between the assault and sample collection. These 

factors create an analytical challenge that requires sensitive and specific methodology. The benefits of this 

assay will be highlighted with case examples. 

Key Words: Benzodiazepines, LC-MSIMS, Screening 

Page 154

A41 

DETERMINATION OF STRYCHNINE IN HUMAN BLOOD USING SOLID PHASE 

EXTRACTION AND GCIEI-MS 

M. Barroso'· I, E. Gallard0 2 , S. Avila l , C. Margalho l , E. Marques I ,M. Lopez-RivaduIla 2 , D.N. Vieira l 

INational Instutute of Legal Medicine, Delegation ofCoimbra, Portugal 

2Institute of Legal Medicine, University of Santiago de Compostela, Spain 

Introduction: Strychnine is the main alkaloid in Strychnos nux vomica, an Indian tree. This plant was first 

introduced in Germany in the XVI century, as a poison to rats and other pests. Although widely used in the 

past, even with pharmaceutical purposes, its use is now limited in many countries. Strychnine is not 

commercialized in Portugal since 1974, but sometimes it is still associated with forensic intoxications, 

because small amounts may remain in storage, particularly in rural areas. The objective of this work was 

the development and validation of a simple and rapid method for the determination of strychnine in human 

blood, employing solid phase extraction (SPE) and GC/EI-MS. 

Materials and Methods: Oasis HLB (30 mg) extraction cartridges were obtained from Waters (Milford, 

MA, USA). Stock solutions of strychnine and papaverine (internal standard) were prepared in methanol, 

protected from light and stored at 4°C until use. 

The sample was submitted to the following procedure: to 500 J.1L of blood were added 2 mL of distilled 

water, and the mixture was centrifuged at 3000 rpm for 5 minutes. The supernatant was then applied to 

previously conditioned SPE columns. After elution of the sample the columns were washed with 1 mL of 

5% methanolic solution, and then dried under full vacuum for 15 minutes. The elution was performed with 

1 mL of chloroform, which was afterwards evaporated to dryness under a gentle stream of nitrogen. The 

dry residue was reconstituted in 50 J.1L of methanol, and an aliquot of 1 J.1L was injected in the GC. 

The GC oven temperature program started at 150°C for 1 minute, then raised by 35 °C/min to 200°C, held 

for 1 minute and finally elevated by 40 °C/min to 270°C, where it was kept for 7 minutes. 

The injector port was set to 200 "C. The mass spectrometer temperature was 280 "C, and it was operated in 

the SIM (Selective Ion Monitoring) mode. The selected ions were 334, 120 and 162 for strychnine; and 

338, 324 and 308 for papaverine. 

Results: The calibration curve was established in spiked blood within a range of 0.10 to 2.50 J.1g/mL, and 

the correlation coefficient was 0.9994. The limits of detection and quantification were respectively 60.27 

and lOO nglmL. The precision (coefficient of variation), calculated at both low and high concentrations, 

was inferior to lO%. Accuracy was superior to 90% for all calibrators. Mean recovery for strychnine was 

90.66%. The method was applied to authentic samples obtained from the Laboratories of Forensic 

Toxicology of the National Institute ofLegal Medicine, Coimbra and Lisbon, Portugal. 

Conclusion: One may conclude that the proposed technique is analytically suitable for the extraction and 

determination of strychnine in blood, since it is linear within the studied range, and presents adequate 

precision and accuracy. Therefore, it can be applied in forensic cases where the compound is involved. 

Keywords: Strychnine, SPE, GClMS 

Page 155

A42 

EXTENDING AND IMPROVING SCREENING AND TARGET ANALYSIS UTILIZING LC-MS 

Tania A. Sasaki'· I and Byron Curtis 2 

IApplied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404; 20ffice of the Chief Medical 

Examiner, 901 Stonewall, Oklahoma City, OK 73117 

GC/MS has long been a powerful and useful technique for analysis of complex mixtures, as well as target 

analyses, because of its inherent specificity and sensitivity. However, the necessity for analytes to be labile 

limits the types of compounds that can be analyzed utilizing this technique and also requires some 

extensive sample preparation, such as derivatization, for successful analysis. However, over the past 15 

years, LCIMS has grown in popularity due to its applicability for analysis of polar molecules, which 

includes most drug and pharmaceutical compounds. LCIMS also has the capabilities to analyze higher 

molecular weight compounds that are not easily examined by GC/MS due to their high boiling points; 

derivatization is rarely, if ever, necessary. Furthermore, chromatographic analysis times are generally 

between 10-40 minutes for LC, which are much shorter than the averagetimes used for GC analysis. This 

paper examines the utility of LC/MS to reduce total analysis times of current protocols, as well as develop 

methods to analyze or screen for compounds that have no current methodology in the forensics field. 

Several types of compounds were studied for target analysis, both quantification and general screening, 

covering a range from warfarins to common pesticides to neurontin. Currently, these compounds have 

been analyzed by either GC/MS or immunoassay. However, the utility of these methods is limited by 

throughput and/or cost. By using LC coupled with a triple quadrupole mass spectrometer, screening of 

over 50 compounds in a single analytical experiment can be accomplished with low ppb detection limits. 

Furthermore, the compounds can be analyzed with minimal sample preparation and short analysis times, 

improving the throughput and greatly reducing expense. 

Key words: Method development, Mass spectrometry, LCIMS 

Page 156

A43 

SIMULTANEOUS DETERMINATION OF SILDENAFIL. VARDENAFIL AND TADALAFIL AS 

ADULTERANTS IN DIETARY SUPPLEMENTS BY LC-ESI-MS 

K. Saisho*, H. Kamakura, M. Kawamura, R. Kikura-Hanajiri, Y. Goda 

National Institute of Health Sciences, I-IS-I, Kamiyoga, Setagaya-ku, Tokyo 15S-S50I, Japan 

In recent years, there is a problem with illegally added prescription ingredients and/or their synthetic 

analogues (as active adulterants) in dietary supplements. Prolonged or excessive consumption of these 

products may cause possible serious health risks to some users. It has been reported that sildenafil (SDF), 

which is therapeutically used for penile erectile dysfunction, is one of the active adulterants in products 

advertising roborant nutrition. Moreover, its structurally similar anti-impotence drugs, vardenafil (VDF) and 

tadalafil (TDF), may be alternative adulterants of SDF. In this study, a simultaneous analytical procedure for 

three anti-impotence drugs, SDF, VDF and TDF (as adulterants in dietary supplements advertising roborant 

nutrition) was developed using LC-ESI-MS. 

These drugs were extracted with the solvent consisted of acetonitrile and distilled water containing I % 

formic acid (4: 1, v/v) under ultrasonication. The separation was achieved on an Inertsil ODS-3 column (2.1 x 

150 mm, 5 Om) at 40°C. The following gradient system was used with mobile phase A(5 mM ammonium 

formate buffer (pH 3.5) I acetonitrile (75:25, v/v» and mobile phase B (acetonitrile); B: 0 % (0-3 min), B 

linear from 0 to 30 % (3-13 min), B: 30 % (13-30 min). The flow rate was at 0.3 mLlmin. LC-MS with ESI 

interface in the positive ion mode was used. The linear regression ofthe peak area ratios versus concentration 

in standard solution was fitted over the concentration range of 10 - 10000 ng/mL (SDF), 5 5000 ng/mL 

(VDF) and 50 - 25000 ng/mL (mF). All calibration curves were obtained with correlation coefficients of 

greater than 0.9990. The recoveries of SDF, VDF and mF spiked a functional food at O.Olmg/mg 

concentrations were 9S.8%, 100.2% and 99.S %, respectively. 

The developed method was applied to the determination of SDF, VDF and TDF in 91 dietary supplements 

obtained from the Japanese market. SDF was identified in seven samples and their contents were in the range 

of24.2 mg 120.9 mg /capsule or bottle. In addition to SDF, homosildenafil (a synthetic analogue ofSDF) 

was also detected in one of the seven samples. TDF was detected in one sample and the content was 0.32 

mg/capsule, while VDF was not found in any sample. Moreover, a new synthetic analogue of SDF, 

hydroxyhomosildenafil, which has never been reported, was also identified in the sample. 

Keywords: Anti-impotence drugs, LC-MS, Dietary supplements 

Page 157

A44 

SCREENING OF CANNABINOIDS, BENZOYLECGONINE AND OPIATES IN WHOLE BLOOD 

AND URINE USING EMIT II PLUS IMMUNOASSAY AND KONELAB 30 

K. Wiese Simonsen*, N. Christiansen, I. Breum MUller, Department of Forensic Chemistry; University of 

Copenhagen; Denmark 

Aims: To present a full validated simple and automatic method for screening of cannabinoids, 

benzoylecgonine and opiates in whole blood and urine for routine use. 

Methods: Blood samples were extracted with acetone before analysis. Urine samples were automatically 

diluted 5 times. EMIT II Plus calibrator levell, 2,3 and 5 were used for calibration. The analysis was done 

with EMIT II PLUS reagents (cannabinoids, cocaine metabolite and opiate assays) on a Konelab 30 

instrument at 340 nm. 

Results: Cut offs in blood and urine are respectively: cannabinoids: 0.0045 mglkg (blood) and 0.050 mg/kg 

(urine); benzoylecgonine: 0.015 mg/kg (blood) and 0.300 mglkg (urine); morphine: 0.020 mg/kg (blood) 

and 0.300 mg/kg (urine). Within day precisions for blood controls spiked at cut off were 5-10% 

(cannabinoids), 10% (benzoylecgonine) and 5-10% (morphine) (n=10). Between day precisions were 6% 

(cannabinoids), 18% (benzoylecgonine) and 12% (morphine) (n=7). Sensitivity was 100% (cannabinoids), 

93% (benzoyJecgonine) and 97% (opiates). Specificity was 86% (cannabinoids), 99% (benzoylecgonine) 

and 95% (opiates). 

Conclusion: A validated method has been described. The method is precise, robust and useful for screening 

of cannabinoids, benzoylecgonine and opiates in whole blood and urine. 

Keywords: Cannabinoids, Benzoylecgonine and opiates, EMIT II Plus immunoassay 

Page 158

A45 

FAST QUANTIFICATION OF ETHANOL IN WHOLE BLOOD SPECIMENS BY THE 

ENZYME ALCOHOL DEHYDROGENASE METHOD; OPTIMIZA TION BY 

EXPERIMENTAL DESIGN 

Lena Kristoffersen·· l , Bjorn Skuterud 1 , Bente R. Larssen 1 , Svetlana Skurtveit 2 , Anne Smith 

Kielland l 

lNorwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse, P.O. Box 

4404 Nydalen, N-0403, Oslo, Norway; 2Norwegian Institute of Public Health, Division of 

Epidemiology, P.O. Box 4404 Nydalen, N-0403, Oslo, Norway 

A sensitive, fast, simple and high throughput enzymatic method for the quantification of ethanol in 

whole blood (blood) on Hitachi 917 is presented. Alcohol dehydrogenase (ADH) oxidizes ethanol 

to acetaldehyde using the coenzyme nicotinamide adenine dinucleotide (NAD), which is 

concurrently reduced to form NADH. Method development was performed with the aid of factorial 

design, varying pH and concentrations ofNAD+ and ADH. The linear range increased and reaction 

end point decreased with increasing NAD+ concentration and pH. The method was linear in the 

concentration range 0.0024-0.4220 g/d!. The limit of detection (LOD) and limit of quantification 

(LOQ) were 0.0007 g/dl and 0.0024 gldl, respectively. Relative standard deviations (RSD) for the 

repeatability and within laboratory reproducibility were in the ranges 0.7-5.7 % and 1.6-8.9 %, 

respectively. The correlation coefficient when compared to head space gas chromatography flame 

ionization detection (HS-GC-FID) methods was 0.9903. Analysis of authentic positive blood 

specimens gave results that were slightly lower than those of the reference method. 

Key words: Ethanol, ADH, Quantitation 

Page 159

A46 

A RAPID METHOD FOR THE ANALYSIS OF ATOMOXETINE IN BLOOD USING GCIMS 

Mike K. Angier*, Russell 1. Lewis & Robert D. Johnson, Federal Aviation Administration, Civil Aerospace 

Medical Institute, Bioaeuronautical Sciences Research Laboratory, P.O. Box 25082, Oklahoma City, 

Oklahoma 73125 

Atomoxetine is a selective norepinephrine reuptake inhibitor (SNRI) prescribed for the treatment of 

attention deficitJhyperactivity disorder (ADHD). It is the first non-stimulant drug approved by the FDA for 

the treatment of this disorder in both adults and children. Since its approval in November 2002, over two 

million prescriptions have been filled. It has been estimated that 3% of the U.S. adult population may have 

ADHD. A review of the current literature has identified no gas chromatographic/mass spectrometric 

(GCIMS) methods for the detection of atomoxetine in the forensic toxicology setting. Therefore, our 

laboratory developed a method for the identification and quantitation of atomoxetine in postmortem blood 

using GCIMS. This procedure incorporates a Varian Bond Elut® Certify solid phase extraction (SPE) 

followed by derivitization with pentafluoropropionic anhydride (pFPA). Derivitization of atomoxetine 

with PFPA, although not required for the GC/MS analysis of this compound, affords both superior linearity 

and sensitivity over the non-derivatized compound. The method described is highly selective and sensitive, 

having a limit of detection of 1 ng/mL for atomoxetine. Atomoxetine was found to have a linear dynamic 

range of 3 - 800 ng/mL on a calibration curve weighted by a factor of l/x. The SPE provided an efficient 

sample extraction yielding recoveries of 40 ± 3% and 51 ± 4% at 25 and 250 ng/mL (n=5 for each group). 

Furthermore, the developed procedure provided superb accuracy and precision. This procedure showed 

intra-day (within day) relative errors of::; 5% and relative standard deviations (RSD) within 2% for both the 

25 ng/mL and 250 ng/mL control groups (n=5 for each group). Using whole blood controls stored at 4°C 

the inter-day (between day) relative errors for the 25 ng/mL control group were 9%, 10% and 3% for days 

2,3 and 7, respectively (n:=:5). The relative errors for the 250 ng/mL control group were 6%, 3% and 4% 

for days 2, 3 and 7, respectively (n=5). The RSDs were all < 5% for both control groups over the 7-day 

period. Based on the day 7 results, it is clear that atomoxetine is stable in blood stored at 4°C for at least 

one week. The method developed proved to be rapid, reliable and sensitive for the identification and 

quantitation ofatomoxetine in blood. 

Key Words: Atomoxetine, SNRI, Forensic Toxicology 

Page 160

A47 

STABILITY OF PLASMA ACETALDEHYDE DETERMINED BY GAS CHROMATOGRAPHY 

POSITIVE ION CHEMICAL IONIZATION-MASS SPECTROMETRY 

Meng Chen"', David M. Andrenyak, David E. Moody and Rodger L. Foltz. Center for Human Toxicology, 

Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112. 

Simultaneous abuse of drugs and alcohol is common. Acetaldehyde is a metabolite of ethanol that 

accumulates when ethanol is taken with disulfiram and in many individuals with genetic variants in alcohol 

dehydrogenase and lor aldehyde dehydrogenase. Analysis of acetaldehyde is considerable difficult due to 

the volatility and various factors, such as enzymatic and nonenzymatic oxidation. Most previously 

published methods require immediate on-site handling of specimens to allow analysis. A goal of this work 

was to develop a method where samples can be collected as plasma, stored and shipped for off-site 

analysis. A novel method was developed for the determination of acetaldehyde in human plasma that 

utilizes a simple liquid-liquid extraction procedure and gas chromatography-positive ion chemical 

ionization-mass spectrometry (GC-PCI-MS). Using acetaldehyde-dt as internal standard, both acetaldehyde 

and acetaldehyde-d 4 were derivatized directly in plasma with 2, 4-dinitrophenylhydrazine in hexane solvent 

at room temperature. The derivatized- acetaldehyde and acetaldehyde-d 4 were extracted into hexane. After 

centrifugation, an aliquot of supernatant was transferred to an autosampler vial for analysis. A GC capillary 

column was used for the separation of the derivatives, which were subsequently ionized with ammonia 

reagent gas and analyzed by MS. The GC oven temperature was initially set at 120'C and increased to 

300'C at 20'C Iminute. Run time for each injection was 10 minutes. MS source and quadruple temperatures 

were set at 200 and 150 'C, respectively. The prominent ions at m/z 242 and 246 for acetaldehyde and 

acetaldehyde-dt. respectively, were analyzed by selected ion monitoring. The lower limit of quantification 

was 0.3 j.tg/mL in 0.5 mL of plasma and the linearity in 4 assays was ~=0.998, over a range from 0.3 to 20 

j.tglmL. When intra- and inter-assay precision and accuracy were evaluated at concentrations of 0.75, 8 and 

15 j.tglmL, mean measured concentrations did not deviate more than 14% from the target and coefficient of 

variance did not exceed 4%. After derivatization, the extracts of acetaldehyde and acetaldehyde-dt were 

found to be stable for up to 117 hrs in autosampler vials at room temperature. This method is simple 

because of: no need for protein precipitation; single step in liquid-liquid solvent extraction; no 

concentration steps, such as drying supernatant and reconstitution. The method was used to study the 

stability of acetaldehyde in human plasma. In conclusion, this simple and reliable method appears to be 

useful in toxicology and other researches. The research is supported by NIDA Contract NO 1 DA-3-8829. 

Key Words: Acetaldehyde, GC-MS, Stability. 

Page 161

A48 

MULTICENTER EV ALUA TION OF THE ROCHE ONLINE® TDM CARBAZEPINE ASSA Y FOR 

ROCHEIHITACHI ANALYZER SYSTEMS 

Mary Jane Coffing* I, Roger L. Bertholf, Martin Kroll 3 , Hilmar H. Luthe 4 , Alain Verstraete 5, Heike 

Sauter 6 , RaifRoeddiger 6 

IClinical Trials, Roche Diagnostics Cooperation, Indianapolis, IN, USA; 2University of Florida Health 

Science Center, Jacksonville, FL, USA; 3Dallas VA Medical Center, Dallas, TX, USA; 4Department 

Clinical Chemistry, Universitiit G6ttingen, Germany; 5Ghent University Hospital, Ghent, Belgium; 

6Clinical Trials, Roche Diagnostics GmbH, Mannheim, Germany 

Carbamazepine is an anticonvulsant and anti-manic drug, used in the treatment of trigeminal neuralgia, 

bipolar disorders, epilepsy, and a wide variety of seizure disorders. Monitoring carbamazepine 

concentrations is essential during therapy in order to ensure achievement of optimal therapeutic effect, 

while avoiding the impact of both subtherapeutic and toxic drug levels. 

The analytical performance of a new, Roche homogeneous microparticle immunoassay for Carbamazepine 

was evaluated at four sites two in Europe and two in the Unites States. Intra- and interassay imprecision, 

lower detection limit, control recovery, and linearity were assessed. Further, the assay method was compared 

to the CEDIA Carbamazepine assay on RochelHitachi 917 and MODULAR P analyzers, Abbott FPIA 

Carbamazepine assay on the Abbott AxSYM and TDx analyzers, and the Roche FP assay on INTEGRA 800. 

Serum vs. sodium heparin plasma comparison was also performed. 

OnLine Carbamazepine intra-assay imprecision showed 8Ds sO.14 J..lg/mL for concentrations up to 4 

J..lg/mL and CVs s3.4 for concentrations >4 J..lg/mL. Interassay imprecision showed SDs s0.15 J..lglmL for 

concentrations up to 4 J..lglmL and CVs s4.1 for concentrations >4 J..lglmL. Analytical sensitivity (lower 

detection limit) to 0.22 J.lglmL and linearity to the 20.0 J..lglmL were observed. Roche COBAS FP control 

materials recovered within range at all sites and competitor control materials recovered within 

manufacturers' ranges with one exception. 

PassinglBablok regression analysis was used to assess method comparison. All comparisons demonstrated 

c I ose agreement between competitive met h 0 d s as note d'mthetable b eow: I 

Methodoiogyllnstrument 

N Slope Intercept Correlation 

x 

y 

Coefficient 

Site 1 CEDIA MODP OnLine MOD P 168 1.055 -0.248 0.993 

8ite2 CEDIA 917-R OnLine 917-R 157 0.982 0.093 0.983 

Site 3 CEDIA MODP OnLine MODP 161 1.059 -0.642 0.972 

COBASFP OnLineMODP 161 1.121 -0.458 0.984 

INTEGRA 800 

Abbott AxSYM I OnLine MOD P 161 1.120 -0.426 0.975 

Site 4 CEDIA 917 OnLine 917 145 1.039 -0.409 0.988 

Abbott TDx OnLine 917 145 1.068 -0.111 0.986 

Comparison of serum vs. sodium heparin plasma produced this regression equation: y 1.l07x - 0.074, 

r=0.982 (n=47). The OnLine Carbamazepine TDM reagent met or exceeded analytical specifications and 

all clinically relevant performance criteria in this evaluation. 

Key Words: Carbamazepine, Automated Method, Multicenter Evaluation 

Page 162

A49 

AN LC-MSIMS METHOD FOR THE QUANTIFICATION OF BUPRENORPHINE AND 

NORBUPRENORPHINE IN BLOOD AND URINE 

Merja Gergov*, Pirjo Tiainen and I1kka Ojanperli 

Department of Forensic Medicine, University ofHelsinki, P.O. Box, 40, FIN-00014 University of Helsinki, 

Finland 

Background: The synthetic opioid buprenorphine has become common in the maintenance therapy of 

opioid addicts in Finland since 1999. The increasing number of samples has required a simple and reliable 

analytical method suitable for routine use. LC-MS/MS provides high selectivity and sufficient sensitivity 

for the quantitation ofbuprenorphine and its metabolite norbuprenorphine in urine and blood. 

Methods: Urine samples were hydrolyzed with ~-glucuronidase enzyme. Sample work-up for blood and 

hydrolyzed urine involved liquid-liquid extraction with ethyl acetate at pH 7. The extracts were run by LC 

on a Genesis CIS reversed phase column using acetonitrile-ammonium acetate mobile phase at pH 3.2. The 

mass spectrometric analysis was performed with a triple quadrupole mass spectrometer equipped with a 

turbo ion spray interface in positive mode using multiple reaction monitoring (MRM). Sufficient 

sensitivity was achieved only by monitoring the surviving parent ions both for buprenorphine (mlz 468.0) 

and norbuprenorphine (mlz 414.2) at the collision energy of 20 eV. Quantification was performed using 

deuterated internal standards and four-point calibration. 

Results: Validation curves were prepared with four replicates at ten concentration levels. The linearity 

criterion was accuracy better than ± 20% at the calibration curve. The method exhibited good linearity 

from 1 to 200 /lg/l for urine (r2 ~ 0.996, quadratic regression) and from 0.2 to 100 !lg/l for blood (r ~ 

0.998, linear regression through zero). The average intra-day precision of real autopsy samples was 3 % 

for both compounds in urine, and 7% for buprenorphine and II% for norbuprenorphine in blood. Criteria 

for the LOQ were: 1) precision better than ± 20%, 2) inaccuracy lower than ± 25% in urine and 20% in 

blood, and 3) signal-to-noise ~ 10. The LOQs for both compounds were I /lgll in urine and 0.2 /lg/l in 

blood. In the study of uncertainty of measurement, systematic errors were not observed. The expanded 

uncertainty of measurement (at the confidence level of 95%) contained only random errors being for both 

compounds 15% and 30% in urine and blood, respectively. A total of six proficiency testing samples 

(UKNEQAS 2003, Labquality 2004) containing buprenorphine were analyzed with the established method, 

and the results were within II% of the consensus or average value. 

Discussion: The present method has been applied to blood and urine samples from more than 420 autopsy 

cases and 670 living patients. Interferences from sample matrix have been detected only very rarely even 

though the surviving parent ions are monitored to obtain adequate selectivity. Successful participation in 

international proficiency testing schemes proves the high reliability ofthe method. 

Keywords: Buprenorphine, Quantification, LC-MS/MS 

Page 163

A50 

V ALIDA nON OF AN EIA-BASED SCREENING ASSAY FOR THE DETECTION OF 

AMPHETAMINE AND MDMAIMDA IN BLOOD AND ORAL FLUID 

Marleen Laloup·' I, Gaelle Tilman l ,2, Gert De Boeck!, Pierre Wallemacq2, Viviane Maes 3 and Nele Samyn l 

INational Institute of Crimina lis tics and Criminology (NICC), Section Toxicology, Brussels, Belgium; 

2Labo Clinical Chemistry and Toxicology, St. Luc University, Hospital-UCL, Brussels, Belgium 

3Department of Clinical Chemistry-Toxicology, Academic Hospital, Free University ofBrussels, Belgium. 

The number of seizures and the use of amphetamine and 'ecstasy' (MDMA) have increased exponentially 

in Belgium since the late nineties. Therefore, screening for these substances in biological specimens has 

become an important part of routine analysis in forensic toxicology laboratories. The use of a reliable 

sensitive immunological assay for the screening of blood and oral fluid samples is less widespread than for 

the preliminary analysis of urine. Such an assay would save cost and labor time in comparison to a more 

specific analysis as gas chromatography-mass spectrometry (GC-MS). 

The objective of this study was to evaluate the suitability of the Cozart® AMP enzyme-linked 

immunoassays (EIA) for the screening of blood and plasma samples, collected with sodium fluoride and 

potassium oxalate as anticoagulant, and oral fluid samples, collected with the Intercept® device. 

Authentic blood samples (n "" 260) were assayed on the EIA plate, using an optimal 1 :5-fold dilution. True 

positive, true negative, false positive and false negative results were determined relative to our routine GC 

MS analysis. The EIA readily detects MDA but shows minimal crossreactivity with MDMA « 0.1%), so 

the interpretation of the GC-MS result of the MDMA-only samples was based on the combined 

MDAIMDMA concentrations. Samples consisted of 100 amphetamine-only positives, 100 MDMAIMDAonly 

positives, and 60 negatives, using the limit of quantitation as the cut-off level for confirmation (10 

nglml). Using these results, receiver operating curves (ROC) were generated and optimal cut-off values for 

the screening assay were calculated. 

For the amphetamine positive samples, the analysis showed an optimal cut-off value at 66.5 nglml 

amphetamine equivalents with a sensitivity of 99.0 % and a specificity of 96.9 %. For MDMA/MDA 

positive samples, 97.0 % sensitivity and 96.9 % specificity were reached at the same cut-off value of 66.5 

nglml amphetamine equivalents. The area under the ROC curve exceeded 0.97. When combining the 

results, the EIA assay is able to predict the presence of either amphetamine or MDMAIMDA in plasma 

samples with 98.0 % sensitivity and 96.9 % specificity at a cut-off value of 66.5 nglml amphetamine 

equivalents. 

A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind 

study. Subjects received placebo or a high (l00 mg) or low (75 mg) dose ofMDMA. Plasma and oral fluid 

samples were collected at 1.5 and 5.5 hours after administration. Preliminary analysis of the oral fluid 

samples indicated a screening cut-off of 51 ng/ml amphetamine equivalents with a sensitivity of 97.9 % and 

a specificity of 98.6 %, using the plasma data for confirmation. LC-MSIMS confirmation of the oral fluid 

samples is in progress. 

In conclusion, these data indicate that the Cozart® AMP EIA plates constitute a fast and accurate screening 

technique for the identification of amphetamine and MDA/MDMA positive blood samples and oral fluid 

specimens collected with Intercept®. 

Keywords: EIA, Amphetamine, Blood 

Page 164

AS1 

SIMULTANEOUS ANALYSIS OF HIPPURIC ACID AND METHYLHIPPURIC ACIDS IN 

URINE FROM PAINT THINNER ABUSERS USING HIGH-PERFORMANCE LIQUID 

CHROMATOGRAPHY-ELECTROSPRAY IONIZATION MASS SPECTROMETRY 

Masatoshi Morinaga*, Forensic Science Laboratory Fukuoka Prefectural Police Headquarters, 7-7 Higashi 

Koen, Hakata-ku, Fukuoka-ken 812-8576, Japan 

A high-perfonnance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) assay 

for the simultaneous analysis of hippuric acid and methylhippuric acids in urine is described. Urine samples 

were collected from abusers sniffing paint thinner including toluene and a small amount of xylenes. These 

samples were centrifuged at 3000 rpm for 10min, then diluted by distilled water 100 times and a 10ui 

aliquot of sample was injected into LCIMS. 1% acetic acid, methanol, THF (90:5:5, VN) was used as a 

mobile phase. The method described allows separation of hippuric acid and methylhippuric acid in less 

than 40 minutes using a stainless steel column packed with octadecyl dimethylsilyiI silica. The separation 

was possible for m-hippuric acid and p-methylhippuric acid. The detection limits of hippuric acid and 

methylhippuric acids were 0.5ug/ml (SIR mode), respectively. The calibration curves were linear in range 

of 1 to 1000ug/ml. Methylhippuric acids were detected from 36 cases of paint thinner abusers and were not 

detected in urines of 10 nonnal subjects. 

• 

Key words: Methylhippuric acids, LCIESI-MS, Paint thinner abuser 

Page 165

A52 

LCIMSIMS AS A ROUTINE METHOD FOR ANALYSIS OF THERAPEUTIC AND ILLICIT 

DRUGS IN FORENSIC SCIENCE 

Pascal Mireault· and France Jobin 

Laboratoire de Sciences Judiciares et de Medecine Legale, 1701 Parthenais, Montreal, Quebec, Canada, 

H2K 3S7. 

Introduction: The aim of this presention was to evaluate the usefulness of Liquid Chromatography coupled 

with Tandem Mass Spectrometry (LC/MS/MS) in routine forensic toxicological casework. Presently, Gas 

Chromatography coupled with Mass Spectrometry (GCIMS) is the most common method used for 

confirmation of drugs and its metabolites. However, this technique has the disadvantage of requiring 

derivatization, because some substances and metabolites are polars and thermolabiles. This drawback can 

be overcomed by using LCIMS/MS. LC/MS interface does not involve heat and offer gentle ionization of 

polar and thermolabile compounds. This technique has been used for routine determination of several 

groups of drugs: opiate, cocaine and its metabolites, amphetamine and other psychoactive phenethylamines, 

benzodiazepines derivatives, ~9-THC and its metabolites and cardiac g\ycosides. 

Experimental: The basic urine or blood were extraction using a solid phase extraction (SPE). 

Chromatographic separation was acheived using a Symmetry (Waters) C-1S or C-S column. Mobile phase 

conditions used a ratio of MeOH and buffer at a flow rate of 1 mLimin. A TSQ-7000 API2 triple 

quadrupole instrument equipped with a APC} ionization source was used in positive or negative mode. 

Results: LC/MSIMS became a very powerful and flexible method for dedicated analyses of substances of 

forensic interest. It has been show that the use of fast LCIMSIMS provide excellent specificity and high 

sensitivity. Spiked urine and blood samples of opiate, cocaine and its metabolites, amphetamine and other 

psychoactive phenethylamines, benzodiazepines derivatives, ~9-THC and its metabolites and cardiac 

gJycosides were analyzed in selected reaction monitoring (SRM) mode to evaluate limit of detection 

(LaD), limit of quantification, linearity and accuracy. LaD obtained range from 0.05 to 2 ng/mL for target 

coumpounds in matrices. The specificity of the method was evaluated with numerous antemortems and 

post-mortem matrices show no significant interferences at the expected retention time oftarget compounds. 

This technique improve the analysis of polars and thermolabile compounds and decrease analysis time. 

Key Words: LC/MSIMS, Biological Matrices, Forensic 

Page 166

AS3 

DETERMINATION OF ETHYL GLUCURONIDE IN URINE BY GAS CHROMATOGRAPHY 


M Wang* 

Department of Biochemistry, Queensland Medical Laboratory, Brisbane, Queensland, Australia 

Ethyl glucuronide (EtG) is a metabolite of ethanol, formed by enzymatic conjugation of ethanol with 

glucuronic acid, and is regarded as a useful bio-marker of recent alcohol consumption. EtG can be detected 

in blood, urine, tissues and hair for an extended time period due to its specific properties of non-volatility, 

water-solubility and general stability in comparison to ethanol. A gas chromatography-tandem mass 

spectrometry (GC-MSIMS) method was therefore developed for the routine determination of EtG in urine. 

Initial extraction involves 3 ml of urine specimen being spiked with 45 ul of EtG-D5 (200 uglml) as 

internal standard and was adjusted to pH 6.0. Following extraction using a Strata NH2 SPE column with 

methanol and water, the extract was evaporated and derivatized with BSTFA (in 1% TMCS and pyridine) 

at 80°C for 30 minutes. After evaporation, the residue was reconstituted in 100 III of acetonitrile. EtG was 

assayed on a Varian Saturn 2000 ion trap GC·MS!MS. The standard curve was linear between 1000 and 

10,000 Ilglml with curve correlation coefficients exceeding 0.99. The recovery of EtG from solid phase 

extraction was approximately 99% at 1000 Ilg/L and 102 % at 8,000 Ilg/L. Measurement of EtG in urine 

makes it possible to monitor ethanol consumption using a direct metabolite. This GC-MSIMS method 

provides a rapid, simple and specific determination of ethyl glucuronide. 

Key words: Ethyl glucuronide, Gas Chromatography -Tandem Mass Spectrometry, Solid Phase Extraction 

Page 167

A54 

SOLID PHASE MICROEXTRACTION GAS CHROMATOGRAPHIC ANALYSIS OF 

ORGANOPHOSPHORUS PESTICIDES IN BIOLOGICAL SAMPLES 

Nikolaos Raikos*·a, lfigeneia Grigoratou b , Georgios Theodoridis b , Heleni Tsoukali a , 

Dimitrios Psaroulis' 

aLaboratory of Toxicology and Forensic Medicine, Medical School; bLaboratory ofAnalytical Chemistry, 

Department of Chemistry, Aristotle University, 541 24, Thessaloniki, Greece 

A headspace solid phase microextraction (HS-SPME) method in combination with GC-NPD was 

developed for the determination of a mixture of organophosphorus pesticides (malathion, parathion, 

methyl parathion and diazinon) in biological samples. Fenithothrion was selected as the internal standard. 

A 85 !-lm polyacrylate (PA) SPME fiber was selected for sampling. Various extraction parameters were 

studied and optimized: salt addition, desorption time, extraction time, extraction temperature. Best 

conditions found were: 0.8 g NaCI added in 3 ml aqueous sample, 20 min sampling at 70 ec, 3 min 

desorption at 230°C, The HS-SPME method in combination with GC-NPD provided satisfactory detection 

sensitivity and liner dynamic range. The method was applied in the determination of the pesticides in 

various biological specimens: human whole blood, plasma, liver, kidney and cerebrospinal fluid. The 

inherent selectivity provided by the NPD detector together with the efficient sample clean-up of the HS 

SPME resulted in clear chromatograms with no interferences. Extraction recovery varied significantly from 

specimen to specimen. Linear response data for these OPPs was obtained with correlation coefficients 

ranging between 0.9866 and 0.9999. High GC signals were observed for diazinon, as a result low limits of 

detection were observed. Limits of detection (LODs) were in the range of 2-10 nglg for diazinon. In 

contrast the signals obtained for methyl parathion and malathion were significantly lower, thus higher 

limits of detection were observed. Estimated LODs ranged from 35 to 55 ng/g for methyl parathion. The 

proposed methodology renders an efficient, cost effective and simple and sample preparation process for 

the determination of OPPs. Most important, the technique overcomes limitations and obstacles of 

conventional methods such as the use of expensive and toxic organic solvents and the application of tedious 

and cumbersome procedures. 

Keywords: Organophosphorus pesticides, HS-SPME, Biological samples 

Page 168

ASS 

EFFECTS OF ASCORBATE DERJV A TIVE ON SERUM HYDROXY RADICALS IN 

ETHANOL-TREATED RATS 

Shinobu Itoh" ',Tomohisa Mori', Akiko Tamagawa 2 , Nobuhiko Miwi, Toshiko Sawaguchi I 

lDepartment of Legal Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, 

Tokyo 162-8666, Japan; 2Department of eel! Biochemistry, Hiroshima Prefectural University School of 

BioSciences, Shobara, Hiroshima 727-0023, Japan 

Objective: It is well known that acute and/or chronic ethanol (EtOH) increases toxicity in human and 

animals, which might be mediated through oxidative stress. The present study was designed to investigate 

the effects of EtOH on serum hydroxy radicals (HR), Furthermore, radical scavenge effect of 

L-ascorbyl-2-phosphate Na (APS) was also examined. 

Materials and Methods: In order to investigate the oxidative stress caused by EtOH in blood, I mllkg b.w, 

of 95% EtOH was administered orally to male Wistar rats under pentbarbital anesthesia, and blood 

sampling from heart were done 5, 10, 15, 20, 30 and 60 min after administration of EtOH, The signal 

intensity of serum hydroxy radicals (HR) was measured using the ESR LJElectron Spin ResonanceD-Spin 

Trap method based on the DMPO 5,5-dimethl-l-pyrroline-N-oxideD-Fenton reaction. ESR apparatus type 

JES-FR30 and recorded in a Krebs-Henselelt buffer containing 100 mM DMPO. APS or saline was then 

administered to EtOH-treated rats, and the signal intensity of serum HR was measured in the same manner 

in an attempt to determine their suppressive effects on EtOH-induced oxidative stress. 

Result: EtOH significantly increased the signal intensity of serum HR about twice as compared with saline 

(P

A56 

AUTOMATED SOLID PHASE EXTRACTION PROCEDURE FOR THE SIMULTANEOUS 

ANALYSIS OF DELTA-9-TETRAHYDROCANNABINOL (THC), ll-HYDROXY-DELTA-9 

TETRAHYDROCANNABINOL (11-0H-THC), AND Il-NOR-DELTA-9-TETRAHYDRO 

CANNABINOL-9-CARBOXYLIC ACID (THCC) IN BLOOD, PLASMA, AND ORAL FLUID BY 

GCIMSIMS 

Paul J. Davis*' \ Nadine Koenigl, Joann Sell I and Dean F. Fritch2, lHealth Network Laboratories, 2024 

Lehigh Street, Allentown, PA, 20raSure Technologies, Inc., 150 Webster Street, Bethlehem, PA 

A forensic automated procedure for the quantitative determination of THC, II-OH-THC, and THCC in 

whole blood, plasma, and oral fluid by GCIMS/MS is presented. Samples are treated with acetonitrile, 

acidified with dilute acid, and all three analytes are simultaneously extracted by solid phase extraction 

using the Caliper Life Sciences Zymark RapidTrace® Solid Phase Extraction Workstation with Varian 

SPEC® C-IS SPE cartridges. The RapidTrace® conditioned the SPE cartridges with methanol, added the 

sample, washed the cartridges with water/methanol, dried the cartridges with nitrogen, and then eluted the 

three cannabinoids with hexane/ethyl acetate. The final extracts were evaporated, derivatized with BSTFA 

with 1% TMCS, and analyzed by GCIMSIMS using the Varian 1200 Quadrupole GCIMSIMS System. 

Quantitation was accomplished using THC-d3 and THCC-d3 as internal standards. Two product ions were 

chosen for each analyte with one product ion serving as the qualifier ion. Retention time had to be within 

I% and the ion ratio of the qualifier product ion within 20% for identification of the analytes. The limit of 

quantitation (LOQ) for THC and II-OH-THC is I ng/mL while the LOQ for THCC is 5 ng/mL. The limit 

of detection (LOD) for THC and II-OH-THC is 0.5 ng/mL, and 2.5 ng/mL for THCC. Both THC and 11 

OH-THC are linear to at least 40 ng/mL While THCC is linear to at least 200 ng/mL. The recovery of all 

three analytes was approximately 50% for all three specimen types tested. At the LOQ for all three 

analytes, between-run precision resulted in a relative standard deviation ofless then 12%. 

Key words: THC, Automated, GCIMS/MS 

Page 170

A57 

ELISA FOR THE SCREENING OF OPIA TES, BENZODIAZEPINES, CANNABINOIDS, 

METHAMPHETAMINE, AND COCAINE METABOLITE IN POSTMORTEM FORENSIC 

WHOLE BLOOD 

Paul J. Jannetto', 1,2, Elvan Laleli-Sahin,,2, Susan Gock', Steven H. Wong"2, and Jeffrey Jentzen l , 2 

'Milwaukee County Medical Examiner's Office; 2Department of Pathology, Medical College of Wisconsin 

In both forensic and clinical toxicology, enzyme-linked immunosorbent assay (ELISA) based screening 

methods are becoming increasingly popular and replacing radioimmunoassay (RIA) methods for numerous 

reasons including: cost, automation, ease of use, and disposal issues. Currently, the Milwaukee County 

Medical Examiner's Office uses the Immunalysis RIA kits to screen whole blood specimens for common 

classes of drugs such as opiates, benzodiazepines, cannabinoids, methamphetamine, and cocaine 

metabolite. In this study, the assay performance of the RIA kits was compared to the ELISA kits from the 

same manufacturer using fifty-one forensic samples. To further validate the ELISA-based assays, the intraand 

interday precision, cross-reactivity, and dose-response curves were analyzed for opiates, 

benzodiazepines, cannabinoids, methamphetamine and cocaine metabolite to determine if the ELISA kits 

were an acceptable alternative to the RIA kits. 

Of the fifty-one samples screened by both RIA and ELISA for opiates, benzodiazepines, cannabinoids 

(THC), methamphetamine and cocaine metabolite, there were a total of five discordant results (3 THC, and 

2 methamphetamine). Gas chromatography-mass spectrometry analysis indicated five unconfirmed positive 

results (false-positives) by RIA (3 cases for THC and 2 cases for methamphetamine). On the other hand, 

the ELISA kits had a 100% positive and negative predictive value for all five assays. The intraday 

coefficient ofvariation (CVs; n == 10) near the immunoassay cutoff concentration (60 ng/mL) was 4.0-7.7% 

for all five assays, while the interday CVs (n 5) at the immunoassay cutoff concentration (50 ng/mL) was 

8.4-10.7%. 

Overall, a comparative assessment of common drug screening assays by RIA and ELISA from the same 

manufacturer indicated some differences in analytical performance. The ELISA based screening kits 

offered acceptable precision, superior specificity/sensitivity and was an economical alternative to the RIA 

kits. The ELISA-based assays are particularly attractive for forensic specimens for several reasons 

including: the ability to use a variety ofsample matrices (e.g. whole blood), small sample volumes reducing 

interferences from some forensic matrices, a long shelf life, and the ability to incorporate automation and 

increase throughput. Based on these criteria, the Milwaukee County Medical Examiner's Office has 

adopted the ELISA-based assays to screen forensic samples for drugs of abuse. All presumptive positive 

immunoassay results are then confirmed by GCIMS. 

Key words: ELISA, RIA, Method validation 

Page 171

AS8 

THE DETERMINATION OF FORENSIC BLOOD ALCOHOLS WITH AN INERT AUTOMATED 

HEADSPACE SAMPLER 

Roger L. Firor, Chin-Kai Meng, Cinthia Cai, Michael Feeney' 

Agilent Technologies, Wilmington, Delaware, USA 

Blood alcohol analysis is a widely used high-throughput application in forensic laboratories. The use of 

static headspace sampling has many well know advantages for determination ofvolatiles in a variety ofless 

than ideal matrices. Blood or other biological fluids are certainly not the cleanest of matrices and therefore 

are well suited for headspace sampling. In terms of GC analysis, reduced inlet and column maintenance, 

better quantitation, and increased throughput are some of the advantages of automated headspace. Dual 

column systems offer an advantage in that the elution order of ethanol and some other common metabolites 

differ on the DB-ALCl and DB-BAC2 stationary phases. This provides added confirmation and a potential 

reduction in possible inferences or coelutions with ethanol. 

A new automated headspace sampler with 70-sample tray and inert flow path is introduced for the 

determination of forensic blood alcohols. The headspace sampler employs a completely inert flow path, 

uniform heated zones, and unique vent line purging capability. When taken together, these important 

attributes lead to a reduction in carryover and improved repeatability. Standard mixtures in water were used 

to demonstrate the analyses. Two headspace-based solutions, based on 0.53mm 10 and 0.32mm ID 

columns, are detailed. Isothermal analyses with cycle times below 5 min are easily achieved with sufficient 

resolution to avoid common interferences. Total system control from the GC Chern Station is possible with 

21 CFR Part 11 compliant software specific for headspace sampling. 

Key words: Blood Alcohols, Headspace, Gas chromatography 

Page 172

A59 

DETERMINATION OF ACONITINE ALKALOIDS BY HPLCITOF-MS: COMPARISON OF 

ELECTRO-SPRAY AND LASER-SPRAY IONIZATIONS 

Rina Kaneko·'], Satoshi Hattori], Shiho Furuta l , Yukari Hirata l , Kanako Watanabe 2 , Hiroshi Seno\ Makoto 

Hamajima l , Akira Ishiil; IDepartment of Legal Medicine, Fujita Health University School of Medicine, 

Japan, 2Department of Legal Medicine, Hamamatsu University School of Medicine, Japan, 3Department of 

Legal Medicine, Aichi Medical University, Japan 

Background: Aconitum species (Ranunculaceae) are widely distributed in Northern Asia and North America. 

Their roots are popularly used in herbal medicines in China and Japan; however, their diesterditerpene-type 

alkaloids are extremely toxic. For example, the LD50 values ofaconitine in mice are 1.8 mg/kg (orally) and 

0.12 mg/kg (intravenously); the estimated minimal lethal dose in man is about 2 mg. Accidental poisoning 

cases by aconite usually happen in Japan. Also, it is sometimes used in suicidal and homicidal cases, 

because of its high toxicity. It is thus important to determine aconitine alkaloids in body fluids with high 

sensitivity. We have succeeded in determining aconitine and its related compounds by HPLC/time-of-flight 

(TOF)-mass spectrometry (MS). Also, we have tried to detect aconitine alkaloids by laser-spray ionization 

and compared its sensitivity with that by conventional electro-spray ionization. 

Methods: Aconitine alkaloids (aconitine, mesaconitine, hypaconitine, and jesaconitine) and 

methyllycaconitine (internal standard; IS) spiked in plasma samples were purified using a BondElut Certify 

HF column. Extracted and dried aconitine alkaloids are reconstituted with 50/lL of mobile phase, which 

consists of70 % of20mM ammonium acetate (solvent A) and 30 % of acetonitrile (solvent B). HPLC was 

employed in the isocratic mode (A:B 70:30) at flow rate of 0.2mLlmin. MS was performed on a JEOL 

(Tokyo, Japan) AccuTOF HPLC-TOF mass spectrometer; the ionization used was electro-spray ionization 

(ESI) in positive ion mode. The MS conditions were as follows: spray voltage; 2.2kV, orifice voltage; 75V 

(for quantitation) or 135V (for identification), nebulizing gas (nitrogen); lOLimin. For the laser-spray 

ionization, a 10 W infrared laser was used. 

Results: All aconitine alka:Ioids and IS are completely separated on chromatogram. In human plasma samples, 

the calibration curves gave good linearity in the range of 5 and 300 ng/mL in all the compounds; their 

detection limits were about 0.3 to 1 ng/mL. Their limits for identification were about 0.7 to 2 ng/mL. The 

intraday CV values were less than 21 % for 200 ng/mL. In the laser-spray ionization, the ion current 

intensity was about 5 times higher compared to that by electron-spray ionization. 

Conclusion: The calibration curves gave good linearity in the range of 5 and 300 ng/mL; aconitine alkaloids 

can be sufficiently detectable at toxic concentrations. Thus this method is useful for quantitation and 

identification of aconitine alkaloids. Laser spray ionization could be also applied to identify trace amounts 

of aconitine alkaloids. 

Keywords: Aconitine, Mass spectrometry, Laser spray 

Page 173

A60 

THE DETERMINATION OF ETHYL GLUCURONIDE IN URINE USING REVERSED-PHASE 

HPLC AND PULSED ELECTROCHEMICAL DETECTION 

Romina Kaushik*, Barry Levine and William R. LaCourse 

University ofMaryland, Baltimore, Maryland, USA 

There is a need for a method to distinguish between ethanol levels detected in biological matrices due to 

alcohol consumption versus ethanol production after death as a result of decomposition. Ethyl glucuronide 

(EtG) is a non-volatile, water-soluble, direct metabolite of ethanol that can serve as a biological marker of 

alcohol consumption. This metabolite can be a marker of acute alcohol consumption even at low levels 

unlike traditional biomarkers. It is an intermediate marker of alcohol consumption, bridging the gap 

between long-term (COT, MCV & GGT) and very short-term (ethanol & HTOL) biomarkers. EtG is a 

highly sensitive and specific alcohol consumption marker that can be detected for up to 80 hours after 

complete alcohol elimination from the body. This has very important clinical and forensic applications. 

Clinical applications for this biological marker would include monitoring patients in treatment for alcohol 

abuse. This marker could improve therapy outcome and quality of life in patients by preventing relapse 

episodes. Furthermore monitoring would increase safety in the workplace and prevent traffic accidents. 

Finally, fetal alcohol syndrome could be avoided by detecting and monitoring EtG in the mother which 

would reduce costs by making therapy more effective. In addition, the metabolite exhibits very high storage 

stability which is important as forensic samples are often stored for extended periods of time. Sometimes 

samples are not analyzed until the case goes to court which could take months or years. In previous studies 

there have been various methods used to detect EtG. These include gas chromatography (GC) coupled with 

mass spectrometry (MS), and liquid chromatography (LC) coupled with MS. GC/MS is available at almost 

all forensic facilities at a moderate cost. However, GC·MS detection of this metabolite requires prior 

derivatization. LC/MS is advantageous because it doesn't require derivatization nonetheless it is an 

expensive technique. For widespread use of EtG as a marker, simpler and less expensive methods are 

necessary. 

The current study involved developing a method for the detection of EtG in postmortem urine samples 

using reversed-phase liquid chromatography with pulsed electrochemical detection. Methyl glucuronide 

served as the internal standard. The mobile phase consisted of 1% acetic acid/water and acetonitrile (98:2), 

with a 600mM sodium hydroxide post-column system attached to enable pulsed electrochemical detection 

(PED). This amperometric detection technique applies alternated positive and negative potential pulses at a 

noble metal electrode. The analyte is oxidized followed by oxidative and reductive cleaning steps. The 

analyte concentration is determined by measuring the electric current resulting from the molecule gaining 

or losing electrons. In order to separate EtG from the biological matrix a solid-phase extraction (SPE) was 

used using aminopropyl columns. EtG was found to have a retention time of 5.3 minutes with LOQ and 

LOD values of 0.4 and 0.1 uglmL respectively. The extraction recovery following SPE was approximately 

50%. This method is specific, reproducible and sensitive. Reversed-phase chromatography enabled a 

simple separation of the analyte without requiring the ion-pairing reagents typically associated with ion 

chromatography. PED is a direct (no derivatization) and affordable detection method. This method is a 

potential tool to clinical and forensic toxicologists for determining alcohol consumption in live and 

deceased individuals. 

Keywords: Alcohol, Biomarker, Direct and affordable analysis 

Page 174

A61 

SIMULTANEOUS ANALYSIS FOR PSYCHOTROPIC TRYPTAMINES AND 

PHENETHYLAMINES USING GC-MS AND LC-ESI-MS 

R. Kikura-Hanajiri*, M. Hayashi, K. Saisho, Y. Goda 

National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 

Many analogs oftry pta mines and phenethylamines are hallucinogenic substances that exist naturally in some 

plants, fungi and animals, but can also be produced synthetically. Some of these drugs are strictly controlled 

by the Narcotics and Psychotrophics Control Law in Japan but many non-controlled analogs have been 

widely distributed as easily available psychotropic substances, especially via the Internet. 

To investigate the trend of these non-controlled drugs of abuse, simultaneous analytical methods were 

developed using GC-MS and LC-ESI-MS for 9 tryptamines/carbolines (AMT, 5-MeO-AMT, 5-MeO-DMT, 

5-MeO-DIPT, 4-0H-DIPT, 4-acetoxy-DIPT, bufotenine, harmine and harmaline), 5 phenethylamines (2C-I, 

2C-T-2, 2C-T-4 and 2C-T-7, including MBDB) of typically non-controlled substances in Japan, and, 

additionally, 4lega\ly controlled tryptamines and phenethylamines originally found in fungi or plants (DMT, 

Psylocin, Psylocybin and mescaline). Moreover, the proposed methods were applied to analyses of these 

drugs in 99 products (purchased at video shops or via the Internet over the past 2 years in Japan), which 

advertised psychotrophic/psychoactive effects. 

The samples (powder, tablets or liquid) were extracted with methanol under ultrasonication. After 

centrifugation, the extracts were filtered thorough a 0.45-0m membrane filter prior to the injection. GC-MS 

analysis was performed within 30 minutes using a DB-5MS capillary column (0.25 mm i.d. x 30 m, 0.25 Om 

film thickness). Regarding the LC-ESI-MS analysis, the separation of the target drugs was optimized on an 

ODS column (Atlantis dC18, 2.1 x 150 mm, 5 Om) in an acetonitrile-IOmM ammonium formate buffer (pH 

3.0) by a linear gradient program and a quantitative analysis was carried out by the monitoring of each 

[M+Ht in the positive ion mode ofESI-MS. 

As a result ofthe analyses using GC-MS and LC-ESI-MS, 5-MeO-DIPT (the synthetic substance known by 

the street name "Foxy") was found in 7 out ofthe 99 products except the products sold as "chemical reagents", 

and AMT, 5-MeO-DMT and harmine were also found in some of the 99 products. These analytical methods 

will be useful for the investigation of the distribution of the non-controlled psychotropic tryptamines and 

phenethylamines in the market. 

Keywords; Tryptamines, Phenethylamines, Non-controlled psychotropic substances 

Page 175

A62 

A FULLY AUTOMATED METHOD FOR THE SCREENING OF BASIS DRUGS IN WHOLE 

BLOOD THROUGH ON LINE SPE-HPLC-DAD 

Rafael Linden* 

Instituto Geral de Pericias, Porto Alegre, RS, Brazil; Centro Universitario Feevale, Novo Hamburgo, RS, 

Brazil 

One of the major tasks in forensic toxicology laboratories, especially those with high workloads, is to 

provide adequate drug screening analysis in a limited amount of time. The purpose of this work was to 

develop a screening method, with reasonable discrimination capabilities, that could work unattended, using 

an aumomated device for solid phase extraction (SPE) coupled to a HPLC-DAD. Postmortem blood 

samples, (free of drugs) were spiked with cocaine, benzoylecgonine, lignocaine, diazepam, nordazepam, 

fluoxetine and sertraJine, until final concentrations of 500 nglmL were achieved. The whole blood samples 

were sonicated, than diluted from 3.5 to 7 mL with carbonate buffer (pH 9.3). The diluted samples were 

centrifuged and the supemadant was used for SPE. The extraction was performed in an Aspec® system, 

using Speed® C18 200mg cartridges, with the following steps: conditioning with 2 mL of methanol and 2 

mL of carbonate buffer pH 9.3, loading of 6.5 mL of diluted samples, washing with 2 mL of 9: 1 carbonate 

buffer:acetonitrile and eluting with 1 mL of 9: I acetonitrile:phosphate buffer pH 2.3. The solvents flows 

and air pushes were optimized. The eluate was injected on line in the HPLC, using a 100 ilL loop. For the 

hplc analysis, a C8 column, 25cm x 4.6 mm, was used in isocratic mode, with a mobile phase composed of 

phosphate buffer and acetonitrile (63:37). The identification of the drugs was made based on their relative 

retention times to imipramine (I.S.) and on a spectral library. Recoveries above 80% were obtained for all 

drugs tested, with extracts sufficiently clean for hplc-dad identification. The method is now being 

expanded to a bigger number of drugs, including a acidic fraction. 

Key words: Solid phase extraction, Drug screening, HPLC-DAD 

Page 176

A63 

SOLID-PHASE MICROEXTRACTION BASED APPROACH FOR ENANTIOMERIC ANALYSIS 

OF AMPHETAMINES 

3 

Sheng-Meng Wang J ,2 and Ray H. LiU 2 . ,4 •• , ICentral Police University, Taoyuan, Taiwan. 2Toxicology and 

Accident Research Laboratory, FAA Civil Aerospace Medical Institute, Oklahoma City, OK, U.S.A. 

3Department of Medical Technology, Fooyin University, Kaohsiung Hsien, Taiwan. 4Department of Justice 

Sciences, University ofAlabama at Birmingham, Birmingham,AL, U.S.A. 

As a solventless approach and with recent advances in the fiber manufacturing technology, solid-phase 

micro extraction (SPME) is now widely studied for its effectiveness for the pretreatment ofvarious categories 

of samples. This study explores a novel SPME approach for enantiomeric analysis of amphetamines, in 

which absorption and derivatization are accomplished in one step. Specifically, 

(S)-(-)-N-(Trifluoroacetyl)-prolyl chloride was adapted as the chiral derivatizing reagent and added directly 

into the sample matrix. Analytical parameters, such as temperature, absorption/desorption duration, and the 

amount of derivatizing reagent, were studied to determine their effects on the yields of analytes on the fiber. 

The derivatization products resulting from this study show excellent desorption characteristics on the 

polydimethylsiloxane-coated fiber adapted for this study. For example, a one-time 5-min desorption leaves 

no detectable carry-over. Optimal operational parameters (absorption: 70°C for 10 minutes; injection: 250°C 

for 5 minutes) cause minimal negative impact on the fiber, allowing repeated used of the fiber for more than 

30 times. This method was evaluated and proved to be effective in (a) quantitative determination of the 

enantiomeric pairs of amphetamine and methamphetamine, in terms of repeatability, linearity, and limits of 

detection and quantitation; and (b) generating case-specimen data comparable to those derived from a 

conventional liquid-liquid extraction approach (Table I). 

/~, 

Table 1. Comparison of enantiomeric composition data resulting from two methods (one-step solventJess 

SPME vs. conventional two-step liquid-liquid extraction/derivatization) 

SPME (concentration in ~/mL) Li~id-liquiifconcentration in ng/mL) . 

Sample d-Methamp. : I-Methamp. -Amp. : I-Amp. d-Met amp .. 1- ethamp. d-Amp. . 

I-Amp. 

1 12,169 1,280 2,477 72 ]0,638 1,564 2,702 63 

2 1,821 219 1,010 42 1,427 146 976 32 

3 7,436 593 1,329 36 7,768 429 1,318 37 

4 13,845 1,960 2,581 94 11,753 1,233 2,545 67 

5 3,766 340 2,675 93 3,310 252 2,763 72 

6 10,745 1,254 3,324 92 9,931 1,315 2,973 79 

7 3,090 704 1,466 78 2,564 591 1,381 79 

8 2,482 5,863 580 403 2,010 6,317 482 538 

9 5,398 486 1,971 50 4,680 435 1,928 43 

10 6,667 608 2,376 84 5,649 543 2,230 66 

11 6,761 947 2,220 78 5,632 449 2,100 73 

12 8,600 972 1,308 90 6,276 950 1,276 58 

Keywords: Amphetamines, Enantiomer, GC-MS 

Page 177 

1

A64 

GC-MS QUANTlTATlON OF CODEINE, MORPHINE, 6-ACETYLMORPHINE, 

HYDROCODONE, HYDROMORPHONE, OXYCODONE, AND OXYMORPHONE IN BLOOD 

Robert Meatherall' 

Department ofBiochemistry, S1. Boniface General Hospital, 409 Tache Avenue, Winnipeg, Manitoba, 

Canada, R2H 2A6. 

A method is described for the simultaneous analysis of seven opiates - codeine, morphine, 6 

acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone in blood samples by gas 

chromatography - mass spectrometry. One milliliter of blood is combined with an internal standard 

mixture containing 200 nanograms of each of the seven deuterated opiates. Two milliliters of acetonitrile 

are added to precipitate the proteins and cellular material. After centrifugation, the clear supernatant is 

removed and the acetonitrile is evaporated. The remaining aqueous portion is adjusted to pH 9 with 

sodium bicarbonate buffer from which the drugs are extracted into chloroform / trifluoroethanol (10: 1). 

The organic extractant is transferred and dried under nitrogen. The residue is reconstituted in dilute 

hydrochloric acid and washed consecutively with hexane, then chloroform. The purified aqueous portion is 

adjusted to pH 9 with bicarbonate buffer and the drugs are again extracted into chloroform / 

trifluoroethanol (10:1). The organic portion is removed from the aqueous fraction and dried under 

nitrogen. The residue is consecutively derivatized with methoxyamine, then propionic anhydride using 

pyridine as a catalyst. The ketone groups on hydrocodone, hydromorphone, oxycodone and oxymorphone 

are converted to methoximes. Hydroxyl groups present at the C-3 and C-6 positions of codeine, morphine, 

6-acetylmorphine, hydromorphone and oxymorphone are converted to their respective propionyl esters. 

After a post derivatization purification step, the extracts are analyzed by full scan gas chromatography 

mass spectrometry using electron impact ionization. 

The method is linear to at least 2000 ng/mL. Day-to-day precision (N 15) at 500 nglmL and 75 nglmL 

were less than 10% for all 7 targeted opiates. Extraction efficiencies at these two concentrations ranged 

from 50% to 68%. For each opiate, the limit of quantitatlon was 10 nglmL, while the limit of detection was 

2 ngimL. 

Keywords: Opiates, Blood, Mass Spectrometry 

Page 178

A65 

A STUDY OF CROSS-REACTIVITY OF SELECTED ANIMAL PROTEINS ON AN ENZYME 

LINKED IMMUNOASSAY FOR RECOMBINANT HUMAN ERYTHROPOIETIN 

Richard Stripp' and Lillian Guia 

John Jay College of Criminal Justice, The City University ofNew York, New York, NY, USA 

The FDA approved recombinant human erythropoietin, otherwise known as rhuEPO, for human use in 

1991. Recombinant human erythropoietin was created for patients with anemia to boost red blood cell 

production, termed erythropoiesis. Alleged abuse due to availability during clinical trials has been reported 

as early as 1987 in human athletes. Administration of rhuEPO to a healthy patient is similar to blood 

doping or training at high altitudes. It increases the level of circulating erythrocytes, increasing the oxygen 

carrying capacity of the blood to reduce fatigue in the athlete competing in endurance sports. This abuse, 

not limited to human athletes, has been reported in horseracing as well. The unique characteristic of 

administration ofrhuEPO to the equine athlete results in an immune reaction to the drug. This results in an 

autoimmune reaction to the horse's intrinsic erythropoietin, causing severe anemia and in some cases death. 

These reactions create antibodies detectable in an enzyme-linked immunoassay (ELISA). Questions have 

been proposed as to the specificity of this assay to react only to the anti- rhuEPO, and not to other intrinsic 

proteins in equine serum. In order to ensure the assay is detecting only the horse's antibody to rhuEPO, we 

have tested the assay against various immunoglobulins and sera from equine and other sources. Reactions 

from all of the 19G's and sera tested and potential cross reactivity between the ELISA rhuEPO plate and 

serum proteins in animals are presented. 

Keywords: rhuEPO, Blood doping, ELISA 

Page 179

A66 

DETERMINATION OF SODIUM AZIDE IN BODY FLUIDS AND BEVERAGES BY ION 

CHROMATOGRAPHY 

Yukari Hirata', \ Saori Ishida l , Hitoshi Tsuchihashi 2 , Makoto Hamajima l , Kanako Watanabe 3 , Hiroshi 

Seno 4 , Rina Kaneko I,Akira Ishii!, IDepartment of LegaJ Medicine, Fujita Health University School of 

Medicine, Japan, 2Forensic Science Laboratory, Osaka Prefectural Police Headquarters, Japan, 3Department 

ofLegal Medicine, Hamamatsu University School of Medicine, Japan, 4Department of Legal Medicine, 

Aichi Medical University, Japan 

Background: Sodium azide, which is widely used as a bactericide in protein samples in clinical or research 

laboratories, is highly toxic. Actually, its oral LD50 value is estimated to be 45 mg/kg, which is only 5 

times greater than that potassium cyanide. In 1998, several mass poisoning cases with sodium azide 

occurred in Japan. It was then regulated as a poison by the Poisonous and Deleterious Substances Control 

Law. It is thus important to determine azide in body fluids and beverages with high sensitivity. We have 

succeeded in determining azide in different materials by ion chromatography. 

Methods: Azide ion in various samples was extracted using a Conway microdiffusion cell. Hydrazoic acid 

was vaporized from 1 mL sample (plasma or beverages) by adding 1 mL of 5% sulfuric acid in the outer 

groove, and absorbed in 250 ~L of 0.1 M NaOH solution in the central round basin. After the cell was 

incubated at 37°C for 30 min, a 20-j..tL aliquot of 0.1 M NaOH was injected to an ion chromatography 

system equipped with a suppressor and a conductivity detector (Dionex DX 500 system). The guard and 

separation columns used were a Dionex AG15 (50 x 2 mm Ld.) and an AS 15 (250 x 2 mm Ld.) column. 

The mobile phase was 38 mM NaOH and the flow rate was set at 0.4 mLimin. 

Results: The retention time of azide ion was about 10 min. In human plasma samples, the calibration curve 

gave good linearity in the range of 50 ng/mL and 10 ~g/mL; its detection limit was about 30 ng/mL. The 

intraday and interday CV values for 5 ~g/mL plasma were 2.8 and 8.8 %, respectively. We have tried 

several internal standards, but the use of an internal standard did not improve the reproducibility in 

quantitation of azide. Also, similar experiments were performed in different beverage samples spiked with 

sodium azide. It gave good linearity in the range of 50 ng/mL and 5 llg/mL; its detection limit was about 

10 ng/mL. 

Conclusion: Combination of the microdiffusion method and a semi-microcolumn has enabled the detection 

of azide with 3 to 4 times higher sensitivity compared to the methods previously reported. Thus, the 

present method can be applicable for clinical and forensic toxicology, because of its simplicity and 

sensitivity . 

Keywords: Azide, Ion chromatography, Microdiffusion method 

Page 180

A67 

A GENERAL SCREENING AND CONFIRMATION APPROACH TO THE ANALYSIS OF 

DESIGNER TRYPTAMINES AND PHENETHYLAMINES IN BLOOD AND URINE USING 

GCIEI-MS AND HPLCIELECTROSPRA Y -MS 

Shawn P. Vorce*, Jason H. Sklerov, Office ofthe Armed Forces Medical Examiner, Division of Forensic 

Toxicology, Armed Forces Institute ofPathology, 1413 Research Boulevard, Building 102, Rockville, 

Maryland 20850-3125 

Recent additions of designer tryptamines and phenethylamines to the Drug Enforcement Administration's 

list of controlled substances necessitate analytical procedures for their detection and quantitation. As 

specific immunoassays are not currently available and cross-reactivities with existing assays are unknown, 

a screening method based on gas chromatography mass spectrometry was developed. The method was 

capable of measuring the pentafluoropropionic derivative of a-methyltryptamine (AMT), N,Ndimethyltryptamine 

(DMT), 4-bromo-2, 5-dimethoxy-~-phenethylamine (2CB), N, N-dipropyltryptamine 

(DPT), 2,5-dimethyl-4-N-propylthio-~-phenethylamine (2C-T-7), and 5-methoxy-N, N 

diisopropyltryptamine (5-Me-DiPT). Separation was optimized to allow tentative identification of 

metabolites, which display common electron impact ionization fragmentation patterns. The screening 

method gave limits of detection between 5-10 ng/mL and demonstrated linearity between 50-1000 nglmL. 

The method was successfully applied to blood and urine samples in suspected AMT intoxications. 

Confirmation of 5-Me-DiPT in one of the subjects' urine was achieved using liquid chromatography/mass 

spectrometry (LCIMS). Quantitation by selected ion monitoring (SIM) yielded a urinary concentration of 

229 nglmL. The method was linear from 25 - 1500 ng/mL with a correlation coefficient of 0.995. The 

limit of detection was 5 nglmL in urine on the LCIMS. Two additional peaks were observed and presumed 

to be metabolic products reported previously as 5-methoxy-N-isopropyltryptamine and 5-methoxy-N, N 

diisopropyltryptamine-N' -oxide. 

Keywords: Tryptamine, Phenethylamines, HPLClES-MS 

Page 181

A68 

MULTI TARGET SCREENING (MTS) FOR 300 DRUGS USING A LINEAR ION TRAP MASS 

SPECTROMETER AND LIBRARY SEARCHING 

Wolfgang Weinmann', I, Claudia A. MUller l , Barbora Maralikova I, Andre Schreiber 2 , Merja Gergov 3 , 

[Institute of Forensic Medicine, University Hospital, AlbertstraBe 9, D 79104 Freiburg (Germany), Phone: 

++497612036828, Fax: ++49 7612036858, Email: Weinmann@ukl.uni-freiburg.de, 2Applied 

Biosystems; Frankfurter StraBe 129B; 64293 Darmstadt (Germany), 3University of Helsinki; Department of 

Forensic Medicine; P.P, Box 40; 00014 Helsinki (Finland) 

A Multi Target Screening (MTS) procedure for drugs in blood and urine for toxicological analysis has been 

developed for the rapid detection and identification of tranquilizers (benzodiazepines), hypnotics, drugs of 

abuse (opiates, cocaine, amphetamines, cannabinoids), antidepressants, neuroleptics, and cardiac drugs 

using LC/MSIMS with a linear ion trap-based tandem-mass spectrometer. In comparison to common 

GC/MS or LC/MSIMS methods used for target analysis, the combination of Multiple Reaction Monitoring 

(MRM) and Enhanced Product Ion (EPI) scan in an Information Dependent Acquisition (IDA) experiment 

with library searching offers both sensitive target analyte detection and subsequent identification by a mass 

spectrometric database in a single LC/MS/MS run. Liquid-Liquid Extraction or Solid Phase Extraction has 

been used for sample clean up. Reversed-phase gradient LC with run-time of 30 minutes and MS/MS 

analysis have been performed in MRM using a Q TRApTM LC/MSIMS system with a TurboIonSpray® 

source. Similar to previous work ofGergov et at. [1] and in contrast to a method presented by Marquet et 

at. [2] , who used scan-mode for the survey scan - in the presented method 300 MRM-transitions have 

been monitored with short dwell times of 5 milliseconds each as survey scan of the IDA experiment. In 

case of a positive detection of an MRM transition, an EPI scan of the precursor ion was triggered as 

dependent scan, yielding product ion mass spectra at three pre-selected collision energies with the high 

sensitivity of the linear ion trap. Obtained EPI spectra were then searched against a newly created product 

ion spectral library, Fit, Reverse fit and Purity fit values are given as results in a library search hit list. A 

ready-to-use acquisition method and mass spectral library with Enhanced Product Ion spectra of 300 

compounds is presented. The developed method was successfully applied to samples of forensic cases, 

Dynamic exclusion of detected MRM transitions was used to detect co-eluting compounds. This was 

especially important for intoxication cases, where concentrations are usually 10 to more than 100 times 

higher than normal therapeutic concentrations. Examples of library searching with identification based on 

fit and purity fit values are given for amitriptyline (at CE of 35eV) and dibenzepine (CE of 50eV) and 

others. Purity fit values above 0.9 were obtained for all cases. The results were confirmed by established 

chromatographic screening methods, The developed method showed high sensitivity and selectivity. A 

further advantage of this method in contrast to the published method of Gergov at al. [1] is that the sample 

has to be injected only once, so less time is needed for the analysis. Conclusions: The developed method is 

useful for the rapid detection and identification of 300 pre-selected target compounds in blood extracts of 

forensic and clinical cases using ESI or APCI. After introduction of retention time windows ("periods") 

with each containing up to 300 compounds the number of target analytes can even be increased to more 

than 1000 compounds per analysis. 

l. Gergov M, Ojanpera I, Vuori E. Simultaneous screening for 238 drugs in blood by liquid 

chromatography-ion spray tandem mass spectrometry with multiple-reaction monitoring. J 

Chromatogr B 2003,795, 41 - 53. 

2. Marquet P, Saint-Marcoux F, Gamble TN, Leblanc J C. Comparison of a preliminary procedure 

for the general unknown screening of drug and toxic compounds using a quadrupole linear iontrap 

mass spectrometer with a liquid chromatography - mass spectrometry reference technique. J 

Chromatogr B 2003,798,9 18 

Keywords: Multi target screening, LC-MS/MS library, Information dependent acquisition 

Page 182

A69 

FORENSIC DRUG ANALYSIS WITHOUT PRIMARY REFERENCE STANDARDS 

Ilkka Ojanpera*, Suvi Laks, Anna Pelander, and Erkki Vuori 

Department of Forensic Medicine, P.O. Box 40, FlN-00014 University of Helsinki, Finland 

Previously, primary reference standards have been considered essential for substance identification and a 

prerequisite for quantitative analysis. However, standards for designer drugs, metabolites or rare substances 

cannot be obtained in a reasonable period of time to allow a prompt forensic investigation. This paper 

reports a new approach in forensic sciences to characterize drugs without the immediate need of primary 

reference standards. 

By liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) or Fourier transform mass 

spectrometry (LC-FTMS), it is possible to measure exact molecular masses on a routine basis. Especially 

benchtop LC-TOFMS instruments, being more cost-effective, are suitable for high-throughput work. 

Modern LC-TOFMS gives a high mass accuracy (approximately 5 ppm) and a moderately high mass 

resolution (5000-10000 FWHM). A target library consisting of exact monoisotopic masses for thousands of 

drugs and metabolites can be created in-house to determine the elemental formulas of sample components. 

This kind of mass list is easily updated by typing recent data from the literature. Even complex biological 

samples can be analysed without complete chromatographic separation of the components. 

Chemiluminescence nitrogen detector (CLND) possesses an equimolar response to nitrogen, and 

consequently it provides a universal means for quantification of nitrogen containing compounds using a 

single secondary standard. As approximately 90% of drugs contain nitrogen, CLND is an attractive tool in 

forensic sciences. However, the technique has not been used in this area thus far. 

In this paper, various critical aspects of LC-TOFMS analysis are discussed. These include internal mass 

scale calibration, dynamic range, sensitivity, library search options, isotope pattern analysis, and utilization 

of urine metabolic patterns. CLND analysis is discussed in terms of sensitivity, linearity, equimolarity, and 

general performance. The merits of LC-TOFMS identification and CLND quantification are demonstrated 

in instances, where the availability of standards is a problem, such as in the analysis of street drugs and in 

the determination of a plasma parent drug/metabolite ratio. 

Keywords: Mass spectrometry, CLND, Drug analysis 

Page 183

A70 

FAST SCREENING AND IDENTIFICATION OF DRUGS USING RETENTION TIME 

LOCKINGIRESUL TS SCREENER BY GC-MS 

Rosa E. De Jesus·, Robert A. Middleberg; National Medical Services, Willow Grove, PA 19090 

A major challenge for all forensic toxicology laboratories is to isolate, detect, identify and quantify the 

wide array of toxicants or drugs that may be present in a case where the cause of death is unknown. It is a 

challenge to screen, in a single analysis, hundreds of the most common toxicants that could be present. 

However, the use of a retention time locking process with GCIMS analysis affords this opportunity. This 

very powerful tool allows for the automated, rapid screening and identification of mUltiple analytes in just 

one injection. This tool combines the two key characteristics for the positive identification of a compound: 

retention time and mass spectra. After maintenance of the chromatographic system (Le. cutting or 

changing the column), the retention times of the analytes can drift, which makes the positive identification 

of analytes more time-consuming. Furthermore, although two or more instruments may be running under 

the same conditions, the retention times of those analytes may not be the same. However, when using 

retention time locking, the inlet pressure is adjusted and the methods are locked to that pressure, resulting 

in consistent retention times of all compounds from run to run. With the use of this technique the event 

times of the method will stay the same, allowing the comparison of real time data with previously acquired 

data. Also the chromatograms can be superimposed, leading to easy observation and comparison of 

patterns. Retention time locking is used then in conjunction with a results screener, where a database is 

created for the locked methods. Results screener contains information of the target and qualifier ions ofthe 

analytes of interest, with their relative intensities, at their expected retention times. Therefore, every 

injection is made using a locked method containing a previously created database, and the system scans for 

the ions produced associated with an analyte in a window at its expected retention time. This allows for 

rapid identification of all compounds, including coeluting analytes. This detection and identification of the 

compounds in the sample, based on retention time and mass spectra, is achieved automatically in justa few 

seconds. The information obtained is then reviewed by the analyst and summarized in a customized report. 

Having no limits in the number of compounds that can be included in the database, retention time locking / 

results screener is a very powerful automated tool for the fast screening and identification of compounds in 

a sample. The use of this tool will be demonstrated as it is utilized for the routine analysis of toxicology 

samples. 

Keywords: Retention time locking; GC-MS drug screening; Drug identification 

Page 184

A71 

USE OF STANDARD ADDITION/STANDARD DILUTION FOR QUANTITATION OF 

TOXICANTS IN UNUSUAL MATRICES 

Erica Horak', Robert Middleberg, National Medical Services, Willow Grove, PA 19090 

At the core of forensic toxicology is the desire to quantitate toxicants from a vast array of biological 

matrices. Often a forensic case involves unusual matrices, including decomposed or embalmed tissues, 

which present complexity to the sample preparation and analysis. Analyte recovery can be variable in these 

types of samples; therefore, alternate approaches to quantitation must be considered. One approach taken is 

to construct a matrix matched calibration curve. However, blank matrix is often not readily available, and 

particularly by the very nature of embalmed or decomposed tissues, a true matrix match does not exist. 

Furthermore, recoveries from the blank matrix and actual case samples may still differ. The standard 

addition/standard dilution (SASD) technique, on the other hand, accounts for variable percent recovery, 

matrix effects, and loss of sample, while the sample itself serves as its own quality control. The SASD 

technique can take any of several approaches. The simplest approach involves establishing a relationship 

between the responses of the analyte in the native sample (blank) with the response of the native sample 

with a known analyte concentration added (spike), without consideration of an internal standard in the 

event one is not available. A second approach includes the use of an internal standard, ideally an isotopic 

analog of the analyte, and establishing the relationship between the response ratios of both the blank and 

the spike (at one or more concentrations). In a third approach, the native sample is divided into four 

aliquots. One aliquot is left untreated and the others are either spiked with various analyte amounts or 

diluted. Each sample is then assayed through the analytical method. A plot of the response ratio data 

versus the amount of analyte added yields a line such that the extrapolated negative x-intercept is the 

concentration of the analyte in the native sample. In a final approach, three measurements can be made: 

that of the blank, the blank spiked at one or more analyte concentration, and the blank diluted by some 

factor. The use of a derived formula relates the relative responses, and spiked addition and dilution 

amounts, to the concentration of analyte in the native sample. In this presentation, the method of standard 

addition/standard dilution as an ideal approach to quantitation of drugs in complex and unusual matrix 

types will be demonstrated from actual forensic case data. Advantages and disadvantages of the technique 

will also be discussed. 

Keywords: Standard addition; Matrix; Quantitation 

Page 185

A72 

COMPARISON OF CHEMICAL DERIVATIVES FOR SYSTEMATIC TOXICOLOGICAL 

ANALYSIS OF AUTOPSY BLOOD USING GAS CHROMATOGRAPHY MASS 

SPECTROMETRY 

Tareq AL-Ahmadi', R.A. Anderson 

Forensic Medicine and Science, University of Glasgow, Glasgow G12 8QQ, UK. 

Aims: Different derivatisation techniques have been developed for drug analysis in whole blood using GC 

MS. The method was validated for six drugs of abuse and their metabolites. 

Method: A mixed standard stock solution was prepared to give a final concentration of I Ilg/ml for each 

analyte and this was used to spike whole blood. Solid phase extraction (SPE) was carried out using the 

polymeric phase Phenomenex Strat-X (60 mg/3ml cartridges containing 331lm particles). Blank blood 

(lml) was mixed with internal standard solution and different concentrations of mixed standard stock 

solution, and phosphate buffer (pH 6, 3.5ml). This was centrifuged for to minutes and transferred to SPE 

columns. The extracts, in methanol, were evaporated to dryness and derivatised using three different 

methods: (a) acylation·esterification with PFPAI PFP-OH (2:1 vlv, 150 Ill); (b) acylation-methylation with 

PFPAI trimethylsilyldiazomethane (TMS) (5:1 v/v, 120 Ill); (c) silylation with MTBSTFA containing 1% 

TBDMSCI (30 Ill). Reaction vials were heated with a microwave oven for one minute. After 

derivatisation, vials for methods (a) and (b) were cooled to room temperature and evaporated to dryness 

under a stream of nitrogen. The derivatised extracts were reconstituted in 50111 of ethyl acetate prior to 

analysis by GC-MS. Extracts derivatised by method (c) were analysed directly. A Thermo-Finnigan Trace 

GC·MS instrument was used in selected ion monitoring (S1M) mode except method (c) which used full 

scan mode. The GC was equipped with an HP5 column (30m x 0.32 mm x 0.25Ilm) from J&W scientific 

and split/splitless injection port at 280°C. The oven temperature was at tOO°C for 2 min, programmed at 

12°C/min to 300°C. Ions monitored for quantitative analysis were as follows: 

Derivative Amp MA MOR BZE THC 

THC 

COOH 

PFPAIPFP·OH 118 204 414 82 417 459 

PFPAITMS 118 204 414 82 417 489 

MTBSTFA 158 172 341 282 371 515 

The method was subsequently applied to 35 forensic autopsy case samples. 

Results: Recoveries for all drugs of interest were found to be over 70%. Limits of detection (LOD) were 

calculated as 3 times the standard error of the regression line plus the intercept. LOD's in blood ranged 

from 0.4 ng/ml to 3.7 ng/ml with PFPA/PFP-OH, O.3ng/ml to 1.4ng/ml with PFPA/TMS and with 

MTBSTFA were from 1.9 ng/mJ to 7.3 ng/mt. Limits of quantification (LOQ) were calculated as to times 

the standard error. They were ::::::12.4 ng /ml with PFPAIPFP·OH, ::::::4.7 ng/ml with PFPAlTMS and ::::::19.8 

ng/ml with MTBSTF A of blood. The case samples analysed were found to contain various drugs of abuse 

and prescription drugs. The different derivatisation techniques gave varying results; however acylation· 

methylation gave the best sensitivity and chromatography. 

Conclusion: A validated, sensitive and specific method for the extraction and quantification of drugs abuse 

in blood is presented. An alternative derivatisation method (acylation-methylation) is proposed which 

gives better sensitivity and improved chromatography for detection and quantification of drugs of abuse 

compared to silylation or acylation alone. 

Keywords: Drugs of abuse, Derivatisation, GC-MS 

Page 186

A73 

PREVALENCE OF GHB IN SUSPECTED DUI CASES IN MIAMI-DADE COUNTY, FLORIDA 

M. Elizabeth Zaney*, WiIJiam D. Gennaro, Gaudelio Saavedra, Bernard W. Steele, H. Chip Walls 

University of Miami School.of Medicine - Forensic Toxicology Laboratory 

According to numerous police and poison control center reports, GHBis a commonly abused substance in 

Miami-Dade County. Therefore, we wanted to study the prevalence of GHB in urine and blood samples 

submitted from suspected Driving Under the Influence (DUI) cases to the University of Miami, Forensic 

Toxicology Laboratory. 

Ninety-one consecutive urine samples received from September-December 2003 (whether positive or 

negative for other drugs) were tested for GHB. In addition, a further 114 urine samples, and 74 blood 

samples which tested negative for alcohol were tested for GHB and other drugs. Urine samples were 

received and stored at 4 'c in containers without preservatives; blood samples were received and stored at 

4'C in gray top vacutainertubes with sodium fluoride as a preservative. 

A method taken from the Journal of Chromatography B, 792 (2003) 83-87 (Marion Villain, Vincent 

Cirimeie, Bertrand Ludes, Pascal Kint=*) was modified utilizing rapid protein precipitation and Iiquidliquid 

extraction using 40 microL of sample, 40 microL of deuterated internal standard, 90 microL of cold 

acetonitrile and high speed centrifugation. The supernatant was then carefully evaporated to dryness and 

the residue reconstituted with 70 microL of BSTF A + 1 % TMCS and heating at 70'C for 25 minutes to 

derivatize. Samples were analyzed by GCIMS with a Hewlett Packard Series 5890 GC coupled to an HP 

Series MSD run in SIM mode. A 5-point calibration curve ranging from 10 mgfL to 200 mg/L was used to 

determine the amount ofGHB present in the specimen. 

Of the total 205 urine specimens tested, 5 tested positive for GHB (2.4% positive) with a concentration 

range of237 mg/L 2147 mg/L (mean 789 mgfL, median = 484 mgfL). The above 205 samples were 

made up of91 consecutive urines of which only 1 was positive for GHB (1.1% positive), and 114 samples 

selected by case history, of which 4 were positive for GHB (3.5% positive). 2 blood samples from the 74 

tested were positive for GHB (2.7% positive) with concentrations of 147 mgfL and 235 mg/L. A summary 

ofthe positive findings can be seen in the Table. 

Our findings would suggest that either GHB is not as prevalent as it is thought, or that users are so impaired 

that they don't attempt to drive a vehicle until metabolism and/or excretion have resulted in below 

detection limit samples, which may account for our low positivity rates. Of interest to note is the 

concurrent use ofGHB and Methamphetamine. 

Case one Urine 

Case two Urine 

Case three Urine 

Case four Urine 

Case five Urine 

(These belong to the 

same defendant) 

Case five Blood 

Case six Blood 

GHB: 2147 mgfL 

Other drue:s present: Ibuprofen 

GHB: 609 mg/L 

Other dru2:s present: Amphetamine, Methamphetamine, Naoroxen 

GHB: 469 mg/L 

Other dru2:s Dresent: Amphetamine, Methamphetamine 

GHB: 237 mg/L 

Other drugs present: Amphetamine, Methamphetamine, MDMA, PPA, 

Pseudoephedrine, Dextromethorphan, Ecgonine methyl ester, Oxazepam, 

Alprazolam, Hydroxy-Alprazo!am 

GHB: 484 mg/L 

Other drugs present: Amphetamine, Methamphetamine 

GHB: 147 mg/L 

Other dru2:s Dresent: Acetone 0.003 g/IOOmL, presumptive positive Amp. class 

GHB: 235 mg/L 

Other dru2s present: Methamphetamine 

Keywords: GHB, GC/MS, DUI 

Page 187

A74 

SEPARATION OF AMPHETATIME AND PIPERAZINE DESIGNER DRUGS BY CAPILLARY 

ELECTROPHORESIS 

Sandra C. Bishop" J, Bruce R. McCord], Samuel R. Gratz 2 , Jill R. Loelige~, Mark R. Witkowski 2 

IOhio University, Department of Chemistry and Biochemistry, Athens, OH, USA; 2U.S. Food and Drug 

Administration, Forensic Chemistry Center, Cincinnati, OH, USA 

The recent emergence of a new class of piperazine-type compounds has brought about the need· for laboratory 

screening methods for both seized drugs and toxicological samples. These piperazine compounds, which include 1 

benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), exhibit comparable physiological 

effects and can be substituted for the classic amphetamine-type drugs. We have optimized a chiral capillary 

electrophoresis (CE) separation that detects a set of 6 piperazine and 4 chiral amphetamine compounds in less than 

23 minutes using a 200 mM phosphate buffer at pH 2.8 with 20 mM hydroxypropyl-f3-cyclodextrin (HPf3CD). A 

liquid-liquid back extraction and a solid phase extraction (SPE) were developed for urine samples. The SPE used an 

altered version ofthe Varian Bond Elut Certify Amphetamines in Urine procedure. In addition to the above standard 

compounds, a series of "clandestine" BZP diHCl samples were also analyzed to assess the ruggedness of the 

method. One ofthe Internet synthetic methods yielded 1,4-dibenzylpiperazine, an unscheduled analog of I-BZP. All 

peak ratios were verified by LC-MS. The novel CE separation was tailored to simultaneously detect piperzine 

compounds in addition to amphetamine-like drugs. Since the reported effects of these two classes of drugs are 

strikingly similar, this method will greatly benefit laboratory analysis where the abused or seized substance is in 

question. Distinct migration time and UV -spectral data were obtained for all compounds of interest. 

Keywords: Piperazine, Amphetamine, CapiHary electrophoresis 

Page 188

A7S 

IDENTIFICATION AND ISOLATION OF FILAMENTOUS FUNGI IN SAMPLES OF 

CANNABIS SATIVA L. 

S.O.S. Cazenave', C. Darini, M.M.R.S. Soares 

Laboratorio de Toxicologia Forense de Campinas, Rua Barao de Parnaiba 322, 1. andar, Centro 13100-000 

Campinas, Sao Paulo (Brasil) 

Cannabis sativa L. (Marijuana) is considered an illegal drug being widely used mostly by young adults. 

The plant dried after harvesting and stored under favorable environment to grow fungi which is spread in 

nature. Since literature lacks studies related to filamentous fungi present in Cannabis sativa L... The 

objective of this study was to isolate and identify filamentous fungi in samples of Cannabis sativa L... 

retained in Campinas area and analyzed in The Laboratory of Forensic Toxicology. Sixty samples were 

used divided as follows: 30 samples with evident contamination and 30 without any traces of 

contamination. First, 5 grams of sample were macerated in 50 ml of sterile distillated water to isolate the 

fungi. Next, samples were filtered and placed in sterile tubes. Volume transferred to sterile Petri dish was 

Iml with 15 ml of Sabouraud Dextrose Agar, subsequent homogenizing and after solidification it was 

incubated in room temperature for five days. If colony growth was found identification was carried out 

with macro and microscopy analysis. Most common species of fungi found were as follows: Aspergillus. 

Fusarium, Penicillium and Scapular/apsis. For this reason, Cannabis sativa L. represents a source of fungi 

contamination as fungi were found even in samples that were not visibly contaminated. These 

mycotoxigenic fungi might cause allergy in hypersensitive individuals, diseases in immune suppressed 

patients, or intoxication through ingestion of contaminated samples, therefore presenting a potential risk for 

users and toxicologists as well. 

Keywords: Fungi, Cannabis sativa, Contamination. 

Page 189

A76 

LC-MSIMS IDENTIFICATION OF ETHYL SULFATE - ANOTHER DIRECT MARKER FOR 

RECENT ETHANOL INTAKE 

Sebastian Dresen*·I, Wolfgang Weinmann\ Friedrich M. Wurse 

IInstitute of Forensic Medicine, University Hospital Freiburg, Alberstr. 9, D 79104 Freiburg, Germany, 

Email: Weinmann@ukl.lIni-freibllrg.de; 

2Psychiatric University Hospital, Wilhelm Klein Str. 27, CH 4025 Basel, Switzerland, Email: 

friedrich. wurst@pukbasel.ch 

Besides ethyl glucuronide (EtG), fatty acid ethyl esters (FAEE) and others, one more direct ethanol 

metabolite - ethyl sulfate (EtS) - has been detected by LC-MSIMS in human urine samples. The structural 

characterization has been performed by LC-MS/MS using electrospray ionization with precursor-ion scan 

and product-ion scan techniques. After the synthesis of a deuterated analogue, the urinary excretion profile 

has been investigated in samples obtained from volunteers up to 44 hours after drinking known amounts of 

alcohol ( 9 and 18 g, respectively). Ethyl sulfate was detectable up to 24 hours in urine, Le., 16 to 22 hours 

longer than ethanol could be detected with a headspace GCIFID method in urine. In summary, the urinary 

excretion profile of EtSwas similar to that of ethyl glucuronide, which is characterized by longer 

detectability than ethanol in urine samples and therefore is a marker for ethanol consumption covering the 

time range of up to approximately three days after severe ethanol abuse. The results of structural 

characterization, way of synthesis of the deuterated analogue, method validation data including analyte 

stability in urine and stock solution are presented. EtS can be used for proof of ethanol intake besides EtG. 

The presented method fulfills· forensic-toxicological guidelines for compound identification by 

chromatographic separation, use of deuterated internal standard, quantitation and identification with 

precursor and two product ions being monitored as target and qualifier ions. 

Key words: Ethyl sulfate, Direct ethanol marker, LC-MSIMS 

Page 190

A77 

DETERMINING THE USE OF N1-ETHYL· GUANINE AS A BIOMARKER· FOR FATAL 

ALCOHOL SYNDROME 

Stacy L. Gelhaus', William R. LaCourse, Janine Cook, Katherine Squibb 

Department ofChemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, 

Maryland, USA 

Background: Fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD) represent the 

largest categories of preventable mental retardation syndromes and birth defects. It is theorized that 

ethanol's toxicity is in part mediated by damage to DNA. In DNA, ethanol's primary metabolite, 

acetaldehyde forms an N2-ethylguanine DNA adduct. The duration of this adduct, its affect on DNA 

replication, and its repair mechanism are currently unknown. A case study is presented to assess N 2 _ 

ethylguanine as a potential alcohol biomarker with possible clinical utility in predicting the risk of 

FASIFASD in infants born to alcohol-using women. 

Methods: The case subject was a 20 year old who reported binge drinking on hard alcohol for the first 5 

weeks of her pregnancy. The separation and quantification of N 2 -ethylguanine from unadducted 

nucleosides was performed by an in-house protocol. DNA was purified from whole blood using a 

commercial genomic DNA purification kit. The isolated DNA was completely hydrolyzed into monomer 

bases using established digestion procedures to liberate purine bases; recovery of the purine bases was 

>96%. The N 2 -ethylguanine DNA adduct was isolated and quantified by reversed phase HPLC with 

isocratic elution and UV/fluorescence detection. Bases were identified by retention time and by standard 

spiking. Base quantitation was by calibration curves ofstandard peak height signals. 

Results: The identification of N 2 -ethylguanine by HPLC was confirmed through standard spiking and 

LCIMS. HPLC precision for the N2-ethylguanine was 0.2% for peak area or 3.9% for peak height. 

Standard curve linearity was to 15 ~mol (r = 0.9999). Preliminary analytical sensitivity was 29 nmol 

(HPLC/uV detection), 2.9 nmol (HPLClfluorescence detection), and 20 fmol (LC/MS). No interference 

was found from free bases. The subject's N 2 -ethylguanine concentration determined by LCIMS at 20 weeks 

gestation was 11.7 pmol/mg DNA, which declined to 8.3 pmol/mg DNA by 30 weeks. Her adduct levels 

are higher than a comparison nonpregnant social drinker whose N 2 -ethylguanine concentration was 6.9 

pmol/mg. Reproducibility of these initial subject results is pending. The subject's fullterm daughter was in 

the 75th percentile for weight, 17th percentile for length and 10th percentile for head circumference, an 

indicator ofFAS. 

Conclusions: Initial studies indicate the acceptability of this method for the separation and detection of 

purines, including the N 2 -ethylguanine. Additional subject data is needed to confirm the adduct's clinical 

utility. 

Public Health implications: Clinical validation ofN2-ethylguanine as a biomarker for alcohol damage will 

fortify the established panel of alcohol biomarkers, aiding in the identification of pregnancies at risk for 

FASIFASD and elucidation ofthe mechanisms of alcohol pathology. 

Keywords: DNA, Biomarker, FAS 

Page 191

A78 

LCIMSIMS DETERMINATION OF LSD, ISO-LSD, AND THE MAIN METABOLITE 2-0XO-3 

HYDROXY-LSD IN WHOLE BLOOD AND URINE 

Sys Stybe Johansen* and Jytle Lundsby Jensen 

Department of Forensic Chemistry, Institute of Forensic Medicine, University of Copenhagen, 

Frederik V's vej 11, DK-2100 Copenhagen OE, Denmark. Phone +45-35326241. Fax: +45-35326085. E 

mail: SSJ@forensic.ku.dk 

Aim: To develop simple LC/MSIMS applications that identify, confirm and quantify rare potent drugs in 

forensic cases. An application with the very potent hallucinogenic drug LSD is shown. 

Method: A liquid chromatography mass spectrometric (LCIMSIMS) method has been developed for the 

determination of LSD in whole blood and urine. Furthermore, determination of the common LSD impurity 

iso-LSD (inactive) and the main metabolite 2-oxo-3-hydroxy-LSD were included. The procedure involved 

a simple liquid-liquid extraction of I g sample containing the analyte and LSD-D3 (internal standard) with 

butyl acetate at pH 9.8. After centrifugation, the organic fraction was removed and evaporated to dryness at 

40°C and reconstituted in 100 III mobile phase. A gradient LC system (HP 1100 system, Agilent tech.) with 

a Zorbax SB CI8 (30 x 2.lmm, 3.5 Ilm) was used to separate the analytes within 10 min. Identification, 

confirmation and quantification were done by positive electrospray ionisation with a triple quadrupole mass 

spectrometer (Quattro micro, Waters) operating in multiple reaction monitoring (MRM) mode. For each 

analyte two MRM's were set up, one for quantification and one as qualifier using one precursor ion and 

two product ions per analyte. The ratio between the responses of the two MRM's was used for 

identification purposes along with the retention time. 

Results: The curves of extracted whole blood standards were linear over a working range of 0.01 to 5.0 

Ilg/kg for LSD and iso-LSD of both transitions. The limit of quantification (LOQ) of LSD and iso-LSD was 

O.ot Ilglkg in whole blood, while LOQ of 2-oxo-3-hydroxy-LSD was 0.5 Ilglkg. The repeatability 

expressed by relative standard deviation (RSD) was better than 10% and the relative accuracy was between 

92-99% for LSD and 89-108% for iso-LSD, respectively. The ratio was determined for each analyte and a 

RSD of max. 10% was confirmed. 

The method was applied for a case investigation involving a 26-year-old man who admitted to have been 

abusing LSD. Blood concentrations of LSD and iso-LSD were determined to 0.27 Ilglkg and 0.44 Ilglkg. 2 

oxo-3-hydroxy-LSD was detected in the urine sample and confirmed the abuse of LSD. The case illustrated 

the importance of analyte separation before MRM detection of a sample. The chromatographic separation 

of LSD and iso-LSD were of importance because the diastereoisomers have identical fragmentation paths 

leading to fragments ofequal m/z ratio. 

Conclusion: The method was proved to be suitable for forensic cases being simple, reproducible and 

selective. However, it was observed that monitoring typical fragments of LSD without chromatographic 

separation is inadequate. After the development of this LCIMSIMS method, the initial screening by RIA 

(radioimmunoassay) for LSD was cancelled because it was too time-consuming and expensive considering 

the low number of investigations/cases per year. 

Keywords: LSD, Whole blood, LC/MS/MS 

Page 192

A79 

LC-MSIMS METHOD DEVELOPMENT FOR A PACLITAXEL ASSAY 

W. Lambert"' I, K. Mortier!, A. Verstraete 2 , and C. Van Peteghem 1 , I Laboratorium voor Toxicoiogie, Ghent 

University, Hareibekestraat 72, B-9000 Gent, Belgium, 2Afdeling Klinische Bioiogie, Ghent University 

Hospital, De Pintelaan 185, B-9000 Gent, Belgium 

The development of a method with liquid chromatography coupled to mass spectrometry (LC-MS) for the 

determination of the anticancer drug paclitaxel in plasma is described. The goal of the method is to produce 

accurate and reproducible data, in a sensitive assay. Therefore, factors that influence reproducibility are 

investigated. The method consisted of a basic Iiquidlliquid extraction of plasma with methyl-t-butyl ether. 

Subsequently, samples were analyzed using a standard narrow bore reversed phase column. MRM 

transitions of pac lit axel and the internal standard (cephalomannine) were monitored. Special attention was 

given to two typical electrospray phenomena: adduct formation and matrix effect. First of all, adduct 

formation was investigated. In the absence ofadditives like formic acid or ammonium acetate in the mobile 

phase, reproducibility of the LC-MS method was decreased, when samples of varying alkali metatcontent 

were analyzed. Already at a low concentration of additive in the mobile phase reproducibility increased to 

an acceptable level. Therefore, a small amount of additive was incorporated in the mobile phase. Different 

mobile phase additives were tested for optimum sensitivity and reproducibility. Secondly, matrix effect was 

examined, especially the influence of the vehicle used in the commercial formulation of paclitaxel (Taxo\): 

Cremophor EL. Ion suppression by formulation vehicles has been reported for e.g. polyethyleneglycol400. 

In view of the application of this method in a pharmacokinetic study with Taxol, investigation of the effect 

ofCremophor EL on the ionization was incorporated in the method development. When an isocratic LC run 

was used, ion suppression was noticed mainly as a result of carry-over to subsequent runs. Changing the 

mobile phase to a higher percentage of organic phase and incorporating a gradient step (column wash) 

resolved the matter. The strategy to evaluate and eliminate adduct formation as well as matrix effects can 

also be applied to LC-MS determinations of other compounds and in fact should become an essential part 

ofa validation protocol. 

Keywords: Paclitaxel, Method development, LC-MS 

Page 193

A80 

QUANTITATION OF PHENAZEPAM IN BLOOD BY GC-MS IN POSITIVE DRUG-DRIVING 

CASES IN FINLAND 

Sirpa Mykkanen*' I, Teemu Gunnar!, Kari Ariniemi 1 , Pirjo Lillsunde 1 , Raissa Krasnova 2 

lNational Public Health Institute, Laboratory of Substance Abuse, Helsinki, Finland 

2Bureau of Forensic Medicine of Moscow Region, Department of Forensic Chemistry, Moscow, Russia 

Phenazepam (1-bromo-5-( ortho-chloropheny 1)-2,3-dihydro-l H-l ,4-benzodiazepin-2-one) is white or white 

with cream-coloured crystal powder that is insoluble in water and slightly soluble in ethanoL After a single 

oral dose of 2 mg, the peak plasma concentrations of 8-15 ng/ml can be attained in 3-4 hours. Effective 

therapeutic concentration for persons with neurosis is in the range of 30-10 ng/ml. Phenazepam is 

comparable with lorazepam in respect of the dose and therapeutic action. 

Phenazepam is not medically used in Finland. However it is misused. Therefore, a reliable and rapid 

method for phenazepam analysis is necessary in Finland. In the present method the phenazepam was 

analyzed together with diazepam, midazolam, alprazolam, nordiazepam, oxazepam, temazepam, 

chlordiazepoxide, nitrazepam, bromazepam and lorazepam and their metabolites. 

The GC-MS procedure contained liquid-liquid extraction and derivatization by N-methyl-N-(Ierlbutyldimethylsilyl)trifluoroacetamide 

(MTBSTFA). Analysis was performed in selected ion monitoring 

(SIM) mode. The developed method was validated and the reliability of the developed method was tested 

on the basis of linearity, accuracy and precision. Phenazepam has been found among drivers suspected for 

driving under influence. Year 2003 there were 20 positive phenazepam cases and the concentrations found 

from Finnish drivers varied from 18 ng/ml to 400 nglml. 

Keywords: Phenazepam, GC-MS, Quantitation 

Page 194

A81 

PHENMETRAZINE OR EPHEDRINE FOOLED BY LIBRARY SEARCH 

Sarah M. Wille*, Carlos H. Van Peteghem, Willy E. Lambert 

Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium 

Phenmetrazine, a central nervous system stimulant, currently abused as an anorectic agent, was positively 

identified by library search after injecting a methanolic extract of an ephedrine containing powder using 

different GC-MS configurations. Phenmetrazine is a controlled substance as described in the UN 

Convention of Psychotropic Substances 1971, while ephedrine is used in over-the-counter medications as a 

vasoconstrictor. However, very recently, the Food and Drug Administration issued a final rule prohibiting 

the sale of dietary supplements containing ephedrine alkaloids because such supplements present a health 

risk. 

GC-MS analysis of a methanolic extract of the herbal product containing ephedrine resulted in a peak with 

a nearly identical retention time as ephedrine but identified as phenmetrazine after a library search. It is 

known that formaldehyde contamination in solvents such as methanol can result in conversion of 

ephedrine-like compounds. Ephedrine reacts with formaldehyde to form 3, 4-dimethyl-5-phenyl-1, 3 

oxazolidine with a similar molecular weight as phenmetrazine (1). The fragmentation pattern is also 

similar because both chemical structures differ only in the position of one carbon unit. Thus, library 

searches alone can lead to erroneous conclusions. Experiments using a HP 7683 cold on-column- HP 

6890GC-HP 5973 MSD configuration and a split injector in combination with a 3400 Varian GC-Finnigan 

Magnum ion-trap MSD demonstrated that high injection temperatures influence the conversion speed. This 

was evidenced by injection of a freshly made ephedrine standard in methanol using injector temperatures 

from 60 up to 300°C. 

Attention should be paid to the possibility of chemical and/or thermal conversion of analytes in a sample 

and/or in the GC-injector, as this can lead to interpretation difficulties and erroneous identification of 

analytes in a toxicological screening. As demonstrated by this case, ephedrine-containing samples are of 

particular interest in view of the judicial implications. 

(I) Lewis RJ et aI., J. Foresic ScL, 45,898-901 (2000). 

Keywords: GC-MS, Ephedrine, Conversion. 

Page 195

A82 

AN IDA MEDIATED LC-MSIMS SCREENING PROCEDURE WITH SEMI-QUANTITATIVE 

POTENTIAL 

Tineke N. Decaestecker l , Pierre E. Wallemacq2, Carlos H. Van Peteghem 3 , and Jan F. Van Bocxlaer" I 

1Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, B-9000 

Ghent, Belgium, 2Laboratory of Toxicology and Special Chemistry, Clinical Hospital St.-Luc, Hippocrate 

Avenue, B-1200 Brussels, Belgium, lLaboratory of Toxicology, Ghent University, Harelbekestraat 72, B 

9000 Ghent, Belgium 

Systematic toxicological analysis (STA) in forensic toxicology comprises general unknown screening 

(GUS) procedures, whether or not restricted to well-defined subgroups, as well as specific confirmation and 

quantitation of individual compounds. Even though in some forensic cases, the substance involved is 

known or strongly suspected, the possibility that other toxic compounds may have contributed to the 

observed biological effect cannot be excluded. Efficient and extensive screening strategies are therefore 

indispensable. This study evaluated an IDA-mediated LC-MSIMS screening methodology, qualitatively as 

well as quantitatively, and shows its scope and limitations in a forensic setting. After all, an LC-MS 

screening strategy could on the one hand reduce the off-line sample preparation mandatory in GC-MS, 

because relatively non-volatile compounds can be analyzed as such, and on the other hand increase the 

range of compounds amenable to MS. IDA is an artificial intelligence based product-ion scan mode 

providing automatic "on-the-fly" MS to MS/MS switching. This laboratory introduced in a preliminary 

communication, as first, the concept ofIDA-based LC-MSIMS screening using a Q-TOFI. By performing 

information dependent scanning at two different fragmentation energies, two collision-induced dissociation 

(CID) spectra for each of the detected compounds are generated. As the MSIMS spectra derive from a 

single precursor ion, a very selective and very specific method was devised. Additionally, advance 

knowledge about the xenobiotics that could be responsible for a certain intoxication is not necessary. 

Limitation of the MSIMS acquisition time to an acceptable minimum resulted in an almost instant switch 

back to the MS mode. As such, this approach provided MS chromatograms that still could be of use for 

semi-quantitative purposes. Since former studies revealed that the IDA intensity threshold, unequivocally 

related to the background noise, seemed to be a critical parameter, the solid phase extraction procedure, the 

liquid chromatographic conditions and the mass spectrometric parameters all were optimized in advantage 

to IDA Optimization of the SPE procedure was performed by means of experimental design. Whole blood 

was preferred as biological matrix because of its relevant toxicological characteristics, although this did not 

facilitate the analysis since the drug concentration in whole blood are relatively low compared to urine 

samples. The influence of two different threshold values, Le. 100eV and 400eV, on the qualitative and 

quantitative results was examined. Finally, the screening procedure we developed was benchmarked on the 

one hand qualitatively against the results obtained from traditional GUS approaches in a number of routine 

toxicological laboratories (37 samples). To that end, immunochemical techniques (EMIT, RIA), HPLC 

DAD, GC-MS and GC-NPD techniques were applied to screen the whole blood samples. On the other hand 

the IDA-mediated screening strategy was compared semi-quantitatively against established LC-MSIMS 

methods (7 samples). From a qualitative point of view the procedure performed exceptionally well: when 

applying a threshold of 100eV, mostly all of the drugs detected by the conventional techniques were 

identified, as well as additional drugs that were not previously reported. The procedure proved well-suited 

for an initial semi-quantitative assessment, as is customary in e.g. forensic toxicology before accurate 

intoxication levels are determined using targeted analytical analysis. 

[l] Decaestecker et aI. Rapid Commun. Mass Spectrom. 2000, 14, 1787-1792. 

Keywords: LC-MSIMS, STA, IDA 

Page 196

A83 

DETERMINA TION OF NALOXONE AND NORNALOXONE IN HUMAN PLASMA BY HIGH 

PERFORMANCE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION 


Wenfang B. Fang' and David E. Moody, Center for Human Toxicology, Department of Pharmacology and 

Toxicology, University ofUtah, Salt Lake City, UT 84112 

Naloxone is a jl-opioid antagonist that has been used clinically for over forty years mainly for the treatment 

of opioid overdose and to reverse narcotic-induced depression following surgery. A more recent 

application is the use of naloxone in a combination tablet containing buprenorphine at a fixed ratio of 4: I 

(buprenorphine: naloxone) for the treatment of opioid dependence. The purpose of the addition of 

naloxone to buprenorphine tablet is to prevent diversion ofbuprenorphine for illicit Lv. use. ~ecause ofthe 

low doses of naloxone used in maintenance therapy, sensitive analytical methods are required. This has 

traditionally proven difficult for 6-keto nor-opiates such as nornaloxone. A highly sensitive method was 

developed to measure naloxone and its metabolite nornaloxone in human plasma to gain further knowledge 

about their metabolism and pharmacokinetics. drNaltrexone and d 3 -oxymorphone were used as internal 

standards for naloxone and nornaloxone. Preliminary experiments demonstrated that solid-phase extraction 

improved the recovery ofnorna lox one to 30% compared to 10% using liquid-liquid extraction. Solid-phase 

extraction was applied for sample preparation using C18 extraction columns and 0.01 M ammonium 

carbonate buffer (pH = 9). High performance liquid chromatography interfaced by electrospray ionization 

to a tandem mass spectrometric detector (HPLC-ESI-MSIMS) was used for quantitation. A XTerra MS 

CI8 3.5 Jlm 2.1x 50 mm column (Waters Corporation, Milford, MA) was used for separation. The mass 

spectrometer was a Finnigan model TSQ7000 Thermo Quest triple-stage quadrupole. Quadrupole 1 was 

set to pass only ions at m/z 328, 288, 345 and 305 that correspond to the MH+ ions of naloxone, 

nornaloxone and their internal standard d 3 -naltrexone and d 3 -oxymorphone. The MH+ ions were caused to 

undergo collision induced dissociation in quadrupole 2 that produced product ions at mlz 310, 270,327 and 

287 respectively, which were then monitored selectively by quadrupole 3. The calibration range was from 

0.075 to 20 ng/mL for naloxone and 1 to 20 ng/ml for nornaloxone with the calibration curve constructed as 

quadratic with llX weighting. The lower limit of quantitation (LLOQ) was determined at 0.075 ng/ml for 

naloxone and 1 ng/ml for nornaloxone. Specificity for naloxone and nornaloxone was determined from 

analysis of blank plasma fortified with internal standard only (3 replicates) and with LLOQ concentration 

(1 replicate) in six different lots of plasma. The primary evaluation was to compare the mean peak area 

ratio of any signal at the retention time of naloxone and nornaloxone to its internal standard for each lot 

with the mean peak area ratio of the six LLOQ samples. Mean ratios relative to mean LLOQ ranged from 

2.68 to 7.72 with a mean of 5.35% for naloxone and from 8.03 to 25.5 with a mean of 16.5% for 

nornaloxone. Intra-run accuracy of the LLOQ was within 5.1 % of target with intra-run precision within 

13.5% for naloxone and within 12% of target and with intra-ran precision within 7.4% for nornaloxone. 

(Supported by NIDA grant ROIDAIOIOO) 

Keywords: Naloxone, Nornaloxone, HPLC-ESI-MS/MS 

Page 197

A84 

SIMULTANEOUS DETERMINATION OF BUPRENORPHINE. NORBUPRENORPHINE. 

BUPRENORPHINE-3-GLUCURONIDE AND NORBUPRENORPHINE-3-GLUCURONIDE IN 

HUMAN PLASMA AND URINE BY LIQUID CHROMATOGRAPHY ELECTROSPRA Y 

IONIZA TION TANDEM MASS SPECTROMETRY: APPLICATION TO HUMAN 

PHARMACOKINETICS 

Wei Huang" \ Elinore F.McCance-Katr and David E. Moodyl, lCenter for Human Toxicology, 

Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112 and 2Division 

of Addiction Psychiatry, Virginia Commonwealth University, Richmond, VA 23298 

Our previously described method for buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-3 

glucuronide (BUPG) and norbuprenorphine-3-glucuronide (NBUPG) (SOFT 2003) has undergone a slight 

modification prior to full validation and use in a pharmacokinetics study. The monitored ions for NBUP 

and its internal standard d 3 -NBUP were changed from 101 (product ion) to 414 (parent ion) and 101 to 417, 

respectively. This provided stronger signals and a resultant lower limit of quantitation (LLOQ) of 0.1 

nglmL for NBUP. 

A full validation using human plasma with a cross-validation to human urine was conducted. The LLOQ 

was 0.1 ng/mL for all analytes in human plasma. For the urine samples, the LLOQ was 0.5 nglmL for 

BUP, BUPG and NBUPG and 2.5 ng/mL for NBUP due to interference in some urine samples. The intraassay 

precision, as coefficient of variation (%CV), at 0.1, 0.25, 25 and 40 ng/mL in plasma did not exceed 

17.5% at the LLOQ or 12.5% at all other concentrations for all analytes; in urine, the %CV did not exceed 

17.4% at the LLOQ or 13.3% at all other concentrations for all analytes. The intra-assay accuracy, as % 

deviation from target, in plasma did not exceed 12.3% at all concentrations for the four compounds; in 

urine the % deviation did not exceed 14% at all concentrations. The interassay precision and accuracy was 

determined at 0.25,25 and 40 ng/mL; %CV did not exceed 7.0% and the % deviation did not exceed 5.6% 

for the four analytes. The method has now been used to determine the pharmacokinetics of the four 

analytes in plasma and urine collected from five patients who had been maintained on a daily sublingual 

does of 16 mg buprenorphine for at least 21 days. Plasma was prepared from blood collected for 13 time 

points from just prior to 24 hours after the daily dose and urine collected for the same 24-hour period. The 

results were as follows: 

Pharmacokinetic parameter BUP NBUP BUPG NBUPG 

Plasma 

Cmax (ng)(mL)"1 4.47::1: 0.54 6.36::1: 1.84 7.19::1:3.27 21.5::1: 4.2 

Tmax(h) 0.85::1: 0.42 1.00::1: 0.47 0.80::1: 0041 1.80::1: 0.27 

AUC (ng)(mL)"I(h) 32.1::1: 3.8 87.6::1: 24.6 25.5::1: 7.9 316::1:172 

24-hour Urine 

Concentration (ng)(mLr l 0.94::1: 0.67 112::1: 58 94.9::1: 27.0 463::1: 153 

Amount (% daily dose) 0.014::1: 0.011 1.89::1: 1.10 1.01::1: 0.42 5.43 ::1:2.46 

Renal Clearance (mL)(min)"1 1.09::1: 0.83 48.7 ::1:23.9 148::1: 43 59.1::1: 6.8 

The current method has been shown to be accurate and precise with sufficient sensitivity to monitor all four 

analytes in human plasma and urine samples. The pharmacokinetic data, for the first time, show the 

magnitude of involvement ofNBUPG in the clearance of buprenorphine. 

Supported by NIDA grants ROI DA 10100, ROI DA 13004, and K02 DA 00478 

Key words: Buprenorphine, Glucuronide metabolites, Pharmacokinetics 

Page 198



Behavioral 

Toxicology 

Page 199

Bl 

COMPARISON OF BLOOD AND BRAIN CANNABINOIDS CONCENTRA nONS IN 11 HUMAN 

CASES. CONSEQUENCES ON TRAFFIC SAFETY. 

Mura p.l', Raul S.B. 2 ,Dumestre V. 3 , Brunet B.l,4, Cavagna v.2, Hauet T.l.4, Kintz p: I Laboratoire de 

Toxicologie, University Hospital, Poitiers, France 2Forensic Toxicology Institute, Strasbourg, France 

3Toxgen laboratory, Bordeaux, France 4, INSERM ERM 324, University Hospital, Poitiers, France 

It is known that cannabis effects on cognitive and psychomotor tasks are proportional to the delta 9 

tetrahydrocannabinol (THC) amount consumed but that such impairments are not directly related to THC 

concentrations in blood. Some studies have also indicated that impairing effects could still be present while 

THC blood concentrations had dropped to a few ng/ml, but this assessment has never been confirmed by 

biological data. However, in a number of circumstances such as traffic safety or homicides, forensic 

toxicologists have frequently been asked to answer this question: according to the blood cannabinoids 

concentrations, was the subject under influence of cannabis In order to document this important question, 

we compared cannabinoids concentrations in blood and brain human samples. 

Blood and corresponding brain corresponding samples were provided from legal autopsies performed to 

determine the cause of death. In 11 cases, cannabinoids were found in blood: these 11 cases were 

consequently included in this study. All samples were stored at -20°C until analysis. THC, 11-hydroxy 

THC (II-OH-THC) and 11 nor-9-carboxy-THC (THC-COOH) were investigated by a gas chromatography 

mass spectrometric procedure derived from the method of Kemp et aL (1). Brain samples were initially 

grinded in pH 7.4 Tris buffer. In 10 cases, the precise location of brain sampling was not known. The 

results, expressed in ng/g, are indicated in the following table: 

I 

I Case number 1 2 3 4 5 I 6 7 8 9 10 

ITHC Blood 0.5 0.6 1.8 2.3 3.0 ! 4.4 11.5

B2 

PROSPECTIVE STUDY ABOUT THE EFFECT OF CANNABIDIOL (CBD) ON THE 

PHARMACOKINETICS OF A 9 -TETRAHYDROCANNABINOL (THC) AFTER ORAL 

APPLICATION OF 10 MG THC AND 5.4 MG CBD IN CANNABIS EXTRACT 

Thomas Nadulskil, Fritz Pragst*\ Andreas Michael Stadelmann 2 , Patrik Roser 2 , Tom Schefte~, Martin 

Schnelle 3 , and E.-M. Fronk 3 : lInstitute of LegaJ Medicine and 20epartment of Psychiatry ofthe University 

Hospital Charite, Berlin, Germany; 31nstitute for Clinical Research, Berlin, Germany 

Besides the psychoactive delta 9-tetrahydrocannabinol (THC), varying amounts of cannabidiol (CBO) are 

contained in hashish and marihuana. Furthermore, cannabis-based medicine extracts and clinical grade 

cannabis contain high quantities of CBO, which frequently equal the percentage of THC. CBO is known 

from literature to modify the effects of THC and to have anti-anxiety effects and anti-psychotic benefits 

and to inhibit the P450 mediated conversion ofTHC to II-OH-THC. Therefore, in the present prospective 

and double blind study with 24 volunteers the concentration vs. time curves of THC and its main 

metabolites II-hydroxy-delta9-tetrahydrocannabinol (II-OH-THC) and II-nor-9-carboxy-delta9 

tetrahydrocannabinol (THC-COOH) as well as of CBO were measured after oral administration of 10 mg 

THC alone or in cannabis extract containing 5.4 mg CBO. Additionally, in 12 volunteers the effect of food 

on the concentration vs. time curves was tested. The analytical method was based on automatic solid phase 

extraction with CIS columns, derivatization with N,O-bistrimethylsilyltrifluoroacetamide (BSTFA) and 

electron impact ionization gas chromatography-mass spectrometry (GC-EIIMS) with deuterated standards. 

The limits of detection were between 0.15 and 0.29 ng/mL For the statistical evaluation, the course of the 

concentration vs. time curves and the areas under the curves (AUC) were compared and the metabolites as 

well as metabolite/drug ratios were included. 

The maximum plasma concentrations after intake of the cannabis extract ranged between 1.2 and 10.3 

nglml (mean 4.05 ng/ml) for THC, 1.8 andl2.3 nglml (mean 4.9 ng/ml) for II-OH-THC, 19 and 71 nglml 

(mean 35 nglml) for THC-COOH and 0.2 and 2.6 nglml (mean 0.95 ng/mg) for CBO. The peak 

concentrations (mean values) of THC, II-OH-THC, THC-COOH and CBO were observed 56, 82, 115 and 

60 min respectively after intake. As a mean slightly higher AUC for THC and slightly lower AUC for 

THC-COOH were measured after application of THC + CBO when compared with THC alone but the 

difference between both kinds of intake was not statistically significant. Therefore, an effect of CBO on the 

pharmakokinetics ofTHC at the studied concentrations could not be proved. 

The data were also compared with the results obtained by Huestis et aL in smoking experiments [1]. Caused 

by the strong first-pass metabolism, the concentrations of the metabolites were increased during the first 

three hours after oral drug intake as compared to smoking. Therefore, the concentration ratio THC 

COOH/THC is discussed as a criterion for distinguishing between oral and inhalative cannabis 

consumption. As a result, the differentiation between both ways of drug use based on this ratio is not 

possible in a practical case, since the exact time of drug intake before sampling is not known, since 

THC-COOH may be present in excess from previous consumption, and because of the large variation of the 

ratios within both groups. However, it is shown that the cannabis influence factor CIF introduced by 

Oaldrup et aL for interpretation of driving impairment by the drug [2] can also be used after oral intake. 

[1] M.A. Huestis, J.E. Henningfield, E.J. Cone. Blood cannabinoids. I. Absorption of THC and formation 

of ll-OH-THC and THCCOOH during and after smoking marijuana. J. Anal. ToxicoL 16:276-282. 

(1992). 

[2] T. Oaldrup, I. Meininger. Begutachtung der Fahrtlichtigkeit unter Cannabis im StrafVerfahren. In: G. 

Berghaus, H. P. KrUger (Eds.): Cannabis im StraBenverkehr. Gustav Fischer Verlag, Stuttgart 1998, pp. 

181-204. 

Keywords: Cannabinoids, Cannabidiol, Oral intake 

Page 201

B3 

EFFECTS AND BLOOD CONCENTRATIONS OF THC, H-OH-THC AND THCCOOH 

FOLLOWING ORAL ADMINISTRATION OF 20 MG DRONABINOL OR OF A CANNABIS 

DECOCTION CONTAINING 20 OR60 MG DELTA9-THC 

A. MenetreyI, C. Giroud I *, M. Augsburgerl, L. Rothuizen 2 , M. Pin t, M. Appenzeller 2 , T. Buclin2, P. 

ManginI ILaboratoire de toxicologie et chimie forensiques, Institut Universitaire de Medecine Legale, 

Lausanne, 2Division specialisee de phannacoIogie et toxicologie cliniques, CHUV, Lausanne, Switzerland 

Smoking and oral ingestion are the most popular methods of Cannabis recreational use. Oral therapeutic 

administration, such as with Marinol® (synthetic THC (dronabinoJ) in sesame oil) was introduced in the US 

to alleviate anorexia in HIV/AIDS patients and to ease nausea in cancer chemotherapy. THC is also 

contained in hemp foods. In Switzerland, there is currently a great concern about the danger of driving 

under the influence of Cannabis. Epidemiological studies have indeed shown that Cannabis alone or with 

alcohol constitute the most frequent group of drugs detected in drivers involved in car accidents or in 

driving under the influence of drugs. The main objective of our study was to assess the effects of an acute 

oral intake of a medium to high dose ofTHC on driving capability. A hemp crude preparation containing 

1.5 % delta9-THC and 4.4 % delta9-THC-A was pre-treated at 140°C for 30 min to obtain a powder with 

4.9 % delta9-THC. From this powder containing 20 or 60 mg of delta9-THC, hemp milk decoctions were 

prepared with a yield of about 80%. A double-blind, ethical committee approved study was carried out with 

eight 20-30 years-old healthy male volunteers who were occasional Cannabis smokers. In 4 successive 

sessions, volunteers were administered a placebo, a decoction made with 20 or 60 mg delta9-THC or 20 mg 

dronabinol capsules in a randomised order. Blood samples were taken before administration and 1,2.5,4, 

5.5, 7, 10 and 24 hours following ingestion. In the same time-period, clinical examination and 

psychological tests were performed with a driving simulator. Blood levels of cannabinoids were determined 

by NCI GC-MS according to a method adapted from Huang et ae with limits of quantification of 0.3 

ng/mL forTHC, 0.8 ng/mL for II-OH-THC, and 0.1 ng/mL for THCCOOH. 

Because of anxiety events, which resolved spontaneously, two volunteers were withdrawn from the study 

after ingestion of the medium dose. Mean maximal concentrations [ng/mL] for THC, II-OH-THC and 

THCCOOH as well as the times [hours] to the maximum blood levels are listed below: 

Preparation 

dronabinol 

20 mg ~9-THC 

milk decoction 

16.5 mg ~9_THC 

milk decoction 

45.7 mg ~9-THC 

Parameter THC II-OH-THC THCCOOH 

I 

Tmax 1 

4 5.5 

Cmax 2.8 3.9 

27.8 

Tmax 

I 

I 

4 

Cmax 3.8 

4.7 27.8 

Tmax 

I 

2.5 2.5 

1 

Cmax I 8.4 12.8 66.2 

I 

The individual results show there was a considerable inter-subject variability. Mean maximum 

concentrations of 11-0H-THC exceeded the corresponding levels of THC. A 2 to 3 fold increase in 

cannabinoid concentrations was achieved following ingestion of the strongest decoction. Almost similar 

levels were measured after intake of the same dose of THC, regardless of the type of preparation (synthetic 

THC or crude plant extract). Obvious effects were observed with the tracking test showing a 50 % mean 

highest performance decrease after drinking the milk decoction containing 45.7 mg THC. Although a 

relatively high dose of THC was taken and significant psychoactive effects and performance reduction 

were noticed, THC and THCCOOH concentrations were much lower than those which are generally 

measured after marijuana smoking. In conclusion, this study shows that oral intake of delta9-THC may 

decrease human performance and exemplify the risks ofdriving under the influence of Cannabis. 

Key words: dronabinol, cannabis, driving under the influence of drugs 

3Huang W et aL J. Anal. Toxicol. 25: 531-537,2001 

Page 202

B4 

REASONS FOR OVERESTIMATION OF THE ROLE OF CYP2D6 IN HUMAN METABOLISM 

OF AMPHETAMINE PRECURSOR DRUGS USING THE DARK AGOUTI RAT MODEL 

Thomas Kraemer', Denis Theobald and Hans H. Maurer: University Hospital, Homburg, Germany 

Background: Female Dark Agouti rats (fDA) have been proposed as a model of the human CYP2D6 poor 

metabolizer phenotype (PM) allowing a preliminary screening for CYP2D6 substrates. This rat model has 

successfully been used by the author's working group to investigate the role of CYP2D6 in human 

metabolism of the new designer drug TFMPP (1-(3-trifluoromethylphenyl)piperazine). However, the 

crucial role of CYP2D6 in N-dealkylation of amphetamine precursors (e.g. fenproporex) to amphetamine 

predicted by this rat model could not be confirmed using recombinant human CYPs and human liver 

microsomes (HLM). Four different CYP isoforms, namely CYPIA2, CYP2B6, CYP2D6 and CYP3A4, 

were involved in this metabolic step with CYP2D6 obviously not being of major importance. Using the 

data on intrinsic clearance for the particular isoforms the percentage of contribution of each isoform to the 

entire microsomal clearance in vitro was calculated for fenproporex. The highest contribution was found 

for CYP2B6 (65 % for R(-) and 72 % for the S(+)-enantiomer) and the lowest values were found for 

CYP2D6 (7 % and 4 % for R(-) and S(+)-enantiomers, respectively). The aim of this study was to elucidate 

the reasons for the failure ofthe rat model. As a characteristic test reaction for CYP2B6 activity (CYP 

isoform with the highest contribution), bupropion side chain hydroxylation was chosen. 

Methods: Data for calculation of intrinsic clearances for fenproporex N-dealkylation were taken from 

previous studies with recombinant CYPs. Bupropion hydrochloride (R,S-2-(tert-butylamino)-3' 

chloropropiophenone; amfebutamone; BU) was administered to Wistar (model of the human CYP2D6 

extensive metabolizer phenotype) and Dark Agouti rats for toxicological diagnostic reasons according to 

the corresponding German law. Urine was collected separately from the feces and analyzed directly after 

sampling. After enzymatic cleavage of conjugates and centrifugation, bupropion and hydroxy-bupropion 

were separated and quantified using an Agilent Technologies (AT, Waldbronn, Germany) AT 1100 series 

atmospheric pressure chemical ionisation (APCl) electrospray LC-MSD, SL version and an LC-MSD 

ChemStation using the A.OS.03 software. Gradient elution was achieved on a Merck LiChroCART® 

column (125 x 2 mm LD.) with Superspher®60 RP Select B as stationary phase and a LiChroCART®1O-2 

Superspher®60 RP Select B guard column. The mobile phase consisted of ammonium formate (5 mM, 

adjusted to pH 3 with formic acid) (eluent A) and acetonitrile (eluent B). Human urine samples from 

volunteers after ingestion of BU were analyzed in the same way. 

Results and Discussion: BU and its metabolites could easily be separated using the applied LC-MS 

conditions. Characteristic fragments in the APCI-mass spectra allowed easy discrimination of the sidechain 

hydroxy from the ring hydroxy metabolites. Bupropion side chain hydroxylation was chosen as 

specific test reaction for CYP2B activity. Indeed, the side chain hydroxy bupropion (HO-BU) was the 

major BU metabolite in human urine samples, whereas it was almost completely absent in urine of the two 

rat strains. CYP2B6 seems to playa major role in human metabolism of amphetamine precursors. BU side 

chain hydroxylation, a characteristic test reaction for this isoform, hardly occurs in either rat strain. As a 

consequence, it could be possible, that for lack of a corresponding CYP2B isoform, CYP2D isoforms take 

over such metabolic reactions in rats leading to an overestimation of the role of CYP2D for human 

metabolism. In addition, it should also be kept in mind, that the lower extent of ring hydroxylation of 

amphetamine precursors under in vitro conditions may also contribute to the differences found between 

animal in vivo models and human in vitro test systems. 

Keywords: Dark Agouti rats; cytochrome P450; amphetamine precursor drugs 

,~,--,,- 

Page 203

B5 

CORRELATION OF SALIVA AND BLOOD ESTAZOLAM CONCENTRATIONS WITH 

BALANCE CHANGES IN SUBJECTS AFTER ADMINISTRATION OF THE DRUG AND/OR 

ALCOHOL 

Maria Kalal> Piotr Adamowicz l , Ewa Chudzikiewicz l , Wojciech Lechowicz l , Ewa Pufal 2 , Wojciech 

Piekoszewski l , Karol Sliwka 2 : IInstitute of Forensic Research in Krakow; 2Department of Forensic 

Medicine ofthe Medical Academy in Bydgoszcz 

In 1992-1999, at the Institute of Forensic Research in Krakow, blood samples taken from 83 drivers 

involved in road incidents tested positive for morphine. Benzodiazepines (BZD) were found in 60% of the 

cases. In 2002-2003, the number of drivers under the influence of drugs involved in accidents was 166. 

There was a high prevalence (65%) ofTHC and amphetamines detected. 17% of the population were under 

the influence of morphine and other medications, and 23% tested positive for BZD. In 2002, 278 drivers 

were questioned about driving a car after intake of a medicinal drug. 134 respondents reported driving a 

car while taking a medicinal drug; 41% of them used BZD. Although the approach to the presence of 

morphine, THC, cocaine and amphetamines in the blood of drivers should without doubt be one of "zerotolerance", 

the situation concerning the legal limit for BZD is not so clear. . 

On the basis of the results both of surveys and laboratory examinations, it can be stated that the BZD's 

most often used in Poland are diazepam and estazolam. Therefore, estazolam was chosen as a model 

substance for analysis of balance changes in persons influenced by the drug. The balance disturbance that 

occurs at the legal limit (0.5 giL) ofalcohol in blood was used as a reference point. 

Concentrations of estazolam in blood and saliva samples were determined by the LC-MS/APCI method 

after LLE. Blood and saliva samples were taken from 25 healthy volunteers who received a single oral 

dose of 1 mg of estazolam. The samples were collected every hour, for four hours. Half an hour before the 

last sampling, volunteers received a dose of ethanol that led to a blood concentration of about 0.5g/1. On 

the next day, the same volunteers received alcohol alone at the same dose, and a blood and saliva sample 

was taken 0.5 h later. 

The LC-MS/APCI method had the following validation parameters [ng/mLl: LOD - 0.33 and 0.63, LOQ 

1.09 and 1.05, LOL - up to 200 and 20 for blood and saliva respectively. 

The dose of estazolam produced average blood concentrations of 21, 24, 24 and 29 ng/mL, and saliva of 

2.8, 1.9, 1.9 and 2.2 ng/mL at the respective sampling points. The correlation coefficient between blood 

and saliva concentrations was 0.50. 

For estimation of balance changes in persons influenced by estazolam, alcohol, and estazolam with alcohol, 

posturography was used. Using the Langevin equation, the diffusion matrix and the friction coefficient 

were calculated. For each of the tested persons, 8 stabilograms were obtained, two (with open and closed 

eyes) for each of the four studied situations:· control (before estazolam or alcohol administration); after 

intake of alcohol alone; 2 h after intake of estazolam alone; as well as after intake of ethanol together with 

estazolam. 

It was shown that the values of the friction coefficient decreased when persons kept their eyes closed, 

1 

from: 9.45 to 7.25 5. , 9.45 to 6.95 5. 1 and 9.83 to 6.77 5. 1 after administration of estazolam, alcohol, and 

alcohol with estazolam respectively. These changes showed a trend when under the influence of estazolam 

(P=0.078) and ethanol (P=0.057), but were statistically significant for estazolam with ethanol (P=0.036) as 

compared to the placebo condition. The values of the friction coefficient after administration of alcohol 

and estazolam did not differ statistically (P=0.677). 

The results show that the friction coefficient can be used as an indicator that allows us to assess if a person 

is under the influence of a medicine that disturbs hislher balance. 

Keywords: Estazolam, Friction Coefficient, Posturography. 

Page 204

B6 

EVALUATION OF THE POST-ROTATIONAL NYSTAGMUS TEST (PRN) IN DETERMINING 

ALCOHOL INTOXICATION 

Majda Zorec Karlovsek*, Joze Balazic: *University of Ljubljana, Institute for Forensic Medicine, 

Ljubljana, Slovenia 

Aims: With a blood alcohol concentration (BAC) legal limit of0.5 glkg (since 1965), Slovenia's road traffic 

legislation has increasingly oriented the work of forensic toxicologists towards quality assurance of 

alcoholometric analyses of BAC and breath alcohol concentration (BrAC). The introduction of a 

Standardised Field Sobriety Test (SFST) for identifying suspected DUl offenders raised our interest to 

evaluate the post-rotational nystagmus test or Tashen test, a part of the physician's examination for 

detecting alcohol intoxication. Post-rotational nystagmus is induced by suddenly stopping the rapid rotation 

of the body; large slow movements of the eyeballs occur in the opposite direction to the direction of 

rotation. 

Performing the test: The person is turned with open eyes in a tight circle 5 times around his axis (while 

sitting on a swivel chair). Time is measured after the chair stops and the person fixes his gaze on an object 

(e.g., a pencil or finger), which the physician holds at a distance of approximately 30 cm from his eyes. 

Materials and methods: The study included the results of 1,006 PRN tests performed during medical 

examinations for DUl cases at the Institute for Forensic Medicine in Ljubljana in the years 1998-2002. 

Cases with a combination of alcohol and drugs were excluded. The evaluation of PRN test results with 

BAC as a reference was based on classification into the following categories and characteristics: true 

positives (TP), true negatives (TN), false positives (FP), false negatives (FN), sensitivity, specificity, 

positive predictive values (PPV), negative predictive values (NPV) and accuracy. 

Results: Measured interval values of post-rotational nystagmus time ranged from 1 to 45 s, mean value 14, 

median 13; BAC values were between 0.0 and 3.16 glkg, mean 1.12 g/kg, median 1.14 g/kg. The values of 

post-rotation nystagmus time and BAC show a positive correlation, which is statistically significant (r = 

0.54,p < 0.01). For the regression line, the following formula was obtained: PRN = 6.0 x BAC + 7.2. 

Raising the threshold values for post-rotational nystagmus time (from 6 to 16 s) shows an increase in 

sensitivity accompanied by a decrease in specificity. The optimal cut-off value of 10 s for post-rotational 

nystagmus time was chosen with the help of a Receiver Operating Characteristic curve (ROC curve) for 

BAC limit 0.5 g/kg (TP=584, FP=43, FN=229, TN=150, sensitivity=0.718, specificity=0.777, PPV= 0.931, 

NPV= 0.396, accuracy=0.730). 

Conclusions: According to the area under the ROC curve, the post-rotational nystagmus test is a fair test for 

predicting alcohol intoxication over 0.5 g/kg. However, as a part of the physician's examination it can 

contribute to the description ofthe clinical state. 

Key words: post-rotational nystagmus, alcohol intoxication 

Page 205

B7 

DETECTION OF COMMONLY ABUSED DRUGS IN URINE OF SEXUAL ASSAULT 

COMPLAINANTS 

Matthew Juhascik 1 *, R.E. Gaensslen\ Christine M. Moore 2 , Paul J. Goldstein', Alice Lindner 4 , Pam 

Greene 4 , Diana Faugn0 5 , Linda Ledral, Barbara Haner 7 , Adam Negrusz 1 : lDepartment of 

Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL, 2United States Drug Testing 

Laboratories, Inc., Des Plaines, IL, 'Epidemiology and Biostatistics, University ofIlIinois at Chicago, 

Chicago, IL, 4Scott & White Memorial Hospital, Temple, TX 5Palomar Pomerado Medical Center, 

Escondido, CA, 6SARC, Hennepin County Medical Center, Minneapolis, MN, 7Providence Everett Medical 

Center, Everett, W A. 

There are approximately 100,000 reported cases of sexual assault in the U.S. every year. It is estimated 

(Bureau of Justice Statistics) that there are more than 300,000 sexual assaults every year, three times the 

number actually reported. Recently, the use of "date-rape" drugs to incapacitate someone has received 

considerable coverage in the media. However, before toxicologists can examine what "date-rape" drugs 

are present in a sexual assault complainant, it is important to know what drugs of abuse are also commonly 

found. This project is designed to estimate which drugs of abuse are found in sexual assault complainants 

through a random sample from four reasonably representative US jurisdictions. Sites include locations in 

Washington, Texas, California, and Minnesota. 

Sexual assault complainants are asked when presenting to the hospital ifthey would like to take part in this 

study. Ifthey agree, consent forms are filled out and the complainant provides a urine sample following a 

protocol approved by the UIC IRB. The sample is then sent to our laboratory for proper handling and 

storage. At the end of the subject recruitment phase ofthe study, 31 sexual assault complainants had been 

recruited at the Texas location, 56 from the California site, 15 from Washington, and 43 in Minnesota, for a 

total of 145. The racial distribution ofthe sample is: 70.3% White, 8.3% Black, 13.1% Latino, and 8.3% 

other/unknown. The ages ofthe complainants range from 18 to 56 with the highest number in the 21-25 

age cohort. 

Urine samples collected from all of the complainants are screened by immunoassay for the following drugs 

ofabuse (values in parentheses are corresponding cut-off values): ethanol (40 mg/dL), amphetamines (250 

ng/mL), opiates (50 ng/mL), PCP (10 ng/mL), cannabinoids (10 ng/mL), methadone (100 ng/mL), 

barbiturates (100 ng/mL), and benzodiazepines (100 ng/mL). Because drug-facilitated sexual assault 

victims may have only been given a single dose, low cut-off values for the EMIT screen were used to 

achieve maximum sensitivity. All presumptive positive samples are confirmed by GC-MS following 

extraction and derivatization if appropriate. To date, 125 specimens have been analyzed. Confirmed 

positives include 13.6% for cocaine, 28% for marijuana, 2.4% for benzodiazepines, 6.4% for opiates, and 

7.2% for amphetamines. These preliminary results suggest that complainants of sexual assault have more 

drugs in their system than the normal population. 

In order to determine if sexual assault complainants are more or less likely to abuse drugs, our results will 

be compared to general population drug use data from NIDA' s Monitoring the Future and the National 

Household Survey on Drug Abuse conducted by SAMHSA. The confirmed positives will also be 

examined by race, age, and geographic location to determine ifany trends are apparent. 

Key Words: Sexual assault, drugs ofabuse, GC-MS. 

Page 206

B8 

TOXICOLOGICAL FINDINGS IN CASES OF ALLEGED DRUG FACILITATED SEXUAL 

ASSAULT IN THE UNITED KINGDOM 

Michael Scott-Ham BSc and Fiona C. Burton PhD*: The Forensic Science Service, London Laboratory, 

109 Lambeth Road, London, SEI 7LP, UK. 

The Forensic Science Service (FSS) is the major provider of forensic services in the UK. It has several 

laboratories throughout the country with two providing forensic toxicology services. These two 

laboratories annually deal with approximately 500 cases of alleged drug facilitated sexual assault (DFSA) 

submitted from various UK police forces. 

This study outlines the results from 1014 cases of claimed drug facilitated sexual assault analysed at the 

Forensic Science Service, London Laboratory between January 2000 and December 2002. As and where 

appropriate, either a whole blood sample or a urine sample from the complainant was initially analysed for 

alcohol and common drugs of abuse. The test for alcohol was by gas chromatography and for common 

drugs of abuse by immunoassay with positive results being confirmed by gas chromatography-mass 

spectroscopy (GC-MS). Common drugs of abuse tested for include cannabis, amphetamine, Ecstasy, 

cocaine, opiate drugs, methadone, barbiturates and the benzodiazepine drugs diazepam and temazepam. All 

urine samples (or blood sample if urine not collected) were further tested for potentially stupefYing drugs 

and their metabolites by either positive or negative ion GC-MS (as applicable). These tests have been 

shown to have the low limits of detection required to detect the drugs of concern. Potentially stupefYing 

drugs included a range of other benzodiazepine drugs (including flunitrazepam) and related drugs such as 

zopiclone, gammahydroxybutyrate (GHB) and a range of chemically basic pharmaceutical drugs such as 

ketamine and other medicinal drugs with sedative properties. The urine samples were also tested for 

trichlorinated compounds (e.g. chloral hydrate) by a colour test. 

The results are interpreted with respect to the numbers of each drug detected. An attempt has been made to 

distinguish between voluntary use and involuntary ingestion by using information provided by the 

investigating police officer (with follow-up discussion where necessary). Furthermore, in those cases 

where alcohol was detected, the most likely blood alcohol level at the time of the alleged incident has been 

calculated. 

Alcohol (either alone or with an illicit or medicinal drug) was detected in 46% of all cases and in 81 % of 

the cases in which the samples were taken within 12 hours of the alleged incident. Of the cases where 

alcohol was detected, 60% had a high back-calculated figure. For the purposes ofthis paper high is defined 

as greater than 150 milligrams per 100 millilitres (%). Illicit drugs were detected in 34% of the 1014 cases 

with cannabis being the most commonly detected drug followed by cocaine, benzodiazepine drugs, opiate 

drugs, Ecstasy, amphetamine and methadone. A number of samples contained more than one illicit drug. 

In 2% of cases, a potentially stupefying drug was detected which had not been admitted and therefore could 

be a genuine DFSA case. GHB was detected in some of these cases but neither flunitrazepam nor its 

metabolites was detected. Although sedative drugs were detected in many more cases, complainants 

admitted prescribed use of these medications. A wide range of non-sedative pharmaceutical drugs were 

also detected. The types and numbers of all these drugs will be presented (together with examples of 

typical limits of detection). 

The results of these studies are in agreement with other studies published in this area. However, this is the 

first study to our knowledge which has attempted to identifY the 'genuine' cases and which discusses in 

detail the significance of the alcohol levels found in cases of this type. 

Keywords: DFSA, flunitrazepam, alcohol 

Page 207

B9 

ABSINTHE ANALYSIS: A STUDY OF TOXIC COMPONENTS OF THE NOTORIOUS 

BEVERAGE 

Smith AC 1 *, Vena J, Wu AH, McLane G; Departments ofPathology and Laboratory Medicine and Medical 

Toxicology, Hartford HospitallUniverity of Connecticut, and Absolute Standards, Inc. 

Introduction: Popular in the 1800's, absinthe is liquor made from the extract of the wormwood plant 

(Artemesia absinthium). The extract had a green hue, which led the drink to be known as the "green fairy". 

Historically color was enhanced with additives such as antimony and copper, which may have influenced 

toxic effects. The drink was popular among artists and writers because its inherent hallucinogenic 

properties known as the absinthe effect. The absinthe effect was known to stimulate creativity and sexual 

desire. Popularity and excess led to a public health problem, and it was banned in the early 1900's in many 

countries. There has been a recent resurgence as this ban has been lifted in Europe under new regulations .. 

In countries were absinthe is still banned, orders can be placed over the internet. The major active 

ingredients are bicyclic terpenes, o.-thujone and /3-thujone, which .are found as diastereoisomers in nature. 

Thujone is a neurotoxic and porphyrogenic compound. The more potent form is o.-thujone, which is 

believed to block the y-aminobutyric (GABA) gated chloride channel. The fractional content of a. and /3 

thujone is dependent upon plant source. CUrrent European guidelines limit thujone content to 10 ppm in 

alcoholic beverages. Analysis was conducted on 5 separate samples to determine thujone and other terpene 

content, as well as analysis of heavy metal and ethanol content. 

Materials and Methods: Five different commercially available absinthe bottles produced in France and 

Germany were analyzed, each claiming to have an infusion of wormwood plant. Extraction was performed 

using chloroform as the organic solvent. GCIMS analysis was performed on the extracts using total ion 

chromatography and selected ion monitoring. A 10 ppm 0.- thujone standard was used to quantifY results 

(Fluka >96%). Samples were treated with nitric acid, and assayed using ICPIMS to determine the metal 

content. Ethanol content was assayed on a Roche Integra chemistry analyzer using the alcohol 

dehydrogenase 

Results: 

0.- thujone (ppm) 

~- thujone (ppm) 

Total thujone (ppm) 

EtOH (label) v/v 

EtOH (measured) v/v 

Copper (ppb) 

Antimony (ppb) 

sample 1 sample 2 sample 3 sample 4 sample 5 

4.0 1.5 3.4 1.3 0.2 

2.1 1.2 3.2 0.0 

6.1 12.7 6.6 ]If 0.2 

55% !55% 60% 45% 55% 

53% 55% 56% 46% 53% 

15.3 71.3 25.8 19.5 19.6 

0 0 6.8 0 0 

A number of other related compounds appeared in the samples, which in addition to thujone, are extracts 

from plant sources used to make absinthe. These were identified with a library spectra match (> 90%). 

Compounds include camphor, menthol, fenchone, methone, and anisaldehyde. 

Conclusion: Modern absinthe does conform to current guidelines in regards to thujone content by our 

analysis. However, much variation was seen between each manufacturer. One sample did have high levels 

of antimony, and all had copper in varied concentration. The clinical toxicity of absinthe with high terpene 

and metal content in the presence of ethanol intoxication needs further research. Although absinthe is still 

officially banned in the U.S., this product is available on the Internet and popularity is rising. In July 2003, 

an article appeared in Maxim magazine describing this recent trend and Internet popularity in the U.S. The 

history of absinthe has shown it to be a public health concern. The high alcohol content combined with the 

effects of thujone may increase the risk of critical intoxication with consumption of modern absinthe. 

Keywords: Absinthe, Analysis, GC/MS 

Page 208

BIO 

LOW BLOOD ALCOHOL LEVELS, ATTENTIVE FUNCTIONS AND BODY SWA Y 

Raffaele Giorgetti, Santo Davide Ferrara*, Massimo Montisci, Rossella Snenghi, Silvano Zancaner, Mirella Libero: Centre of 

Behavioural and Forensic Toxicology, University of Pad ova, Italy 

This double-blind study aimed at examining the effects of low blood alcohol levels (BAC) with placebo control and random 

cross-over distribution. Psychomotor performance and body sway were studied in 16 healthy volunteers after intake of low 

doses of ethyl alcohol (0.5 glkg). Neurosensory and psychomotor functions were assessed by a Divided Attention Test (DAT) 

together with Short-Term Memory and Response Competition Tests (RCT). Changes in body sway were recorded by a 

stabilometer. Tests were administered at 30, 60, 120 and 180 minutes after alcohol intake. Tests were repeated with a wash-out 

period ofone week. BAC was monitored by breathalyser and analysis of blood levels. 

Statistical analysis showed that., as regards the RCT, the number of errors did not increase in a statistically significant way (p = 

0.9071) in any of the experimental phases after alcohol intake (Table I). Only the time effect (learning effect) reached 

statistical significance. 

TABLE I- Response Competition Test 

Treatment Time Treatment x Time 

F T p-value F I p-value F I p-value 

Errors 0.001 1 0.9071 10.61 I

Bll 

HUMAN PERFORMANCE FORENSIC TOXICOLOGY IN TURKEY 

S. A. Akgur*l, P. Ozturk l , S. Cengiz 2 , A. Yemiscigil l 

lEge University Faculty of Medicine, Department of Forensic Medicine, Bornova, Izmir, Turkey. 

2Institute of Legal Medicine and Forensic Science, Istanbul, Turkey. 

Human performance toxicology, also referred to as behavioral toxicology, is a branch of forensic 

toxicology concerned with the relationship between the presence of a drug and associated behavioral 

changes. Forensic toxicologists have become interested in human performance toxicology because it is a 

natural extension of their interest in the medicolegal implications of drug use, misuse, and abuse. This 

branch needs new educational and analytical approaches and developments in our country. The aim of this 

study is to describe the present status, progress and applications of human performance toxicology in 

Turkey. 

Forensic Toxicology in Turkey is carried out in the Forensic Medicine Council of the Ministry of Justice 

and Forensic Medicine Departments and Forensic Medicine Institutes of Universities. Forensic Toxicology 

is practiced almost exclusively as a result of investigation of fatalities. As a result, postmortem forensic 

toxicology is the main topic in the Forensic Medicine Council. 

In most countries Forensic Toxicology has progressed with the advances in analytical chemistry but the 

same progress has not been achieved yet in forensic chemistry in countrywide of Turkey except Istanbul 

Ankara and izmir. Forensic Medicine Departments that provide education in forensic medicine are also 

attempting to develop the fields of forensic sciences. There are several forensic toxicology education 

programs in universities and institutes, including Ege University. 

Ege University, in cooperation with the Police Department, is applying on-site (saliva) drug tests in traffic 

cases. The positive results will be· confirmed by Enzyme Multiplied Immunoassay (EMIT) and Gas 

Chromatography! Mass Spectrometry (GCIMS). Most countries have legislation covering driving under 

the influence of drugs (DUID). Most have an impairment approach but not an analytical approach. Several 

European Union (EU) member states have recently introduced or are preparing 'per se' laws on DUID. 

Driving under the influence of drugs is prohibited by traffic legislation in our country also, but 

unfortunately the analytical procedure is not yet effectively used. The Police Criminology Laboratories do 

not analyze biological specimens for drugs. In routine applications, the impairment approach is only related 

with driving under the influence of alcohol. 

At present, the breath alcohol tests performed by police are routinely applied to motor vehicle operators 

driving under the influence of alcohol. The allowed ethanol limit for driving under the Turkish Road 

Traffic Act is O.5gfL. In alcohol testing, the first suspicion of the police officer is followed by an on-site 

breath analyzer test and under certain conditions it is followed by the blood test for alcohol in Council of 

Forensic Medicine cases. Blood Alcohol Determination is performed by Conway- micro diffusion, 

immunoassay techniques and gas chromatography. Recently, the Forensic Medicine Chemical Analysis 

Specialty Office has made an effort for analytical toxicological research projects to include GC, GCIMS, 

High Performance Liquid Chromatography (HPLC) and Head Space Gas Chromatography (HS-GC). 

More collaborative projects on educational and laboratory practices will help us to achieve improvements 

in Forensic Toxicology in Turkey. 

Keywords: Human, Performance Toxicology, Turkey 

Page 210

B12 

DRUGS OF ABUSE IN PORTUGAL; A STATISTICAL REVIEW 

S Avila l "" A Castanheira, M Barroso, C Margalho, E Gallardo, S Santos, E Marques, D N Vieira: 

National Institute of Legal Medicine-Delegation of Coimbra Portugal 

Introduction: Drug abuse and its eventual association to fatal intoxications, or other violent deaths, is 

responsible for a great number of requests for drug testing at the Portuguese National Institute of Legal 

Medicine. With approval of recent legislation in 1998, which establishes procedures for conducting drug 

screening requested by driving enforcement authorities, it has been possible to develop a new approach to 

the evaluation of drug consumption linked to driving in situations which did not result in fatal accidents. 

Objectives: The authors present a retrospective study involving cases from 2003, from the centre of 

Portugal. Drug testing in post mortem blood was performed in all cases requested by the pathologist, as 

well as in those cases of individuals aged less than 50 years old, when the autopsy was inconclusive. The 

authors also evaluated the incidence of drugs ofabuse in deaths related to driving accidents, and discuss the 

actions of the pathologist, as well as of the law forces, in these situations, regarding the request of the drug 

screening. 

Methods: The samples tested were always whole blood. After screening by enzyme-linked immunoassay 

(EIA by Coda, Bio-Rad), the positive samples were submitted to a procedure involving solid phase 

extraction followed by confirmation and quantification analysis using a HP 6890 gas chromatographer 

equipped with a HP 5973 mass selective detector (GCIMS) using electron-impact ionization. Cut-off values 

for the most common drugs are established by law: 100, 150,80 and 1000 ng/mL for opiates, cocaine and 

metabolites, cannabinoids and amphetamines, respectively. The limits of quantification (LOQ) are less than 

these cut-off values for all drugs analysed. 

Results: The authors present the overall results oftesting for drugs of abuse in post mortem samples, with a 

description of the circumstances of driving accidents. 261 analyses for drug testing in post mortem blood 

were performed and 18 were positive (6.9%). 61 (23.4%) of the cases were due to driving accidents and 

only I of these was positive for drugs of abuse. Law enforcement authorities requested 163 analyses for 

drug testing. There were 10 (6.1%) positive results. In 6 of these positive cases, the information was 

sufficient to establish links to driving accidents. 

Conclusion: Since the law that regulates drug screening in driving cases is very recent, it is still difficult to 

make reliable conclusions about the influence of these substances on driving, and on the incidence of 

accidents. On the other hand, law enforcement authorities do not consistently request drug testing, because 

the law is not clear enough in this respect. The pathologists also use variable criteria to request testing, 

therefore, screening tests for drugs of abuse have only been performed on a small percentage of the total 

fatal accidents. 

Key words: Drugs of abuse, Driving, Portugal 

Page 211

B13 

CONCENTRATION OF DRUGS IN BLOOD OF SUSPECTED IMPAIRED DRIVERS 

Marc Augsburger*\ Nicolas Donze 2 , Annick Menetreyl, Clarisse Brossard l , Frank Sporkert\ Christian 

Giroud l and Patrice Mangini : I Institut Universitaire de Medecine Legale, Lausanne, Switzerland; 2 

Consilia, Sion, Switzerland 

Blood has been undoubtedly considered to be the only suitable specimen for the evaluation of driving 

impairment due to drug consumption. In order to gain more information about the type and the 

concentrations of drugs used by drivers arrested for driving under the influence of drugs (DUID), we 

analyzed 440 blood samples. All official DUID cases submitted by the Justice Department during a period 

ranging from 2002 to 2003 concerning living individuals were considered. This study included 400 men 

(91%) and 40 women (9%). The average age of the drivers was 28±1O years (minimum 16 and maximum 

81). One or more psychoactive drugs were found in 89% of blood samples. For the majority of cases (51% 

from 440), mixtures (from 2 to 6) of psychoactive drugs were detected in blood. The most commonly 

detected drugs in blood are cannabinoids (54%), ethanol (42%), cocaine (12%), benzodiazepines (11%), 

amphetamines (8%), opiates (8%) and methadone (6%). Among these 440 cases, THCCOOH was found in 

54% {mean 35 nglml (1 to 215 ng/ml)), ~9-THC in 49% {mean 5 ng/ml (I to 35 ng/ml)), ethanol in 42% 

{mean 1,28 g/kg (0.14 to 2.95 g/kg)), benzoylecgonine in 12% {mean 515 ng/ml (29 to 2430 ng/ml)), free 

morphine in 8% {mean 19 nglml (1 to 111 nglml)), methadone in 6% (mean 165 ng/ml (27 to 850 nglml)), 

MDMA in 6% {mean 388 nglml (1O to 2480 ng/ml)), free codeine in 5% {mean 5 ng/ml (I to 13 ng/ml)), 

cocaine in 5% {mean 109 ng/ml (15 to 560 ng/ml)), midazolam in 5% {mean 56 ng/ml (20 to 250 nglml)), 

nordiazepam in 4% {mean 492 ng/ml (30 to 1560 ng/ml)), amphetamine in 4% {mean 63 nglml (10 to 183 

nglml)), diazepam in 2% {mean 279 nglml (80 to 630 nglml)) and oxazepam in 2% {mean 614 nglml (165 

to 3830 ng/ml)). Other drugs, such as lorazepam, zolpidem, mirtazapine, methaqualone were found in less 

than 1 % of the cases. Propositions for a new zero-tolerance law will suggest that the presence of scheduled 

drugs like amphetamine (2:20 ng/ml), methamphetamine (2:20 nglml), MDMA (2:50 nglml), MDE (2:50 

ng/ml), cocaine (2:20 nglml), free morphine (2:20 nglml) and ~9-THC (2:2 ng/ml) is sufficient for 

prosecution, regardless whether the capacity of driving of the person was impaired. If we apply these 

conditions to these cases, 60% ofthe drivers tested in this study would be prosecuted. 

Keywords: drugs, alcohol, driving 

Page 212

B14 

EFFECTS OF OPIOIDS ON METHAMPHETAMINE-INDUCED STEREOTYPICAL 

SELF-INJURIOUS BEHAVIOR IN MICE 

Tomohisa Mori"', Shinobu Itoh, Toshiko Sawaguchi : Department of Legal Medicine, Tokyo Women's 

Medical University, Tokyo, Japan 

Psychostimulants as well as other abused drugs, such as morphine and heroin, induce several behavioral 

effects in rodents, which is believed to be mediated by the activation of the dopaminergic system. 

Furthermore, relatively high doses of psycho stimulants induce stereotypical self-injurious behavior (SIB) in 

rodents. The combination ofpsycho stimulants and opioids is quite common and is reportedly used to produce 

a more intensely pleasurable "rush". Therefore, some investigators have sought to characterize the 

interactions of these psychostimulants and opioids. However, the effects of opioids on dopamine-related 

behavior, especially psychostimulant-induced SIB, remain to be fully elucidated. The present study was 

designed to investigate the effects of j.l.-, 0- and K-opioid receptor agonists (morphine (5.0 - 20 mg/kg, s.c.), 

SNC80 (1.25 - 5.0 mg/kg, s.c.) and U50,488H (1.0 - 10 mg/kg, Lp.), respectively) and buprenorphine (0.125 

- 2.0 mg/kg, s.c.) on methamphetamine-induced SIB. After the administration of methamphetamine, SIB, 

especially skin-picking behavior or self-biting around the chest, was measured for 3 min at 15 min intervals. 

A score of 0 was given for inactivity of SIB-like behavior, 1 for very mild SIB (less than 1 min), 2 for at least 

1 min of SIB, 3 for continuous SIB through 3 min. Methamphetamine (20 mg/kg) induced SIB in 6 out of7 

ddY mice, while· in the combination test, the opioid receptor agonists significantly attenuated 

methamphetamine-induced SIB. The relative potency of these opioids in attenuating 

methamphetamine-induced SIB was morphine> buprenorphine > U50,488H » SNC80. These results 

suggest that the stimulation of j.l.- and K- (particularly j.l.-) opioid receptors exerts an inhibitory effect on 

stereotyped high-dose methamphetamine-induced SIB in mice. 

Keywords: Self-injurious behavior; Methamphetamine; Opioid 

Page 213

B15 

LONG-TERM ROADSIDE SURVEYS FOR DRUNKEN DRIVING IN FINLAND 

M Portman l .., A Penttila\ K Kuoppasalmil, P Lunetta 2 ,P Nevala 3 : lNational Public Health Institute 

Helsinki 2Department offoren sic Medicine, University of He\sinki 3The Mobi\e Police, Helsinki ' 

Numerous reports have shown that the rate of drunken driving declined worldwide in the 1980s. 

Nevertheless, drunken driving is still a considerable factor in fatal motor vehicle accidents. In 2002, 415 

persons were killed in road traffic accidents in Finland (population 5.2 millions). In these accidents the 

number offatalities involving drunken drivers was 91 (21.9%), whereas 12 persons (2.9 %) were killed in 

accidents involving other intoxicants than alcohol. Annual roadside surveys have been carried out since 

1978 in the province ofUusimaa in southern Finland to determine the incidence ofdrivers who had been 

drinking. The statutory limit of blood alcohol concentration for drunken driving in Finland is 0.5 0/00 

(glkg). The police can make a demand for a driver's breath sample to be tested for the presence of alcohol 

even in the absence ofobvious signs ofdrunken driving. Annually a total ofabout 30000 drivers have been 

breath tested. The surveys have been carried out using the same protocol and, therefore, the results obtained 

from year to year are comparable. The surveys take place on three Tuesdays and three Saturdays during 

both spring and autumn. These days are chosen to represent one weekday and one day of the weekend. 

Every survey consists of4-5 roadblocks, each lasting 30-40 minutes. The roadside survey team includes 

one chief inspector, 8-14 police officers, one physician and a few assistants. The driver of every motor 

vehicle is breath tested with an Alcometer PST-MIR (Lion Laboratories Ltd., Cardiff, U.K.) or an 

Alcosensor IIIR (lntoximeters Inc., Saint Louis, Missouri, U.S.) screening device. The incidence of 

drunken drivers has been 0.2 % since the beginning ofthe 1990s. The percentage of drivers with an alcohol 

concentration below the statutory limit has been 0.4 % during the 1990s and since 2000 a rising trend has 

been observed. In 2003 the frequency was 1.01 %. A possible explanation for the observed rise is that the 

attitude, especially of young drivers, has changed, resulting in an increase in the number of cases involving 

low blood alcohol concentrations. More and more young drivers recognize that while dining in restaurants 

or attending parties they can consume one to two beers or ciders while their blood alcohol concentration 

can remain safely below the statutory limit of0.5 0/00. The coming years will show whether the recent 

significant increase in Finland of drivers with low alcohol content is a permanent trend or not. The taxes of 

alcohol beverages were substantially lowered in March this year, which may well contribute to 

maintaining, or accelerating this trend. 

Keywords: drunken driving, roadside survey, breath testing 

Page 214

B16 

AMELIORATION OF LEAD TOXICITY WITH VITAMIN C AND SILYMARIN SUPPLEMENTS 

Shalan, M.G.*l; Mostafa, M.S.2; Hassouna, M.M; Hassab EI-Nabi, S.E. J , EI-Refaie, A.4: lBiological and 

Geological Sciences Dept., AI-Arish Faculty of Education, Suez Canal University, Egypt. 2Clinical 

Pathology Dept., National Liver Institute, Menoufiya University, Egypt. 3Zo010gy Dept., Faculty of 

Science, Menoufiya University, Egypt. 4Pathology Dept., National Liver Institute, Minoufiya University, 

Egypt. 

The aim ofthe present study was to investigate the impact of the combined administration ofvitamin C and 

silymarin on lead toxicity. Male albino rats were subdivided into 3 groups; the first was a control group, the 

second received lead acetate in the diet as 500 mg/Kg diet daily, the third received the same lead acetate 

dose and supplemented with vitamin C (I mg/IOO g body weight) and silymarin (I mg/lOO g body weight) 

by gastric tube 3 times per week. Blood samples were taken after 2,4 and 6 weeks oftreatment. Significant 

lead-induced elevations in serum AL T, AST, GGT and ALP activities were observed after different periods 

of treatment. However serum LDLc was decreased. The intensities of RNA and apoptotic fragments of 

DNA were measured as optical density by the Gel-pro program. Lead acetate decreased the intensity of 

DNA at 6 weeks and induced apoptotic DNA fragments reversibly with time. After two weeks of lead 

administration dilation and congestion of terminal hepatic veins and portal vein branches were observed. 

Lead also induced hepatocyte proliferation without any localized distribution among zones 1,2,3. Portal 

inflammatory infiltrate with disruption of the limiting plates (interface hepatitis), steatosis, apoptosis and 

mild fibrosis were detected especially by sixth week of lead administration. Combined treatment of leadexposed 

animals with vitamin C and silymarin showed marked improvement ofthe biochemical, molecular 

and histopathological findings. These experimental results strongly indicate the protectiVe effect ofvitamin 

C and silymarin against toxic effects of lead on liver tissue. 

Keywords: Lead, vitamin C, silymarin, liver, serum 

Page 215

B17 

DFSA-REPORTED INCIDENTS IN THE UK AND THE MELLANBY EFFECT 

Mark Tyler* : Culham Science Centre, Abingdon, United Kingdom 

A study of 41 cases of drug-facilitated sexual assault (DFSA) in 2003 from 2 separate areas of the United 

Kingdom has shown some interesting trends. The vast majority (38) of complainants were female, 28 were 

under 30 years of age, and 30 of the alleged incidents occurred between 21:00 hours and 03:00 hours. 

Perhaps more significantly, 35 of the cases were alcohol-related. Apart from drugs of abuse (mainly 

cannabis) found in 16 cases, none ofthe so-called DFSA drugs (Rohypnol, GHB, sedatives) were detected. 

These findings echo previously published data (EISohly et aI, 1999) indicating that alcohol is the common 

denominator in these cases. 

The intensity of the CNS effects of alcohol are proportional to the blood alcohol concentration (BAC), as 

described by Dubowski in 1997, but as noted by Mellanby in 1919, the effects are far more pronounced as 

the BAC is rising as compared to when the BAC is falling. In addition, as the rate ofalcohol ingestion rises, 

the greater the degree of intoxication (Moskowitz et al 1977). 

The number of reported "DFSA" cases in the UK has risen dramatically over the past 5 to 10 years. During 

a similar period, the UK has seen a steep increase in the availability and popularity of 'alcopop' drinks 

(bottles of brightly coloured drinks typically containing vodka and lemonade or other fruit flavours, 

designed to appeal to younger drinkers), plus a wider variety and choice of strong beers, and larger 

measures of wine and spirits in public houses. There is now government concern and a rising awareness of 

"binge-drinking" amongst young people in the UK. All of these factors may just be coincidence, but it 

would appear that changing drinking habits and greater CNS intoxication associated with a rapid BAC rise, 

might be responsible for the majority ofthese DFSA allegations. 

Key words: DFSA, alcohol, Mellanby 

Page 216

·Scientific Session 


Clinical and 

Environmental. 

Toxicology 

Page 217

Cl 

THE VOLUNTARY DETOXIFICATION PROGRAMME - A SURVEY OF THE EFFICIENCY 

V.Vorisekl' ,J.Cizek 2 , V.Palicka l 

I Institute ofClinical Biochemistry and Diagnostics, University Hospital and Medical Faculty of Charles 

University, Hradec Kralove; 2Detoxification Centre, Department of Psychiatry, University Hospital, Hradec 

Kralove, Czech Republic 

Aims: The object of this survey was to evaluate the effectiveness of our detoxification strategy for future 

planning and corrections on the basis of the analytical results. Methadone and buprenorphine programme 

applied to opiate and amphetamine abusers in the detoxification centre for north-east area of the Czech 

Republic ( Hradec Kralove county) is based on the voluntary and liberal principle. However a common 

problem related to this approach is a relapse to drug abuse. Detection of the key analytes 

(methylamphetamine, amphetamine,MDMA, MDA, 6-MAM, morphine, codeine, acetylcodeine and 

cannabinoids, especially ll-nor-delta-9-tetrahydrocannabinolic acid) in urine specimens is the decisive 

cause of definitive or temporary discharge from the treatment according to the individual status of patient 

and all case consequencies. 

Methods: A total of 3464 urine specimens randomly collected from 85 patients admitted to programme ( 70 

males, 15 females, aged 19 to 43) over an 8-year (1996-2003 ) period were screened for illicit drug use, 

especially for opiates and amphetamines. The screening analyses were carried out by immunochemical 

methods (EMIT II) and full-automatic HPLC-UV procedure ( REMEDi HS Drug Profiling System ). The 

confirmations were performed by GC-MS (Magnum MAT Finnigan). A total of 25600 tests were done. 

Extraction procedures before GC-MS acquisition were carried out by solid phase extraction devices with 

reagents containing ethyl acetate, acetone, methylene chloride, 0.01 M acetic acid, methanol and 

ammonium hydroxide. Bond Elute CertifY II columns were used for the extraction of amphetamines and 

opiates after enzymatic hydrolysis with p- glucuronidase at 60°C for 3 hours. Positive samples for cannabis 

afterdeconjugation with lIN potassium hydroxide were extracted on SPEC CI8 AR columns. 

Amphetamines and opiates were detected simultaneously in one analysis in the form of 

pentafluoropropionyl derivatives (PFPA reagent).Cannabinoids were derivatized with BSTFA + TMCS 

(99+1) mixture. Samples for evidence of psilocine abuse were extracted on SPEC MPI mixed disc and 

derivatized with acetic anhydride in pyridine in the ratio of 3:1 (LOD IOng/mL). The chromatographic· 

separations were achieved on a DB5ms fused-silica capillary column (30m x 0.25mm x 0,25J.lm film 

thickness) using helium as carrier gas at lml/min flow rate. 

Results: It was found that the commonest illicit drug detected in this programme was cannabis ( 20%) 

followed by opiates (9.9 % ) and amphetamines (9 %). Other classes of illicit drugs (cocaine, LSD and 

psilocine) were not detected. 

Conclusions: Specimens tested positive for opiates and amphetamines showed decreasing tendency during 

the whole period of the detoxification programme but specimens tested positive for cannabinoids were 

stable over time. It seems to be problem concerning the number of positives because positive results for 

cannabis caused temporary discharge from treatment. In addition to this problem we have to admit that GC 

MS such as Magnum does not able to offer sufficient analytical sensitivity for LSD analysis. A cut-off 

value of LSD immunochemical assay is 1 ng/ml but LOD for our GC-MS procedure was 25 ng/ml after 

silylation with BSTFA + TMCS, 99 + 1). However, methylamphetamine and heroin users mostly prevailed. 

Therefore LSD did not belong to main drugs of abuse in this controlled group of abusers. Positive results 

for LSD above a cut-off value were not obtained. Nevertheless in spite of these facts the analytical data 

derived from this survey give relative satisfaction and demonstrate the high level of effectiveness of this 

manner of drugs of abuse therapy. 

Keywords: drugs of abuse, substitution therapy, detoxification programme 

Page 218

C2 

PARADOXICAL RESULTS FROM SCREENING FOR URINARY 6-AM AND OPIATES WITH 

DIFFERENT IMMUNOASSA YS IN A HEROIN SUBSTITUTION TREATMENT POPULATION 

OlofBeck and Michael Boettcher* 

Department of Medicine, Division of Clinical Phannacology, Karolinska University Hospital, Stockholm, 

Sweden. * Arztpraxis rur Medizinische Mikrobiologie, Labordiagnostik und Hygiene, Dessau, Gennany 

Identification of 6-Acetylmorphine (6-AM) in urine samples is regarded as an unequivocal proof of recent 

heroin abuse. Due to its short elimination half-life of about 30min, 6-AM normally cannot be detected for a 

longer period than 24 hours after heroin consumption. In addition, it is generally assumed that the 

metabolite morphine and its glucuronidated fonns can be detected much longer. Therefore, the presence of 

6-AM in urine samples at detectable levels is only expected together with high total morphine 

concentrations. 

Since the availability of the 6-AM CEDIA (Microgenics) we started to test opiate-CEDIA (Microgenics) 

positive samples with the 6-AM assay before GCIMS analysis for 6-AM is conducted. This is done 

semiquantitatively on a Hitachi 911 with calibrators at 0, 5, 10 and 20nglmL applying a cutoff at 5nglmL. 

Quantitative results within the measuring range of the 6-AM CEDIA mostly correlate well with the data of 

GCIMS but are discrepant when increased amounts of free morphine are present in the sample. This crossreactivity 

of free morphine (~0.06%) can even lead to false positive results especially for samples from 

patients substituted with morphine. 

As we always screen urine samples from dihydocodeine substituted patients with the 6-AM CEDIA instead 

of opiate-CEDIA we wanted to learn if there are any other cross-reactive drugs increasing the 6-AM 

CEDIA result or can lead to false positives. We therefore run the 6-AM test together with the opiate 

CEDIA and opiate-DRI (Microgenics) on every urine sample from substituted patients on 49 consecutive 

workdays (3696 samples). No false positive results were found with the 6-AM assay. However, in 29 

(0.8%) samples from 23 different patients (9 females, 14 males) paradoxical results were observed with 

positive 6-AM CEDIA results (>5ng/mL) and negative (cutoff 100nglmL) or low positive results 

«400ng/mL) in the opiate tests. The DRI and CEDIA opiate immunoassays gave similar results. 

Interestingly all the samples analysed were confinned to be 6-AM positive with only small or undetectable 

amounts of free morphine. There were only two discrepant samples where the concentration of 6-AM 

found with GCIMS (9.3ng/mL, 2.5ng/rnL) was significantly lower than expected from the 6-AM CEDIA 

assay (both samples >20ng/mL). An additional cross-reactive substance or perhaps cross-reacting 6-AM-3 

glucuronide may be assumed. Ongoing investigations with LC-MS-MS will look for 6-AM-3-glucuronide 

and morphine-3- and morphine-6-glucuronide in all 29 samples. 

Conclusion: Positive screening results for urinary 6-AM as a proof for very recent heroin abuse should be 

looked at very carefully and always in the context oftotal morphine concentration. 

Keywords: 6-AM, opiates, CEDIA 

Page 219

C2 

..~ 

no. 

substitute, dose 

[mg/day] 

creatinine 

[mg/dL] 

6-AMCEDIA 

>5 ng/mL 

Opiates-CEDIA 

[ng/ml) 

Opiates-DRI 

[ng/ml] 

"tree" 6-MAM GC/MS 

[ng/ml] 

free Morphine GC/MSI 

Ing/ml] . 

1 M14 66 16.0 41 42 18.5 1.5 

2 M4 91 >20 197 148 30.0 7.9 

3 M6 15 >20 85 54 20.7 20 212 212 9.3 37.0 

5 M13 14 >20 81 60 25.0 14.0 

6 M8 14 >20 125 119 35.3 41.5 

7 M17 52 >20 80 69 58.6 3.1 

8 M7 0 >20 217 203 n.t. n.t 

9 M5 106 >20 114 77 19.7 20 354 308 45.9 20 115 107 n.f. n.f. 

26 M16 14 >20 72 49 n.f. n.f. 

27 M3.5 33 >20 80 55 nJ. n.t. 

28 M4 74 >20 169 151 n.t. n.f. 

29 P2 132 >20 113 75 n.f. n.t. 

M = Methadone 

P = Polamidone 

B = Buprenorphine 

n.t. = not yet 'finished 

Page 220

C3 

ALUMINUM TESTING IN TRACE-METAL FREE CONTAINERS 

Lee M. Blum*, Ela Bakowska, Gulo Gigolashvili 

National Medical Services, Inc., Willow Grove, PA 

The testing of biological samples for elemental analyses can be quit challenging. Specimen collection is a 

critical aspect of these tests. Proper specimen collection procedures are necessary in ascertaining an analytical 

result that is truly reflective of the amount of determinant in the specimen. Contamination of the specimen is a 

considerable problem especially in the analysis of trace metals. The results of analysis of blood and urine 

specimens can be misleading if proper precautions are not taken to minimize sample contamination. Because of 

the prevalence of aluminum in our environment, the collection of specimens without precautions can lead to 

misinterpretation. Contamination may come from the patients themselves, the procedures and devices used in 

the specimen collection, and/or the laboratory performing the test (including particles and dusts in the air or 

reagents used in the analysis). Specimen collection tubes are a known source of contamination. The source of 

the contamination may be from the glass, the stopper, and/or the specimen preservative in the tube. Aluminum 

testing in biological samples is important for assessing occupational/environmental exposures and for clinical 

monitoring of renal dialysis patients, among others. The accumulation of aluminum in the body can increase the 

risk of neurotoxicity ("dialysis encephalopathy"), bone disease and renal failure. This study examined the 

effectiveness of specially manufactured trace-metal free specimen collection tubes in the determination of 

aluminum. Specimens were collected consecutively in trace-metal free tubes containing no preservative 

(serum), and trace-metal free tubes containing either heparin (plasma) or EDTA (plasma) as preservatives. The 

aluminum concentrations were determined under identical conditions by inductively coupled plasma/mass 

spectrometry (ICPIMS) with a reporting limit 0{5 mcg/L. 

A summary of the serum and plasma aluminum findings (mcg/L) is listed below: 

I 

Serum Aluminum I Heparinized Plasma EDTA Plasma 

I 

i Patient 

Aluminum 

Aluminum 

I A 8 6 II 

B 11 13 11 

C 5 7 57 

D 8 7 6 

I E 7 13 110 

F 8 19

C4 

THE ENVIRONMENTAL IMPACT OF LEAD, CHROMIUM, ZINC AND CADMIUM PRESENT 

IN RUBBISH DUMP 

C.H. Colangelo*I,2,3 ,L.A. Ferrari 1,3 ,J.C. Chaumeil l , C.A. Seminario Correal, S. Georgeri l 

1. Laboratorio de Toxicologfa y Quimica Legal * Direcci6n General de Asesorias Periciales - Suprema 

Corte de Justicia de la Provincia de Buenos.Aires Calle 41 e/119 y 120 La Plata (1900) - Buenos 

Aires Republica Argentina .,2. University of La Pampa - Argentina, 3. University of Moron Argentina 

Lead, Chromiun, Zinc and Cadmiun concentrations in differents environmental matrixes (soils, drinking 

underground waters, surfaces waters and edibles fresh vegetables such as celery and lettuce) collected at 

selected areas near a rubbish dump located at Buenos Aires Province in Argentina were analyzed. 

The above mentioned samples were conditioned for their analysis by atomic absorption spectrophotometry. 

Soils and vegetables samples were first treated by microwave digestion with nitric acid, and then metal 

concentrations were determinated by aspiration in flame of air* acetylene technique. 

Water samples were analyzed by using a graphite oven. 

The following table shows the metal concentrations obtained for the different samples: 

Samples 

Lead Chromium Zinc Cadmium I 

ppb 1>pb ppb 

i 

..I!Pb 

Underground water 1 (e) 140 70 23

C5 

DETECTION OF NITRAZEPAM USING ION MOBILITY SPECTROMETRY 

G. Jayashanker*, N.V.R.Kiran, A.K.Srivastava, M.Afzal, R.K.Jain,P.Samikannu, S.Sudhakar, R.K.Sarin. 

Central Forensic science laboratory, MHA, Govt. Of India, Ramanthapur, Hyderabad-India 500013. 

Ion Mobility Spectrophotometer (IMS) has been used as a rapid sensitive and selective technique for the 

detection of Nitrazepam in body fluids. Nitrazepam an tranquillizer is often misused/abused in criminal 

activities like theft, robbery, rape etc. The detection of specific compound in body fluid determines the drug 

administered in such cases. The standard routine procedures are time consuming. In this study IMS was 

used as rapid technique to detect Nitrazepam. The IMS utilizes the fact that a mixture of different ions may 

be separated into individual components as they travels at different velocities [mobilities] under the 

influence ofan electric field, through a tube containing the drift gas. 

The case of a lady being sexually assaulted and found unconscious was brought to the hospital by police for 

treatment. The clinical symptoms of being drugged could be observed and the stomach wash collected and 

sent to the forensic lab for testing. The routine drug screening was followed and simultaneously a portion 

of the sample was subjected to IMS screening. The ion mobility of standard Benzodiazepines like 

Diazepam, Flurazepam, Nitrazepam were carried and the drift time and Ko value was noted. 

SINo Sample Drift time Ko 

1. Calibration 9.044 1.86 

2. Diazepam 13.79 1.213 

3. Flurazepam 16.44 1.023 

4. Nitrazepam 16.55 1.016 

5. Sample 16.55 1.016 

I 

I 

I 

I 

The exhibit was analysedand the drift time and Ko value could be matched with Nitrazepam. The standard 

screening procedure of Acidic and basic fraction analysis by GC-MS also confirmed the Nitrazepam. The 

standard solution of diazepam was studied for the limit of detection with 50 ng solution. The selectivity of 

the method for the drug identification can be only compared with the standard samples ion motilities 

analysed sequentially with samples as the drift times ofdifferent compounds may overlap on the same. The 

IMS technique could detect the Nitrazepam even in the gastric lavage directly put on the sample holder of 

IMS. The interference from the direct analysis had no shift in the peaks. The IMS was found to save time, 

simple and easy technique to detect drugs of abuse. The procedure can be used to screen for the presence 

of Benzodiazepine group of drugs in routine analysis and is an additional analytical corroboration in the 

analysis ofdrugs. 

Key words: IMS, Toxicology, Drug-facilitated sexual assault 

Page 223

C6 

MEPROBAMATE OVERDOSE: A ONE-YEAR EVALUA TION OF REPORTS 

Fabienne Pehourcq 

Laboratoire de Pharmacologie, Universite Victor SegaJen, 33076 Bordeaux, France. 

Meprobamate is a mild sedative-hypnotic drug used in therapy since the 1955s. An overdose with this drug, 

even taken alone, may cause severe or even fatal respiratory depression, hypotension, shock, and heart 

failure. This drug is widely known in France, particularly for use in suicide attempts. 

One hundred and fifty-three cases of meprobamate intoxication were admitted to the emergency room of 

our hospital during one year. In these patients, the plasma concentrations of meprobamate measured by gas 

chromatography after liquid-liquid extractions were above the therapeutic level of 15 mgIL. Ninety-seven 

intoxicated patients (63%) were female. The age of the patients studied was 14-75 years (mean ± SD, 42.2 

± 12.7). The mean age for females (43.7 ± 13.1) was significantly greater than that for males (39.6 ± 11.6) 

(p< 0.05). Most (58%) of the patients arrived in the emergency room during the time period 6:00 PM to 

6:00 AM. Among these 153 hospitalized patients, meprobamate was detected 95 times at concentrations 

above the toxic level of 50 mgIL. Thirty-two patients (21 %) presented with concentrations in the range of 

100-200 mgIL; Six cases of overdose were described with meprobamate levels greater than 200 mgfL; and 

among them, three voluntary intoxications were fatal (1.96%). One third of the cases turned out to be 

combined intoxications, 82% of the patients also took benzodiazepines. Most poisonings took place in the 

first quarter ofthe year. 

In comparison with older studies, the pattern of meprobamate poisoning does not appear to have change in 

France over the last 25. years. An overdose with this drug, even when taken alone, produces intoxication 

that is often serious and sometimes fatal. The questionable efficacy of meprobamate as a selective antianxiety 

agent and its potential for life-threatening intoxication are important drawbacks to the clinical use 

ofthis drug. 

Keywords: meprobamate, overdose, epidemiology 

Page 224

C7 

METHCATHINONE: A NEW POSTINDUSTRIAL DRUG 

H.Belhadj-Tahar 1 ' ,N.Sadeg 2 

1. Groupe Sante Recherche Toulouse, 35 rue Bernard de Ventadour, 31300 Toulouse, France. 

2. Laboratoire Claude Bernard, Centre Hospitaiier Rene Dubois, 95303 Pantoise, France. 

Methcathinone is an illicit drug also known as ephedrone, which is a methyl derivative of cathinone, a 

stimulant found in the « khat» plant, Catha edulis, and which can be easily manufactured by oxidation 

from pseudoephedrine. Target consumers of this drug seem to be well-educated people aware of the risks 

and precautionary measures 1 • Therefore, it is difficult to diagnose and to cure properly and it becomes even 

more complicated as there are misleading symptoms related to intoxication by cathinone derivatives of 

synthetic or natural (derived from the khat) origin. Thus far, reports of methcathinone intoxication 

documented and based on reliable analyses are rare since it is not systematically detected. This paper 

describes a case of reiterated coma due to an overdose of pure methcathinone dissolved in alcohol and 

mixed with bromazepam. 

Case report: At 10 pm, a 29-year-old woman was admitted in emergency department from Paris suburbs 

for a coma of toxic origin. Medical history showed that it is the second episode occurring under similar 

circumstances and at one month interval. In both cases, the patient took Lexomil® dissolved in alcohol. 

Besides, the family indicated that the patient presented signs of depression, incoherent behavior. She is an 

amphetamine-like drug consumer. Clinical examination revealed a Glasgow coma score 9 and symmetrical 

reactive pupils with mydriasis. The patient presented a polypnoea. Her blood pressure was 93/53mmHg. 

The rest ofthe examination was unremarkable. 

Biological check-up: the ionogram and the blood gas analyses were normal, the blood alcohol level was 

1.67 gIL. Urinalysis revealed benzodiazepines, pH 5.4, along with a high concentration of amphetamines 

dosed by FPIA (Axsym, Abbott - USA) and identified by HPLC (Remedi, Biorad USA) as follows: 

methcathinone (17.24 mglL), ephedrine (11.60 mglL) and methylephedrine (1 LIO mglL). Serum analysis 

by HPLC revealed concentrations of bromazepan (8.89 mglL), methcathinone (0.50 mgIL) and 

methylephedrine (0.19 mgIL). The patient was kept in quiet room: the hemodynamic and neurological 

functions evolved quite favourably in the next hours. 

Discussion: This case illustrates a coma mainly due to a combined bromazepam and methcathinone 

intentional intoxication. This coma is not only ascribable to bromazepam and ethanol. Indeed, on one hand, 

the blood alcohol level at 1.67 gil, the absence of anionic and basic deficit related to an acetate outbreak 

eliminates the hypothesis of ethylic coma and, on the other hand, the paradoxical presence of polypnoea 

instead of respiratory distress eliminates the hypothesis of a coma due to benzodiazepine overdose. 

Therefore, we can affirm that cathinone altered typical clinical symptoms of bromazepamlalcohol 

intoxication, namely hypotension and respiratory distress. Methylephedrine detection at infratoxic doses 

could be a chemical precursor resulting from the alkylation of primary amine for monomethylamine 

(pseudoephedrine) and dimethylamine (methyl ephedrine) synthesis (Belhadj-Tahar H. et al. Therapie, 

2003,59: 265-271). It seems to be chemical tag of a fraudulent origin. 

Conclusion: This case describes an example of a new addictive behaviour of "well educated" people 

involving the intake of methcathinone, a postindustrial psychostimulant intentionally combined with anticonvulsant 

benzodiazepine. 

Keywords: methcathinone, ephedrone, illicit drug 

Page 225

C8 

. ANALYSIS OF METHYLPHOSPHONIC ACID AND ISOPROPYLMETHYLPHOSPHONIC 

ACID, A METABOLITE OF SARIN NERVE GAS, BY TANDEM GC MASS SPECTROMETRY 

AND POSITIVE CHEMICAL IONIZATION 

Eric Phillips, Jessie Crockett Butler*, Trisa Robarge, and Meredith Conoley. Thermo Electron, 2215 Grand 

Ave Pkwy, Austin, TX 78728-3812 

The two primary metabolites of the nerve agent sarin are methylphosphonic acid (MPA) and 

isopropylmethylphosphonic acid (IPMPA). They are analyzed by GCMS after derivatization with BSTFA. 

In positive chemical ionization mode using isobutane, the MH+ ion ofthe derivatized compound is isolated 

and then fragmented into product ions for structural elucidation. Blank samples of urine, cleaned up by 

solid phase extraction, were spiked with commercially available MPA and IPMPA. The linearity and 

detection limits of the method were determined in urine using IPMPAd7 as an internal standard. Multiple 

scan events were set up to perform sequential Full Scan and MSIMS; The linear fit for MPA-TMS was R2= 

1.000 (Full Scan) and 0.9982 (MSIMS/MS) and for IPMPA-TMS, R2 = 0.9990 (Full Scan) and 0.9978 

(MS/MS) with a precision of5.1% RSD for the internal standard (IPMPA-d7-TMS). The precision for the 

internal standard for 60 replicate injections of spiked urine was 9.5 %RSD. The instrument of analysis was 

the external source quadrupole ion trap with a temperature programmable injector. Mass Frontier software 

was used to predict the MS/MS product ions for assistance in the selection of a precursor ion of the 

derivatized target compounds. 

Keywords: Ion trap, MSIMS, nerve gas 

Page 226

C9 

A PROPOSED SCHEME FOR FOXY METABOLISM 

JM Wilson*\ F McGeorge l , R Meatherale. 

l.Dept of Clinical Pathology and Emergency Medicine, William Beaumont Hospital, Royal Oak, MI USA. 

2. St. Boniface General Hospital, Winnipeg, Manitoba CAN. 

We report an emergency room admission following an ingestion of 5-Methoxy-N,N-diisopropyItryptamine, 

5-MeO-DIPT or FOXY. The subject was a 23 year old Caucasian male presenting 3 hours post ingestion 

of a capsule provided by an acquaintance and initially described as "acid". This ingestion was preceded by 

intake of 4 beers. Approximately thirty minutes before presentation the subject experienced four episodes 

of vomiting followed by tactile hallucinations and paranoia, primarily related to a suspicion of being 

poisoned. He denied auditory and visual hallucinations or other neurological symptoms. Vital signs 

revealed a temperature of 31 "C, pulse 16 bpm, respirations 18 per min and blood pressure of 135170 mm 

Hg. Pupils were midpoint and reactive with normal neurological, motor, reflex, cardiovascular and 

gastrointestinal function. Blood and urine chemistry and hematology results were not revealing. Urine 

toxicology screening reported presence of 5-MeO-DIPT and suspected metabolites. The subject received 

activated charcoal, intravenous fluids, three additional hours of supportive care with resolution of his initial 

condition and was discharged without complaints or subsequent readmission. 

Urine toxicology testing involved initial screening by ToxiLab® (Varian Inc, Lake Forest CA) followed by 

confirmation by electron impact GC/MS. Toxi-A revealed three spots at RF 2.4, 4.6,6.1 (S 1 all blanch, S2 

4.6 tan, others faded, S3 all UV absorption, S4 all brown). Extraction for confirmation was preceded by 

combination of 2 mL of urine, 0.5 mL of 1 N sodium hydroxide, 500 ng of internal standard (SKF-525-A) 

and 5 mL of dichloromethane. Mixing, separation of the organic layer, evaporation under an air stream at 

45 "C in a water bath was followed by reconstitution with 25 ilL of ethy I acetate. One ilL of the extract 

was injected for GCIMS analysis on a Hewlett Packard 5912A Mass Selective Detector equipped with a 30 

m, 0.25 mm id DB-5® (Agilent Technologies, Wilmington DE) capillary gc column with a 0.25 Ilm film 

thickness using splitless injection. Oven temperature was initially 120 "C for 3 min, raised to 225°C at 10 

"C per min and held for 5 min, followed by a second temperature increase to 300 "C at 15°C per min and a 

3 min hold. 

Electron impact GCIMS data revealed four chromatographic peaks (RT 15.86, 18.76, 20.13, 21.07) with 

molecular ions of 232, 274, 260, and 290 m/z, respectively. These have been tentatively identified, in 

chromatographic order, as 5-MeO-NIPT, the N-desalkyl metabolite, 5-MeO-DIPT, the parent substance, 5 

OH-DIPT, the O-desmethyl metabolite, and 5-MeO-DIPT N-oxide, the ring oxidation product of the parent 

substance. Identifications were based on comparative spectra with literature sources and, in the case of 5 

OH-DIPT, corroborative CIIMS with 5 % ammonia in methane, and ethylation with ethyl iodidelTMAH. 

Assuming that peak heights reflect urine concentrations, the relative magnitude of parent substance and 

metabolites were 5-MeO-DIPT>5-0H-DIPT>5-MeO-DIPT N-oxide>5-MeO-NIPT. Detection and 

identification of these metabolites permits the characterization of FOXY metabolism as three parallel 

oxidative pathways involving each ofthe molecule's non-carbon sites. 

Key words: 5-Methoxy-N,N-diisopropyltryptamine, metabolism, FOXY 

Page 227

CIO 

EFFECTS OF CHRONIC ACONITINE ADMINISTRATION ON ITS CONCENTRATION IN LIVER, 

KIDNEY, HEART, AND BLOOD OF MICE 

Kentaro Wada*, Makoto Nihira, Hideyuki Hayakawa, Yukari Tomita, Makiko Hayashida" Youkichi Ohno. 

Department ofLegal Medicine, Graduate School of Medicine, Nippon Medical School, Tokyo, JAPAN 

Aims: Aconitum alkaloids are well known for their acute and high toxicity, for example, in the causation of severe 

arrhythmias leading to death. Aconitine, one ofthe major Aconitum alkaloids, is a highly toxic compound from the Aconilum 

species. The use ofAconitum alkaloids has been known since ancient times, over 2000 years in Asia as a homicidal agent, 

sometimes as poison for arrowheads. Even today, aconites are sometimes used as homicidal or suicidal agents. Until today, 

several cases of murder have been reported using single-dose ofa large amount ofAconitum alkaloids in Japan. In 1995, a 

man murdered was subjected to autopsy in which Aconitum alkaloids were administered repeatedly over a period ofmonths, 

in Saitama Prefecture, Japan. Although there are various studies reported on the single-dose effect ofaconitine, no reports are 

available on the long-term effects of aconitine, probably due to its high toxicity. Therefore this study was conducted to 

investigate the influence ofchronic administration ofaconitine in experimental animal models. 

Methods: A total volume of 1.0 mg/kg/day was administered to the experimental animal models. The lethal dose 50% 

(LD50) of aconitine for mice is 1.8 mg/kg (orally, single dose) and 0.308 mg/kg (intraperitoneally, single dose). The male 

ICR (Institute ofCancer Research) mice were divided into 2 study groups: "acute group" (day 0: 0, 15,30,60,90, 120 min, 

1440 min = 24 hours) and "chronic group" (days 1,3,7,1O,15,19,and 22), according to the time when the animals were 

sacrificed. The experiments were conducted according to the guidelines of the Ethical Committee on Animal 

Experimentation of Nippon Medical School (Tokyo, Japan). We determined the concentration of aconitine and its 

metabolites (benzoylaconine and aconine) in organs and blood with gas chromatography/selected ion monitoring (GC/SIM). 

In addition, we concurrently recorded the electrocardiogram (ECG). 

Results: Fifteen min after administration on day 0, the early aconitine administered group (acute group) revealed peak organs 

and blood concentration levels of aconitine with gradual decrease, thereafter. The concentration of aconitine in organs and 

blood (from day 0 to day 22; 90 min after the last administration of aconitine) gradually decreased according to repeated 

administration, whereas benzoylaconine and aconine increased. ECGrevealed various types of arrhythmias (for example, 

ventricular fibrillation, ventricular tachycardia, torsade de pointes, atrioventricular block, and bundle branch block). 

However, the frequency ofarrhythmias remarkably decreased with time and repeated administration of aconitine. 

Conclusions: In this study, 2 facts were revealed. First, the frequencies offatal arrhythmias remarkably decreased to day 22. 

Secondly, the organs and blood concentration of aconitine (90 min after the last administration of aconitine) gradually 

decreased and its metabolites (benzoylaconine and aconine) increased until day 22. These 2 facts have raised the possibility 

that the activity of drug metabolism increased due to long-term administration of aconitine. In the case of long-term 

administration of aconitine, it is very important to determine not only the concentration ofaconitine but also its metabolites 

(benzoylaconine and aconine) in the organs and blood from the viewpoint offorensic toxicology. 

Keywords: aconitine; chronic administration; GC/SIM 

Page 228

ell 

ABDOMINAL COMPLICATION OF INHALED METHAMPHETAMINE 

Wenceslo Kiat* and Irma Makalinao 

National Poison Control and Information Service, University of the Philippines-Philippine General 

Hospital Ward 14-A, Taft Avenue Manila, Philippines 1000 

Methamphetamine toxicity continues to be the leading cause of referral to the poison center. Despite the 

abundance of information regarding its cardiovascular and central nervous system toxicity; abdominal 

complications related to inhaled methamphetamine are not commonly encountered and anticipated. We 

report a case of a 33 year old female, a known methamphetamine abuser for the last five years who 

consulted the emergency room for acute onset of severe abdominal pain and dysuria noted a day prior to 

consult Gynecologic problem was ruled out. Abdominal ultrasound showed bile sludge in the gallbladder. 

She underwent emergency exploratory laparotomy under general anesthesia. However, intraoperative 

findings were normaL The initial urine methamphetamine level done on the fifth day after the last used 

showed 2157 ng/ml. This report suggests that methamphetamine toxicity could present as "acute abdomen" 

and should be anticipated by emergency room physicians. 

Key words: Inhalation, Methamphetamine, Abdomen pain 

Page 229

el2 

TOXICOKINETICS, RECOVERY, RESIDUES AND CYTOTOXICITY OF ACTP-ESTER IN 

THE GOAT 

C.R. Sahu*" Biplab Bagchi 

Department ofZoology, Kalyani University, Kalyani, West Bengal, India 

ACTP- Ester (Triclopyr butyl) marketed by Dow Elanco, USA is a selective herbicide used widely in 

agricultural field. In order to study the toxicokinetic, total recovery and cytotoxicity study of ACTP- Ester 

and its two potential metabolites - triclopyr acid (M]) and 3,5,6- trichloro-2 pyridinol (M2), the compound 

ACTP- Ester was administered orally 396 mglkg body wt. to Black Bengal Goats (Capra capra). The 

control groups were however treated with the same amount of carboxymethyl cellulose (CMC). 

Blood samples were collected before (0 hr) and after 0.25,0.50,0.75, 1,2,3,4,6,8,12,36,48,60,72,84,96, 

120, 144 and 168hr post administration (Pd) and were prepared for HPLC analysis following standard 

protocols. Toxicokinetic parameters of ACTP Ester were determined from computerized interactive curve 

fitting programme of respective blood level time profile and data were analyzed as per the standard formula 

using various disposition kinetic parameters. Gross lesions for different tissues like liver, kidney, lung, 

brain etc were recorded for ACTP-Ester induced cytotoxicity in goats sacrificed on 4,5,6 and 7-day pd. An 

adequate blood level of ACTP-' Ester was detected as 4.94 ± 0.43 at 0.25 hr (pd). The concentration (C B 

max 25.15 ± 2.62 j.lg Iml was recorded at 6 hr followed by a slow decline and minimum (C B min 3.52 ± 

0.23 j.lg Iml) was detected at 60 hr pd. Kinetic behaviour of ACTP- Ester in goats followed a "two 

compartmental open model kinetics". The blood level for M] and M2 was 6.52 ± 0.89 and 2.61 ± 0.13 

. j.lg/ml respectively. The lower absorption rate constant suggested slow absorption of ACTPEster from 

GJ.tract. The CI H value, which is equivalent to CI B value suggested that major route of elimination of 

ACTP-Ester be directed through liver. Very poor CI g value suggested that urine is the minor route of 

excretion of the compound. The concentration of both metabolites appears zigzag fashion, which might be 

due to variable quantity as well as rate of metabolism in every unit of time. 

The recovery percentage of ACTP-Ester from faeces, G.I.tract content, urine and tissues were respectively 

65.38, 63.34, 66.10 and 66.24 in goats sacrificed on 4, 5, 6 and 7-day pd. Liver and lung showed gross 

malformations than other tissues due to the treatment. 

Keywords: Toxicokinetic, residue cytotoxicity ACTP, goat 

Page 230

C13 

A HOMOGENEOUS ENZYME IMMUNOASSAY FOR THE DETECTION OF 

OXYCODONE AND OXYMORPHONE IN URINE 

Lakshmi Anne* Ming-che Wang, Vani Bodepudi, Scott Casseday, Manolito Datuin and Daryush 

Mirlohi; Microgenics Corporation, 46360 Fremont Boulevard, Fremont, CA. 

Oxycodone is an opioid analgesic derived from thebaine. It is structurally similar to codeine with a higher 

abuse potential than morphine. Oxycodone is prescribed for the treatment of moderate to severe pain, 

chronic pain or terminal cancer pain and is available as OxyContin® or in combination with acetaminophen 

or aspirin. Oxycodone is metabolized to oxymorphone, an active metabolite that has the same analgesic 

potency as oxycodone. Other metabolites of oxycodone include noroxycodone and noroxymorphone, which 

are inactive. The majority of oxycodone is excreted in urine within 24 hours as free oxycodone, conjugated 

oxycodone and oxymorphone. Increased use of oxycodone has resulted in an increase in oxycodone abuse 

and abuse-related deaths. None of the currently available opiate immunoassays has the sensitivity to detect 

oxycodone or oxymorphone at therapeutic or abuse levels. The objective of this study is to develop a 

homogeneous immunoassay for the specific detection of oxycodone and oxymorphone at therapeutic as 

well as abuse levels in urine. 

Microgenics DRI® Oxycodone Assay is a dual cutoff assay using 100 nglmL and 300 ng/mL oxycodone as 

cutoff calibrators. The assay uses a highly specific monoclonal antibody that can detect both oxycodone 

and oxymorphone. The assay is based on competition between drug labeled with glucose-6-phosphate 

dehydrogenase (G6PDH) and free drug from urine sample for a fixed amount of antibody binding sites. In 

the absence of free drug from the sample, the specific antibody binds the enzyme labeled drug causing a 

decrease in enzyme activity. This phenomenon creates a direct relationship between the drug concentration 

in urine and enzyme activity. The enzyme activity is determined spectrophotometrically at 340 nm by 

measuring its ability to convert NAD to NADH. 

The reagents and calibrators are liquid ready-to-use. The dynamic range of the assay is 0 to 1000 ng/mL. 

The performance ofthe assay was evaluated on the Hitachi 717 analyzer. The within-run and total precision 

(CV) for the cutoff calibrators and ± 25% controls is

C14 

COMPARATIVE STUDY ON TOXIC MANIFESTATIONS INDUCED BY INGESTION OR INJECTION OF COMMONLY 

USED DISINFECTANTS AND SURFACTANTS 

Yoko Hieda*l, Yuying Xue', Koji Takayama', Yoshio Tsujin0 2 , Junko Fujihara l , Haruo Takeshita l - 'Department of Legal Medicine, Shimane 

University School of Medicine, Shimane, Japan, "Department of Dermatology, Shimane University School of Medicine, Shimane, Japan 

Background and aim: Accidental or intentional ingestion or injection of household products sometimes occur due to their easy accessibilities, 

but the toxic manifestations have not been well characterized when they are internally administered since these products are not developed for 

medicines. We previously evaluated the toxic and kinetic properties of Osvan® (benzalkonium chloride) that is widely used as a cationic 

surfactant and disinfectant, in which we showed that (I) different toxicological progression and manifestation appeared in administration via 

femoral artery (FA) among intravascular administrations even though the blood concentration profiles were similar, and (2) the degree of 

toxicity correlated with the peak blood concentrations in orally dosed (PO) rats (Toxicol Lett 148: J13-123, 2004). The aim ofthis study was to 

evaluate whether or not the difference in toxic manifestations among routes of administration observed in Osvan® were specific only to cationic 

surfactant, any kinds of surfactants, or any kinds of disinfectants. 

Materials and Methods: The test drugs involved Osvan® (cationic surfactant and disinfectant), Hyamine® (cationic surfactant and disinfectant), 

Tego® (zwitterionic surfactant and disinfectant), linear alkylbenzene sulfonate C I" (LAS,,) (anionic surfactant), VOlpo®20 (nonionic surfactant), 

Maskin® (non-surfactant and disinfectant), Ethanol (non-surfactant and disinfectant) and saline (control). Fifty-five male Sprague-Dawley rats 

'were administered one ofthe test drugs orally, intravenously via jugular vein (JV) or intraarterially via FA. Two to four different doses of drug 

were examined for each drug, and varied between 3 to 150 mglkg for a intravascular dose based on individual LD 5Q and 250 or 1250 mglkg for 

a oral dose except for ethanol. The dose ofethanol was set up at 0.3-1.5 glkg and 1.5-1.5 glkg for a intravascular and oral dose, respectively. The 

conditionofrat was observed for 24 h after a dose and then the rat was sacrificed. The cardiac blood and tissue samples were collected for assay 

and histoIogical examination. The rats that died before 24 h were autopsied immediately after death to collect samples. 

Results and discussion: Toxic manifestations were different among the routes of administrations within the same test drugs except for Volpo"'20 

and saline. The toxic peak appeared soon after the dose following JV administration, while toxic effects developed with the lapse of time 

following higher doses ofFA and ,PO administrations, though the degree ofeffects or time-course symptoms varied among drugs. Necrotic-like 

symptom developed around the injecting side of leg following FA administration in cationic or twitterionic surfactants and Maskin®, while it' 

appeared on opposite side ofleg inLAS 12. The FA-dosed rats had higher blood myoglobin concentrations compared to JV- or PO-dosed. rats and 

they hardly urinated after a dose, suggesting that kidney was highly affected in FA administration. In PO administration, all high dose of 

surfactants except for Volpo"'20 died at 5-20 h after a dose, while all non-surfactant disinfectants survived for I day at the same dose, suggesting 

that ionic surfactants have greater toxicities in PO administration. Severe damage of internal membrane of alimentary tract observed in ionic 

surfactants indicated that the ionic surfactant is critical chemical property to induce toxic effects in PO administration. The overall toxic degree 

based on the dose-size, except ethanol, could be ranked as strong as cationic surfactant =Maskin > zwitterionic surfactant =anionic surfactant 

> nonionic surfactant. These results suggested that (l) generally ranked stronger disinfectants have higher toxicities when they are 

intravascularly administered, (2) toxic manifestations are different between JV and FA administrations but with having similar trend in any kind 

of disinfectants and surfactants, and (3) ionic surfactants have stronger toxicities compared to nonionic surfactant or non-surfactant when they 

are orally administered. 

Keywords: surfactant, disinfectant, toxicity 

Page 232

CIS 

RAPID DETERMINATION OF CAUSATIVE AGENT USING DETACHED ROOF OF BULLA IN CHEMICAL BURNS OR 

DERMAL EXPOSURE 

Yoshio Tsujino' J , Yoko Hieda l , Haruo Takeshita 2 , Eishin Morita' - JDepartment of Dermatology, Shimane University School of 

Medicine, Shimane, Japan, 2Department of Legal Medicine, Shimane University School of Medicine, Shimane, Japan 

Background and aim: Liquid specimens such as blood or urine are commonly used in forensic examinations, while skin samples are 

rarely used. Recently we have been studying the percutaneous absorption of chemicals and have shown that skin analysis is useful in 

identil'ying dermal exposure to petroleum products. Only small amounts of skin «0.03 g) are required for analysis since lipophilic 

chemicals, such as aliphatic hydrocarbons used to identil'y the petroleum product, remain in the skin (Forensic Sci Int 133, 141-145, 

2003). However, most living patients do not agree to have a skin biopsy for chemical analysis. We. present a case ofa patient who was 

exposed dermally to some solvents where we determined the causative agent without skin biopsy. 

A case: A 73-year-old woman was first aware of slight redness with soreness on her anterior right thigh one evening. When she took off 

her trousers 3 h later, the redness had spread over her rigbt thigh up to her abdomen with some erosion. When she visited our hospital 

next morning, extensive erythema with bulla and erosion appeared over 10% of her body.. These first- and second-degree bums were 

thought to be caused by exposure to some unidentified organic solvents. 

Examination: The lesions were first washed thoroughly with saline. To examine the lesions pathologically, and tojdentil'y the causative 

agent, a skin biopsy was recommended but not agreed upon with the patient. For an alternative specimen, small pieces of detached roof 

ofbulla. which are usually taken off, were collected for analYSis during topical treatment with a steroid ointment. For rapid examination 

to estimate the causative agent, a part of the collected bulla (0,01 g) was put into small amount ofn-pentane in a glass tube, sonicated in 

an ultrasonic bath for I min, and then I iii oin-pentane was analyzed using a GC-MS system (HP5890)in a scan mode (mlz 30-400) 

. equipped with a capillary column (HP-5, 0.25 mm i.d. x 30 m, 0.25 lim thickness), The column temperature was set at 50°C for I min, 

then increased I O°C/min up to 280°C and held for 10 min. The temperature ofinjection port and ion source was set at 270°C and 280·C. 

respectively. 

Results: In the rapid analysis, typical kerosene components (aliphatic hydrocarbons with carbon number 9-16 and some aromatic 

hydrocarbons) were detected based on the retention times, mass spectra and pattern of the peaks. The causative agent was determined 

kerosene. Detailed analysis performed later using the remained collected roof of bulla and blood sample (0.5 ml) utilizing liquid-liquid 

extraction confirmed kerosene components in the both samples. No other chemicals causing inflammatory response were detected. 

DisclISsion: The.patient may have had been exposed to kerosene while filling a kerosene stove at home about 5 h before first becoming 

aware of her skin irritation. Subepidermal bulla, often observed in second-degree bums is a phenomenon separating epidermis from 

dermis, indicating that the detached roof of bulla in this case was composed mostly of epidermis. We recently reported that lipophilic 

chemicals tend to be trapped in lipophilic stratum corneum in epidermis (Int J Legal Med 118,41-46,2004). This suggests that a skin 

biopsy, which removes skin to the subcutaneous fat depth. and causes significant patients pain, is not necessary when petroleum products 

are involved. Analysis ofonly 0.0 I g of detached roof of bulla with simple sample preparation is a useful diagnostic method for dermal 

exposure to petroleum products both in clinical and forensic fields, 

Key words: chemical burns, bulla, GC-MS 

Page 233

C16 

A CASE OF TETRODOTOXIN POISONING CAUSED BY INSERTION OF A PUFFER FISH INTO 

THE VAGINA 

Mikio Yashikil*,Akira Namera l , Kengo Nishioka 2 , Manami Nishida l , Kojiro Kimura l 

lDepartment of Legal Medicine, Graduate School of Biomedical Sciences, Hiroshima University and 

2Department ofAnesthesia, Chuden Hospital, Hiroshima, Japan. 

We report an unusual case of sudden heart and pulmonary failure in a 33 year-old young woman due to 

insertion ofa puffer fish into the vagina. While traveling on a boat, the woman felt unwell while having sex 

with a man who had inserted a puffer fish into her vagina. Upon arriving at port, she became unconscious and 

then went into cardio-pulmonary arrest. She was immediately transported to a hospital where she received 

cardio-pulmonary resuscitation. Following an intravenous injection of2 mg ofepinephrine, her heart rate and 

blood circulation recovered. Few days later, she began spontaneous breathing, however, her level of 

consciousness continued at a low stage (JSC 200). She remained in a vegetable state for two months. 

Following her transfer to another hospital, she died of sequential pneumonia two months later. A serum 

specimen collected upon admission and a urine specimen collected two days post-admission were found to 

contain 58 nglg and 65 nglmL of tetrodotoxin (TTX). TTX in serum and urine was hydrolyzed. to 

2-amino-6-hydroxymethyl-8-hydroquinazoline (C 9 -bas)e by NaOH. C 9 -base was purified by CI8"SPE 

cartridge and analyzed by GC/MS after TMS derivatization. It is known that some species of puffer fish 

release TTX from the skin. In this case, a toxic concentration ofTTX was rapidly absorbed by the victim's 

vaginal membrane, distributed throughout her body causing her cardio-pulmonary arrest and eventual death .. 

Key words: puffer fish, tetrodotoxin, vagina 

Page 234

C17 

CHANGES OF GENE EXPRESSION BEFORE AND AFTER ACETAMINOPHEN INJECTION 

DETECTED BY DIFFERENTIAL DISPLAY 

2 

Mizuho Namiki I, Tomohisa Mori 2 , Shinobu It0 , Toshiko Sawaguche 

I Dept.of Emergency Medicine,Tokyo Women's Medical University,Tokyo,Japan 

2Dept.ofLegal Medicine,Tokyo Women's Medical University,Tokyo,Japan 

Objectives; Acetaminophen is a familiar antipyretic analgesic. While the mechanism by which 

acetaminophen causes chemical damage has been fully explained, the molecular biological dynamics that 

takes place in the damaged liver has not been elucidated. The current study was conducted to advance the 

molecular biological understanding in the dynamics of the gene expression during the subsequent hepatic 

damage by acetaminophen. 

Materials and Method: I)Preparation of mice with acetaminophen-induced hepatic damage. Following 

fasting for 24 hours, 250 mglkg body weight of an acetaminophen solution was administered via a 

peritoneal route to mice to serve as a model for a drug-induced hepatic disorder, while the same amount of 

a physiological saline was also given via the peritoneal injection to the control animals. Both groups fasted 

for an additional 24 hours thereafter. Under chloroform anesthesia, laparotomies were performed. 

Following blood specimen collection from the heart, the liver was excised. 2) Serum AL T and AST 

determinations were measured by using an autoanalyzer (Hitachi-7450). 3) The excised livers were frozen 

with liquid nitrogen and pulverized, from which RNA was collected by using Trizol. The product was then 

treated with DNAse and processed with phenol chloroform for cDNA synthesis. The DNA was amplified 

by using a primer from the Fluorescence Differential Display Kit (Takara). 4) The second PCR and 

acrylamide gel electrophoresis were conducted by employing the fluorescence differential display method. 

After a band pattern was visualized by a molecular imager FX, the bands with eminent differences were cut 

out, from which the amplified products were recovered for cloning. 5) The homology was confirmed 

between each base sequence that had been obtained from the Web site NCBI BRAST and the known mouse 

cDNA. 

Results: The model for a drug-induced hepatic disorder displayed numerous small white blotches, which 

were visible with the naked eye, around the hepatic portal system; and the results of blood chemical 

analyses indicated AST and AL T levels that were significantly higher (P

C18 

EMBLICA OFFICINALIS (FRUIT) INHIBITION OF LIVER FIBROSIS INDUCED BY CARBON 

TETRACHLORIDE AND THIOACETAMIDE 

SA Tasduq, P. Kaiser, H.S. Maheshwari,R.K. Johri. 

Phannacology Division, Regional Research Laboratory, Jammu and Kashmir, India. 

The study was carried out to investigate the antifibrotic effects of hydro-ethanolic extract of a reputed 

Indian medicinal plant Emblica officinalis fruit on liver fibrosis induced by carbon tetrachloride (CCI 4 ) and 

thioacetamide in rats. Liver fibrosis was assessed by measuring levels of hydroxy proline, lipid 

peroxidation (LPO) based on malondialdehyde (MDA) production, microsomal drug metabolizing enzyme 

CYP450 2EI measured as aniline hydroxylation, along with the antioxidant enzyme systems as catalase 

(CAT), Glutathione peroxidase (GSH-Px), Glutathione (GSH). Liver Na+ K+ ATPase and y-glutathione 

transpeptidase (y-gt) were studied to assess the cell membrane disintegration together with serum enzyme 

activities (AST, AL T, ALP, Bilirubin). In both models of chemically induced liver fibrosis, the levels of 

hydroxyproline, LPO, CYP450 2EI, Na+ K+ ATPase, y-gt along with serum enzumes remained significantly 

increased with suppression of anti-oxidant defence enzymes. Treatment with a hydro-ethanolic extract of 

Emblica officinalis significantly reduced the levels of hydorxyporline, MDA, , Na + K+ ATPase, y-gt and 

serum enzymes. The anti-oxidant defence enzymes were restored to normal values as well. Thus this study 

finds Emblica officinalis as a useful alternative source oftreatment in chronic liver fibrosis. 

Keywords: Liver fibrosis, Emblica oificinalis, rat 

Page 236

C19 

A CASE OF SURREPTITIOUS NON-FATAL MERCURY POISONING 

Alphonse Poklis*. Department of Pathology, Medical College ofVirginia Campus at Virginia 

Commonwealth University, Richmond, VA 23298-0165 

A case of non-fatal mercury poisoning disclosed suspicion of criminal poisoning and resultant toxicology 

testing is presented. A 46 yr-old, white male Fire Chief with episodes of chest pain and diaphoresis, 

presented in the emergency department with a one month history of fatigue, generalized weakness, 

headaches, "inability to focus at work" and "hardly able to walk up stairs", but denies shortness of breath. 

He had a prior history of episodes of chest pain and diaphoresis. Physical examination and chest X-rays 

were unremarkable, serum chemistries and enzymes including CPK and LDH, and thyroid were normal. He 

displayed an EKG on normal rhythm. His hospital course was uneventful and he was discharged after four 

days. Five weeks later, the chief once again presented in the emergency department with a four day history 

of: "chills and shakes", a fever of 102 0 F, back pain, headaches, increasing fatigue and muscular weakness, 

tingling in his fingers and toes and a palpable tender left axilla lymph node. Once again the physical 

examination was unremarkable and laboratory test normal with the exception of a serum creatinine of 1.5 

mg/dL and a urine protein of 60 mg. His hospital course was uneventful and he was discharged after three 

days. Three weeks after discharge while eating at work a lunch provided by his assistant-chief, the chief 

began vomiting violently. Suspicion of poisoning was raised by the comments of co-workers. Therefore, 

coffee prepared the next morning for the chief by his assistant was saved and given to the police. Initial 

screening of the coffee for toxic metals by the Reinsch Test resulted in a classic "silver mirror" indicating 

the presence of mercury. The coffee and a 24 hour urine specimen obtained from the chief were then 

analyzed in a "cold vapor" Mercury Analyzer System (Perkin Elmer Corp.). The urine contained 20ug of 

mercury/gm of creatinine. While executing a search warrant at the assistant-chiefs home, the police found 

over 128 reagent chemicals and explosives, including several mercury compounds. The assistant admitted 

adding mercurous nitrate periodically to the chiefs coffee, to sandwiches the day the chief was violently 

vomiting, and to doughnuts the day of the chiefs first emergency room admission. The motive for the 

poisoning was that the assistant had been passed over for promotion. Three years prior to the presented 

case, a former chief working with the assistant, was retired with "complete disability" due to a 

neuromuscular disease of unknown etiology characterized by slow mental functioning, peripheral 

neuropathy and chronic nephritis! This case exemplifies the difficultly of diagnosing the rather diffuse and 

non-specific signs and symptoms of surreptitious chronic mercury poisoning. 

Keywords: Mercury poisoning, criminal poisoning, mercury analysis 

Page 237

C20 

INVESTIGATION OF THE ACCUMULATION OF 2,4-D1CHLOROPHENOXYACETIC ACID 

(2,4-D) IN RAT KIDNEYS 

Handan Aydm* : University Istanbul, Faculty of Veterinary Medicine, Department of Pharmacology and 

Toxicology, 34310 Avcllar, Istanbul, Turkey 

A study was conducted to assess the accumulation in the kidneys of 2,4-dichlorophenoxyacetic acid (2,4 

D), the most widely used herbicide in the world, and its metabolite 2,4-dichlorophenol (2,4-DCP). 2,4-D is 

eliminated in humans and animals mainly through renal excretion. Male and female Sprague-Dawley rats 

were treated with 2,4-D that was introduced in their drinking water for 30 days. Group A (control group) 

was fed a normal diet, while Group B was treated with 50 ppm, and Group C 100 ppm 2,4-D. In addition, 

2,4-D was given daily as an oral dose, combined with their drinking water, consisting of 25 ppm in Group 

D and 50 ppm in Group E. Levels of2,4-D and its metabolite 2,4-DCP in the kidneys were measured using 

an HPLC method. It was observed that, though the administered doses of 2,4-D did not produce 

significant toxic effects, its metabolite in particular was present at high levels in the kidneys. 

Keywords: 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4-dichlorophenol (2,4-DCP); HPLC 

Page 238

e21 

RAPID, SIMPLE AND V ALIDA TED GC-MS ASSAY FOR DETERMINATION OF DRUGS 

RELEVANT IN DIACNOSIS OF BRAIN DEATH IN HUMAN BLOOD PLASMA 

Frank T. Peters', Julia Jung, Thomas Kraemer and Hans H. Maurer 

Department ofExperimental and Clinical Toxicology, University of Saarland, D-66421 Homburg (Saar), 

Germany, frank.peters@uniklinik-saarland.de 

Background: Before declaring the brain death of a patient (e.g. prior to explantation of organs), a ~umber 

of requirements have to be fulfilled. One of them is the exclusion of effective plasma concentratIOns of 

drugs which might mimic brain death, especially ofthose typically administered in intensive Cl"e medicine. 

Recommendations for toxicological analysis in the context of the diagnosis of brain death have recently 

been published (Hall bach et a\., T1AFT Bulletin 34, 2004, 14-16), which include a minimum consensus on 

the relevant analytes (thiopental, pentobarbital, methohexital, phenobarbital, diazepam, nordiazepam, and 

midazolam). The proposed limits of quantification correspond to one half of their lowest therapeutic 

concentrations. Therefore, the aim of the presented study was to develop and validate a rapid and simple 

assay for determination ofthe above mentioned drugs in human plasma samples. 

Methods: After addition of 50 J.1l of internal standard solution (4.0 mg/l pentobarbital-ds, 2.0 mg/l 

methohexital-ds, 40.0 mg/l phenobarbital-ds, 0.8 mg/l diazepam-ds, and 0.8 mg/l nordiazepam-ds in butyl 

acetate) and 50 J.11 of butyl acetate to 200 III of plasma, the samples were extracted for 2 min on a rotary 

shaker. After phase separation by centrifugation (1 min, 10000 g), 2 III of the organic phase (upper) were 

injected into the GC-MS system (Agilent, GC-MSD 5973). The analytes were separated within 10 min by 

gas chromatography (HP-l column, 12 m x 0.2 mm LD.) and detected by mass spectrometry. The mass 

spectrometer was operated in the full scan mode for identification and in the selected ion mode (S1M) for 

quantification (target ions ml= 156, 161, 172,247,252,204,209,256,261,242,247,310). Validation was 

performed according to a minimum consensus on method validation in this context currently developed by 

the Clinical Toxicology Committee of the GTFCh. This included evaluation of selectivity, calibration 

model, precision and accuracy. Furthermore, the results for accuracy and precision obtained with six-point 

and one-point calibration were systematically compared. Finally, the applicability of the described assay 

was tested by analysis ofreal samples from brain death cases. 

Results: The analytes were fully separated and sensitively detected. No interfering peaks were detected in 

blank plasma samples from ten different sources. The assay was linear from 0.25 to 10 mg/I for 

pentobarbital and thiopental, from 0.125 to 10 mg/l for methohexital, from 2.5 to 50 mg/I for phenobarbital, 

from 0.05 to 2.5 mg/I for diazepam and nordiazepam, and from 0.01 to 0.5 for midazolam. Using six-point 

calibration, accuracy data (in terms of bias) ranged from -17.9% to 23.7%. Within-day and intermediate 

precision data (expressed as CV) ranged from 1.4% to 6.4% and from 2.6% to 7.0%, respectively. Using 

one-point calibration with a calibrator close to the center _of the. linearity range, accuracy data (in terms of 

bias) ranged from -11.6% to 29.7%. Within-day and intermediate precision data ranged from 1.3% to 6.2% 

and from 2.6% to 9.6%, respectively. Recoveries ranged from 85% to 109%. The acceptance criterion 

defined by the Clinical Toxicology Committee of the GTFCh (99% confidence interval of measured mean 

within ±50% oftarget value) was easily fulfilled for all analytes, even with one-point calibration. The assay 

was successfully applied to analysis ofreal brain death cases. 

Conclusion: The described assay allows rapid, fast and reliable determination of analytes relevant in the 

diagnosis of brain death. Systematic studies showed that the assay can be performed with one-point 

calibration. This is an important advantage, because it keeps the workload low for the usually single cases 

and because results are needed quickly in this context. 

Keywords: brain death, GC-MS, validation 

Page 239

e22 

NEONATAL ABSTINENCE SYNDROME IN METHADONE-EXPOSED INFANTS IS ALTERED 

BY LEVEL OF TOBACCO EXPOSURE 

Robin E. Choo*·, Marilyn A. Huestis·, Jennifer R. Schroeder·, Hendree E. Jones b , 

·IRP, NIDA, NIH, Baltimore, MD, bDept. Psychiatry and Behaviora\ Sciences, lHU School of Medi.cine 

Baltimore, MD 21224, US 

' 

In utero exposure to tobacco has been associated with lower birth weight infants, pre-term births, smaller 

head circumference, intrauterine growth retardation and increased fetal morbidity; however, few studies 

have examined the neurobehavioral effects of prenatal tobacco exposure. This report describes the effect of 

prenatal tobacco exposure on neonatal abstinence syndrome (NAS) for infants born to methadone (mean 

dose 77.0 mg/day ± 19.4, range 40-110) maintained mothers. Twenty-nine pregnant women and their 

infants participated in this IRB approved study. Smoking histories were obtained by maternal self-report at 

enrollment into the study and at delivery. Sixteen women, light smokers (LS), reported cigarette 

consumption of 10 or less cigarettes/day (mean 8.4 ± 2.3) and thirteen women, heavy smokers (HS), 

reported smoking 20 or more cigarettes/day (mean 

21.5 ± 5.5). The onset, peak and duration of NAS 

were examined. Infants born to mothers in the HS group had significantly higher (p=0.014) NAS peak 

scores of 9.8 ± 4.8 as compared to 5.6 ± 3.8 as seen in the LS group. There was a significant difference 

(p=0.016) in time to peak between the HS group (113.8 ± 90.0 h) and the LS group (37.8 ± 33.8 h). The 

duration ofNAS showed a trend towards statistical significance (p=0.054) between the HS (mean 9.5 ± 7.3 

days) and LS (mean 5.1 ± 4.6 days) grolJps. These data showed that infants born to mothers in the HS 

group had a 57% higher NAS peak score, took 33% longer to peak and had a 54% increase in NAS 

duration as compared to the LS group. These results demonstrate the need for future studies to examine the 

role of tobacco exposure as a variable in the examination of opioid associated NAS. 

Keywords: Prenatal, Methadone, Nicotine, NAS, Outcomes 

Page 240

C23 

BIOMONITORING OF EXPOSURE TO CHEMICAL WARFARE AGENTS 

D. Noort*, MJ. van der Schans and H.P. Benschop 

Division of Chemical & Biological Protection, TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA 

Rijswijk,The Netherlands 

Methods to analyze chemical warfare agents (CWA) and their decomposition products in environmental 

samples were developed over the last decades. On the other hand, methods for such analyses in biological 

samples have only recently become available. Retrospective detection of exposure to CW A can be useful 

for various applications. With regard to the "Homeland Defense" program, it can be envisaged that rapid 

diagnostic methods can play a pivotal role in case of a terrorist attack with CWA. In the same context, 

confirmation of non-exposure of worried citizens is of utmost importance. Also, such methods can be used 

for forensic analyses in case of suspected terrorist activities ("chemical fingerprints"). It is self-evident that 

these methods will also be highly valuable from a military point of view, e.g., to establish firmly to which 

chemicals casualties have been exposed to, which is a starting point for adequate medical treatment, or for 

health surveillance of workers in destruction facilities of chemical warfare agents. This presentation will 

focus on a number of specific methods currently available for verification of exposure to the most common 

CWA, i.e., nerve agents and mustard agents. 

There are basically four methods to diagnose an exposure to a nerve agent: 

1. cholinesterase inhibition measurements 

2. analysis ofhydrolysis products, e.g., alkyl methylphosphonic acids 

3. analysis of generated phosphofluoridates after treatment of blood with fluoride ions ("fluoride 

reactivation") 

4. mass spectrometric analysis of phosphylated peptides after enzymatic digestion of modified 

cholinesterase. 

For mustards, there are three distinct methods to assess an exposure: 

1. mass spectrometric analysis of low molecular urinary metabolites 

2. analysis of DNA adducts by means of mass spectrometric or immunochemical methods. 

3. mass spectrometric analysis of protein adducts, e.g., to hemoglobin and albumin. 

This presentation will focus on methods that are based on the analysis oflong-Iived protein adducts, i.e., on 

methods 3 and 4 for nerve agents and on method 3 for mustards. Advantageously, protein adducts are stable 

and therefore detectable weeks or even months after the exposure, in contrast to DNA adducts and urinary 

metabolites which are excreted much more rapidly. The developed technology will be described briefly and 

examples of real exposure incidents will be presented. 

Acknowledgements: This presentation covers work that was funded by the US Army Medical Research and 

Materiel Command, by the Bundesministerium der Verteidigung, InSan I 3, Germany, and by the 

Directorate of Military Medical Service ofthe Ministry ofDefense, The Netherlands. 

Keywords: chemical warfare agents, adducts, diagnosis 

Page 241

C24 

ATTEMPTED SUICIDE BY INGESTION OF CHLORPYRIFOS: IDENTIFICATION IN SERUM 

AND GASTRIC CONTENT BY GC-FID/GC-MS 

Maria A. Martinez l , Salome Ballesteros 2 , Carolina Sanchez de la Torre l , Antonio Sanchiz\ Elena 

Almarza 1 , and Alejandro Garcia-Aguilera} 

IChemistry Department, National Institute of Toxicology and Forensic Sciences, Ministry of Justice, CI 

Luis Cabrera 9, 28002 Madrid, Spain. 2Spanish Poison Control Center, National Institute of Toxicology 

and Forensic Sciences, Madrid, Spain 3Emergency Service, Hospital Rafael Mendez, 30800 Lorca, Murcia, 

Spain 

The case history, toxicological findings, and poisoning characteristics of an attempted suicide by ingestion 

of a chlorpyrifos formulation are reported along with a description of the validated analytical method. 

Chlorpyrifos, an organophosphate pesticide introduced by Dow Chemical Company in 1965 as a 

broadspectrum insecticide, is the active ingredient in many commercial insecticide formulations available 

in Spain. Organophosphate insecticides inhibit cholinesterase activities which causes accumulation of 

acetylcholine at synapse, and as a result an overstimulation of neurotransmission occurs. The mortality rate 

of suicide poisoning is usually high and therefore early diagnosis and appropriate treatment is often life 

saving. Other compounds can be present in organophosphate formulations and are responsible for part of 

the toxicity of these commercialized products. In fact, aromatic hydrocarbons, such as toluene and/or 

xylenes, and other additives could increase the risk of toxicity after ingestion of pesticide formulations. 

Numerous cases of acute nonfatal and fatal poisoning because of the inhalation or ingestion of chlorpyrifos 

have been reported in the literature. However, there is a lack of chlorpyrifos poisoning cases published 

where analytical findings were included. 

A I5-year-old female teenager went to the emergency room after the ingestion of a product from a bottle 

marked with a label "Poison". On admission she was obtunded, with normal vital signs and a strong smell 

of solvent. Therapeutic measures included the application of decontamination procedures, oxygen and 

gastric protectors. She had a good outcome with mild CNS depression and bradycardia. Two hours after 

ingestion biological samples were collected in the emergency room and sent for analysis to our laboratory 

with instructions to investigate the presence of solvents. The serum and gastric content contained 5.3 and 

9.4 j.lg/mL of unmetabolized chlorpyrifos, 4.6 and 6.9 j.lg/mL of toluene, and 2.5 and 7.9 Jlg/mL of butyl 

acetate, respectively. The toxics were isolated after liquid-liquid extraction of 3 mL ofsample with 1 mL of 

diethyl ether using n-octyl-benzene as internal standard. The simultaneous determination of chlorpyrifos, 

toluene, and butyl acetate were performed using the combination of gas chromatography with flame 

ionization detector (GC-FID) for screening analysis, and gas chromatography-mass spectrometry (GC-MS) 

for confirmation of the obtained results. Both gas chromatographs were equipped with methylsilicone 

capillary columns. The previous GC-FID screening analysis was used for quantitation of toluene and butyl 

acetate, and GC-MS SIM mode was used for quantitation of chlorpyrifos, using serum calibration curves in 

the range of 0.l-5 j.lg/mL. Limits of detection were 25, 36 and, 23 ng/mL for chlorpyrifos, toluene, and 

butyl acetate, respectively. Absolute recoveries were more than 90 %, intra-assay precisions less than 5 %, 

and linearity up to 5.0 Jlg/mL for all the analytes. 

The method provides an excellent and rapid tool for use in cases of unknown poisonings allowing the 

simultaneous determination of a wide variety of pesticides and additives, including petroleum distillates, in 

the performance of systematic toxicological analysis in forensic and clinical laboratories. 

Keywords: Chlorpyrifos, Poisoning, GC-FID, GC-MS 

Page 242 1

C25 

PROPYLENE GLYCOL IN EXTREMELY HIGH ION GAP LACTIC ACIDOSIS 

Robert L. Fitzgerald* and David A. Herold 

V A Healthcare System San Diego and University of California-San Diego, San Diego, CA. 

A 31-year-old male presented to the ED disoriented in acute distress. His admission laboratory data were 

remarkable for the degree of acidosis present. An arterial blood gas measurement showed his pH to be 6.85 

(reference range 7.35 to 7.45) with a pC02 of 14 mm Hg (reference 35 to 45) and a lactic acid of 30 

mmollL (reference range 0.7 to 2.1). Additional labs included an ethanol of217 mg/dL, and a measured 

osmolality of 376 mOsmikg (270 - 310). Taking the effect of ethanol into account, the patient had an 

unexplained osmol gap of 65 mOsmikg. In addition to the unexplained osmol gap the patient had an anion 

gap of 50 mEq/L (reference range 10 to 20), the L-Iactate of 30 explained this gap. Based on the 

unexplained osmol gap, the acidosis, and a high anion gap, stat methanol and ethylene glycol were ordered. 

Negative finding for methanol and ethylene glycol prompted a search for other causes of hyperosmolality. 

Discussion of the case with the toxicology laboratory revealed the presence of a large peak on the gas 

chromatographic tracing from a volatiles screen that was not reported initially. This peak represented 40 

mgldL of propylene glycol. Either ethanol or propylene glycol can result in a high anion gap lactate 

acidosis. The profound acidosis and extremely high L-Iactate was a result of the patient drinking for 5 days. 

While the osmol gap could not be completely explained, the high anion gap could be explained by L-Iactate 

and the implied D-Iactate resulting from the propylene glycol metabolism. 

Based on the laboratory data, the patient was treated with intravenous bicarbonate and IV fluid replacement 

in an attempt to correct the acidosis. Despite 4 ampules of bicarbonate, the patients arterial blood gas pH 

did not increase significantly nor was there a significant increase in urinary output. Due to the impending 

renal failure and acidosis, the patient was hemodialyzed. Four hours of hemodialysis corrected his 

acidlbase imbalance. Unfortunately, after hemodialysis, the patient developed acute respiratory distress 

syndrome and required ventilation assistance. The patient also became septic and was treated with 

antibiotics. After a twelve day hospital stay which included intermittent hemodialysis for renal failure, the 

patient recovered and was discharged in apparent good health. 

This presentation will review the common causes of hyperosmolality in clinical toxicology and the 

diagnostic pathway for identifying various intoxicants that cause high anion gap metabolic acidosis. The 

importance of communicating directly with the laboratory to help identity unusual causes of combined 

osmol/anion gap metabolic acidosis will be emphasized. 

Key words: propylene glycol, metabolic acidosis, ethanol 

Page 243

C26 

CORRELATION BETWEEN METABOLIC ACIDOSIS AND CLINICAL PARAMETERS IN 

DIETHYLENE GLYCOL (DEG) POISONING VICTIMS 

L.A.Ferrari*! ,L. Giannuzzi 2 

ILaboratory of Toxicology and Legal Chemistry, Buenos Aires Court of Justice. 41 y 119, (1900) La Plata, 

Argentina. laferrari@unimoron.edu.ar 

2Catedra de Toxicologla y Qulmica Legal. Facultad de Ciencias Exactas. Universidad Nacional de La Plata, 

47 y 115 (1900). Argentina.leda@biol.unlp.edu.ar 

This work analyzes fifteen victims of a massive intoxication that took place in Argentina in 1992 as a result 

of the intake of pro polis syrup: a popular medicinal agent used in the 90s for upper respiratory system 

infections. Diethylene glycol (DEG) was found as the responsible agent which caused metabolic acidosis, 

anuria, renal failure and death in the 15 studied victims. 

DEG poisoning cases were classified into three groups according to the survival time of the victims, 

namely: 1) those who survived up to 3 days; 2) those who survived from 3 to 5 days; and 3) those who 

survived from 5 to 21 days. 

A methanolic fraction extracted in a Soxhlet system with subsequent concentration and purification was 

obtained from viscera and blood. Gas Chromatography and FlO detector (GClFlO) methodology was 

performed. On the other hand, samples of the propolis syrup from each of the victims was studied through 

NMR and quantified by GClFlO, using ethylene glycol as internal standard. Finally, each clinical history 

from victims were studied in detail: anionic Gap (AG), Excess base (EB) and pH. 

Patients belonging to group 1 showed the highest AG values and the lowest (EB) values as well as the 

major severity in their clinical manifestations. Correlation between pH and EB was ~ =0.68, 0.99 and 0.55 

for groups 1,2 and 3 respectively. In 3 out of 15 fatal cases studied, DEG could be isolated from viscera 

and blood. The concentration ratio (DEG)viscer.l (DEG)blood ranged 1.45 - 1.55 with a coefficient correlation 

of ~ = 0.96. In the other victims, DEG could not be detected. This could be due to the long survival period 

of the deceased victims from the syrup ingestion to death, and to the fact that putrefYing mechanisms could 

be operating. 

Results showed that the syrup samples contained DEG in a 24% to 65.0% (p/v) range. A proper correlation 

between the amount ofDEG ingested and the anionic Gap (r2=0.63-0.78) could be observed in the victims 

studied, according to theoretic amount of syrup ingestion (5 -20 ml). So, the lethal dose for humans in this 

episode was found to vary from 0.019 to O.l74mg DEGlKg corporal weight. These results could contribute 

to the understanding of DEG toxicity parameters On the other hand, they could provide data concerning 

lethal dose in humans. 

Keywords: Diethylene Glycol, poisoning, lethal dose 

Page 244

C27 

PHARMACEUTICAL IDENTIFICATION AND QUALITY CONTROL TESTING AT A MAJOR 

MEDICAL CENTER 

Carl E. Wolf* and Alphonse Poklis. Department of Pathology, Medical College of Virginia Campus at 

Virginia Commonwealth University, Richmond, VA 23298-0165. 

Clinical Toxicology laboratories are often presented with the problem of identifying the contents of 

pharmaceutical products. These preparations come into question in a variety of situations: the preparation is 

found outside of it's "controlled" environment, the inappropriate or wholly unexpected response of a 

patient to the administration of the preparation, or suspicions of drug diversion or abuse by a health care 

provider. Medical institutions generally address the issue of drug abuse by health care workers through preemployment 

urine drug testing, random drug testing, and/or "for cause" drug testing after an incident has 

occurred. However, such programs seldom, if ever address issues of pharmaceutical compounding or 

tampering. 

We have developed a simple HPLC method to routinely identify and quantitate pharmaceutical 

preparations for over 15 different drugs, including opiates, synthetic opiates, local anesthetics, and 

midazolam. The method uses a Beckman ODS column (4.6 x 250 mm), and an isocratic mobile phase 

consisting of acetonitrile: perchloric acid: water (33:0.134:67) for drug separation. Drugs are detected and 

quantitated using a UV detector at either 206 or 280 nm. In the past 6 years, we have analyzed thousands of 

pharmaceutical products for content and concentration. Most of these products were analyzed for 

concentration, to verify proper compounding by our hospital pharmacy or local pharmacies. 

Several cases will be presented of typical findings from products that have not been tampered with, as well 

as, instances of mistaken compounding and drug. diversion. Cases include: preparations from patient 

controlled anesthesia, IV bag solutions, rescue squad drug boxes, and improper compounding of pediatric 

preparations for c1onidine, and hydromorphone. 

Keywords: Drug Diversion, Pharmaceutical Testing, Narcotics 

Page 245

C28 

METHAMPHETAMINE DETECTION IN URINE OF CHILDREN CO-EXISTING WITH 

CLANDESTINE METHAMPHETAMINE MANUFACTURING 

Ann Marie Gordon*, Barry K. Logan, PhD 

Washington State Toxicology Laboratory, Forensic Laboratory Services Bureau, Washington State Patrol, 

2203 Airport Way South, Seattle, WA 98134. 

In 2000, the Washington State legislature enacted the "Drug Endangered Children's Law" to protect 

children found at clandestine methamphetamine manufacturing sites. The law mandates that children 

found at these sites be placed in protective custody and allows for a mandatory two-year enhancement on 

any "Unlawful Manufacturing of a Controlled Substance" conviction when children are present at the 

clandestine lab site. In March 2002, the legislature passed a follow-up bill mandating that any person 

allowing hislher child to be present at a methamphetamine lab, be charged with a felony child 

endangerment charge, regardless of whether they were charged with the manufacture of methamphetamine. 

The Washington State Toxicology Laboratory is tasked with providing forensic toxicological services for 

the coroners and medical examiners and all police agencies within the 39 counties of the state. We began 

receiving samples (typically urine) as part ofthe Drug Endangered Child program in 2000, without advance 

notice and without a suitable protocol for appropriate testing. Over the past two years, we have coordinated 

with two police agencies to develop a suitable sample collection and testing protocol. 

The Washington State Toxicology Laboratory received 26 samples, from February 2002 through February 

2004, collected from children for investigation of drug endangerment; 5 diapers, 1 serum and 20 urine 

samples were submitted to the laboratory for analysis. The ages ofthe children ranged from 4 months to 16 

years of age, mean and media age of7 years, half of the subjects were female and 23 of the 26 cases (88%) 

were positive for methamphetamine. 

In most of the cases (20 of the 23 positives), the concentrations of methamphetamine and amphetamine in 

the samples were relatively low, between 0.02 mg/L to 0.2 mgIL methamphetamine. In these cases, it is 

reasonable to state that the children ingested methamphetamine but may not have exhibited any associated 

effects. Of the remaining three cases, two exhibited signs of methamphetamine intoxication at the time of 

their encounter with the police and were treated in the hospital for their symptoms. The first, a 6-monthold 

male, had a serum methamphetamine concentration of 0.12 mg/L and the second child, a 2-year-old 

male, had urine concentrations of 13.59 mgIL methamphetamine and 0.9 mg/L amphetamine. The third 

case with high drug concentrations was a 16-year-old male with urine methamphetamine of 0.05 mgIL and 

amphetamine of 18.5 mgIL. 

Children living in a methamphetamine-manufacturing environment are endangered by many associated 

factors, the toxicity of the associated chemicals used in the manufacture, inherent fire danger, neglect and 

are at a higher risk for sexual abuse. The presence of methamphetamine is an indicator that the children 

have been in unsafe environment and this data can be used to support a case of child endangerment. Child 

endangerment charges have been filed in at least 12 of the cases; in one case the defendant was convicted 

of illicit methamphetamine manufacture and he received a sentence enhancement for the child 

endangerment charge. 

Key Words: Methamphetamine, Children, Clandestine 

Page 246

C29 

AN EVENT ASSOCIATED WITH FATAL 2,5-DIMETHOXY-4-BROMOAMPHETAMINE 

OVERDOSE 

Marie Balikova* 

Institute of Forensic Medicine and Toxicology, 1 st Medical Faculty, Charles University in Prague, 

121 08 Prague 2, Czech Republic, (mbali@lfl.cuni.cz) 

2,5-dimethoxy-4-bromoamphetamine (DOB) is a strongly acting hallucinogen with effective dose estimate 

for an 80 kg man being 2 mg (I). In the event reported here, two men were provided a capsule with white 

powder containing an unknown substance in order to test its effects as a new LSD like hallucinogen. They 

shared the content of a capsule and consumed it orally followed with strong rapid hallucinations and 

vomiting, unconsciousness and coma lasting several days. After unknown time elapsed since the 

application, they were admitted to the hospital in coma state without response to analgesic irritation. The 

man AX 28 years (113 kg) survived. The man BX 29 years (65 kg) experienced convulsions, metabolic 

acidosis (pH 6.6) and died after six days. Immediately after the admission to the hospital, gastric content, 

blood and urine specimens were sampled and sent for toxicological examination. Alcohol in blood samples 

was not found. Cedia urine screening indicated the presence of THCOOH in both cases, cocaine and 

metabolites in AX urine, and no signal corresponding to amphetamines presence was detected. Finally, 

GC-MS method for unknown drugs discovered the presence of DOB in gastric content and urine samples 

of both persons. GC-MS targeted analysis for acetylated DOB confirmed its presence in blood of both 

persons and quantitative analysis provided the concentration values in serum 13nglml (AX - survived) 

and 19 nglml (BX - deceased). This report both on nonfatal and fatal DOB overdose cases is based on clear 

toxicological evidence, moreover it is completed with antemortem DOB blood serum levels and probably 

it is the first sign ofDOB occurence in the Czech Republic. 

Reference: (1) A. Shulgin, A. Shulgin: PiHKAL: A Chemical Love Story. Transform Press, Berkeley CA, 

1998 

Keywords: 2,5-dimethoxy-4-bromoamphetamine, DOB overdose, fatal intoxication 

Page 247

C30 

SIMULTANEOUS DETECTION OF STIMULANT LAXATIVES AND DIURETICS IN HUMAN 

URINE USING GC-MS AFTER ENZYMATIC CLEAVAGE OF CONJUGATES AND 

EXTRACTIVE METHYLATION 

J. Beyer', A. BierI, F.T. Peters and H.H. Maurer 

Department of Experimental and Clinical Toxicology, University of Saarland 

0-66421 Homburg (Saar), Germany,jochen.beyer@uniklinik-saarland.de 

Background: Laxatives and diuretics are widely abused for various reasons. Surreptitious abuse of both is 

often associated with eating disorders or Milnchhausen syndrome. Concerning laxatives, there is also 

habitual abuse because of chronic constipation, whereas diuretics are also abused in doping. Such abuse 

may lead to serious disorders like chronic diarrhea, hypokalemia, dehydration, and disturbance of acid-base 

balance. Because of the heterogeneity of these side effects and their similarity to symptoms of 

gastrointestinal or renal disorders, a toxicological screening for laxatives and diuretics should be part of the 

differential diagnosis of such syndromes. This may also help to avoid extensive and expensive diagnostic 

work. 

Methods: After accelerated enzymatic cleavage of conjugates (glucuronidase/arylsulfatase, EC no. 

3.2.1.3113.1.6.1, 100000 Fishman units per mL, 50 cC, 90 min), the drugs and their metabolites were 

isolated from 2 mL of urine by extractive methylation. For details of extractive methylation see Maurer HH 

et aI., JAT 25,2001,237. The extract was reconstituted in 50 I-lL ethyl acetate and 2 J..lL were injected into 

the GC-MS (Agilent GC-MSD 5972). Analytes were separated on an HP 1 column (12 m x 0.2 mm 1.0.) 

and detected by mass-spectrometry in the EI full scan mode. They were screened by using reconstructed 

mass chromatography using selective ions and identified by visual and computerized comparison of the 

peak underlying mass spectra with the corresponding reference mass spectra (PMW _tox4). 

Results: The assay allowed the detection of the diphenylmethane laxatives phenOlphthalein, bisacodyl and 

picosulfate (the latter two via their common metabolites bisacodyI diphenol, methoxy bisacodyl diphenol 

and dimethoxy bisacodyldiphenol) as well as of anthraquinone laxatives contained in plants like senna, 

cascara, rhubarb, frangula and aloe via their common metabolite rhein. Furthermore, the diuretics 

acetazolamide, bemetizide, bendroflumethiazide, bumetanide, butizide, canrenoic acid (also main 

metabolite of spironolactone), carzenide, chlorothiazide, chlortalidone, c1opamide, cyclopenthiazide, 

cyclothiazide, diclofenamide, etacrynic acid, etozolin, furosemide, hydrochlorothiazide, indapamide, 

mefruside, metolazone, piretanide, poly thiazide, tienilic acid and xipamide could be detected as well as the 

uricosurics benzbomarone, probenecide and sulfinpyrazone, which are relevant in doping control. Cleavage 

of conjugates was a prerequisite for sensitive detection of the laxatives and/or their metabolites, because 

these analytes are excreted mainly as conjugates. For analysis of diuretics, enzymatic hydrolysis is not 

necessary, because they are mainly excreted unchanged. The limits of detection (LOD, SIN 3) ofthe tested 

drugs and their metabolites lay in the range of I to 500 nglmL, 90% within 5 to 100 nglmL. Recoveries 

were determined for a limited number of analytes (bisacodyl diphenol, butizide, furosemide, 

hydrochlorothiazide, phenolphthalein, piretanide, probenecide, rhein, sulfinpyrazone, and tienilic acid) 

representing the different groups of the structurally heterogeneous analytes and ranged from 33 to 99 with 

coefficients of variation from 4.2 to 21.1 % (determined at 5 x LOD). Applicability studies showed that at 

least the given drugs and/or their metabolites were detectable over periods of 24 to 72 hours after 

administration of the lowest therapeutic dose (picosulfate, 5 mg; furosemide, 40 mg; hydrochlorothiazide, 

15 mg; spironolactone, 25 mg; or senna plant extract containing 7 mg ofsen nos ide B) to one young healthy 

volunteer each (after informed consent). Furthermore, the extractive methylation (without the enzymatic 

cleavage) has proved to be suitable for simultaneous detection of other acidic drugs (Maurer HH, J. 

Chromatogr. B 733, 1999, 3). 

Conclusion: The presented GC-MS procedure allowed simultaneous identification and differentiation of 

stimulant laxatives, diuretics and/or their metabolites in urine. This procedure should be applicable for 

diagnosis or differential diagnosis of an abuse ofthe described drugs or for doping control. 

Keywords: Diuretics, Laxatives, GC-MS 

Page 248

C31 

DETECTION OF NEW MINOR METABOLITES BY LC/MS AND CHARACTERIZATION BY 

LCIMS/MS AFTER COMPELLED INGESTION OF COCAINE 

L. Humbert l , R. EljaoudLI, F. Orisel 2 , T. Kargar-OriseI 2 , D. Mathieu 3 , M. Lhermitte i 

I Laboratoire de Biochimie & Biologie Moleculaire, H6pital Calmette, 59037 Lille Cedex France. 

2 Waters Corporation, European Headquarters & Waters S.A.S., Rue Jacques Monod, 78280 Ouyancourt 

France. 3 Urgence respiratoire et reanimation medicale, H6pital Calmette, 59037 Lille Cedex France. 

A well-known cocaine addict presented spontaneously to the emergency service of the Lille Hospital 

(France) and reported that he was forced to ingest a large quantity of a white powder. Surprisingly, the 

patient did not present any intoxication symptoms but was placed under medical observation. Immunoassay 

screening of a blood sample did not reveal the presence of any drugs and immunoassays performed on 

urine led to positive results for cocaine only. LCIMS was used to confirm the immunoassay results and the 

anticipated metabolites of cocaine were found. In addition, many other minor metabolites were detected 

and characterized by LCIMSIMS. 

Materials and methods: The urine sample was extracted at alkaline pH using a chloroform/isopropyl 

alcohol mixture (9: 1) and evaporated to dryness. 100 ,.d of mobile phase was added to the extract and 20 J.l.1 

injected onto the LCIMS system. Chromatographic separation was performed using a Waters Alliance 

HPLC equipped with a Waters Xterra MS C18 column (ISO x 2.1, 3.5 J.l.m). Mass detection was achieved 

using a Waters ZQ mass spectrometer operated in the electrospray mode in both positive and negative ion 

polarities. Multi-functional full scan acquisitions were performed at different cone voltages. Well-known 

urinary metabolites of cocaine were identified through their fragmentation patterns and an additional 

LCIMSIMS analysis was performed to elucidate the structure of other analytes previously detected in MS 

mode that could correspond to new non-documented metabolites. Exhaustive LC/MSIMS study (neutral 

loss, parent and daughters, MRM) was performed using a Quattro Premier (Waters) operated in 

electrospray combined to modified chromatographic conditions leading to greater retention for the polar 

compounds and subsequent better separation efficiency. 

Results: The LC/MS analysis of the gastric content revealed the presence of cocaine. Subsequent LCIMS 

analysis of the urine samples led to the detection of many minors metabolites The oral ingestion was 

confirmed by the positive identification of characteristic metabolites such as m- and p 

hydroxybenzoylecgonine and norbenzoylecgonine l which has also been found metabolised as N 

hydroxynorbenzoylecgonine. It was possible to change the selectivity ofthe chromatographic separation by 

using a Xterra column at basic pH (10 mM ammonium bicarbonate) allowing the separation of several new 

polar metabolites which usually coelute when analysed in acidic conditions. 

Conclusion: Thanks to the spontaneous presentation of the patient, the delay between urine sample 

collection and cocaine oral absorption was very short thus allowing the detection of many minor 

metabolites of cocaine not usually found because of their very low concentrations. Combining the 

sensitivity and the selectivity of tandem mass spectrometry with enhanced chromatographic separation, the 

expected major metabolites were unambiguously identified and many other new metabolites were detected 

and assigned to compounds such as N-hydroxynorbenzoylecgonine, methoxyecgonine and norecgonine 

methyl ester. 

References: 1. Klette KL, Poch OK, Czarny R, Lau CO. Simultaneous OC-MS analysis of m- et p 

hydroxybenzoylecgonine and norbenzoylecgonine: a secondary method to corroborate cocaine ingestion 

using nonhydrolytic metabolites. J. Anal toxicol. 2000 Oct ;24(7) : 482-8. 

Keywords: Forced ingestion, cocaine, metabolite, mass spectrometry 

Page 249

C32 

URINARY EXCRETION RATES OF KETAMINE AND NORKETAMINE FOLLOWING 

THERAPEUTIC KETAMINE ADMINISTRATION: METHOD AND DETECTION WINDOW 

CONSIDERATION. 

Piotr Adamowicz·, Maria Kala 

Institute ofForensic Research, ul. Westerplatte 9, 31-033 Krakow, Poland 

Ketamine is widely used in veterinary medicine. Its medical application in humans is limited to children because 

in adults it induces severe psychedelic episodes. In recent years, ketarnine has been (ab)used by teenagers as a 

recreational and club drug because of its hallucinogenic or stimulant effects. Ketarnine is also (mis)used as a 'daterape' 

drug - to induce amnesia in unsuspecting victims. In a typical scenario, ketarnine is surreptitiously added by 

the perpetrator to the alcoholic beverage ofan unsuspecting person, who is subsequently sexually assaulted while 

under the influence ofthis substance. Many victims do not report the incident until several days after the event. 

This situation creates a demand for sensitive analytical methods to reveal the presence of the drug and/or 

metabolites in biological specimens collected from the victim. The second very important parameter in drug 

testing for forensic purposes is the detection window - how long after drug administration a person tests positive 

for the drug or metabol ite. 

Sensitive gas chromatography-mass spectrometry negative chemical ionization (NCI-GC-MS) and liquid 

chromatography-mass spectrometry atmospheric pressure chemical ionization (APCI-LC-MS) methods for the 

simultaneous quantification of ketarnine and its major metabolite - norketamine in urine were developed and 

validated. These methods were used to study the elimination ofketamine and norketamine in urine collected from 

six hospitalized children (age 4-13 years) who had received a single intravenous dose ofketarnine as an anesthetic 

for short surgical procedures. The doses ranged from 0.75 to 1.59 mglkg. Individual urine samples were collected 

every day or once every two days for 4-16 days.,~ 

Target analytes were isolated from urine samples after enzymatic hydrolysis (with B-glucuronidaze) followed by 

solid phase extraction (HCX column). Ketamine-D 4 and norketamine-D 4 were used as internal standards. For 

NCI-GC-MS procedure, extracts were derivatized with HFBA. The monitored negative ions for ketamine 

derivative were (mlz) 226 and 357, for norketarnine, 383 and 399, and for norketamine-D 4 , 387 and 403. APCI 

LC-MS analyses were carried out without analytes derivatization. Pseudomolecular ions of (mlz) 224 and 228 

(for norketarnine-Do and -D 4 ), 238 and 242 (for ketamine-Doand -D4) were monitored. 

The NCI-GC-MS assay had an LOQ of 20 nglmL for ketamine and of 50 pg/mL for norketamine, and 

displayed LOL across a concentration range of 20-1000 nglmL and 50-I 500 pg/mL for parent drug and 

metabolite respectively. LOQ and LOL for the APCI-LC-MS method were 2 nglml and 2-2000 ng/mL for 

both compounds. For the NCI-GC-MS, mean inter-day precision for ketamine and norketamine ranged 

from 21.4-30.4% and 16.8-23.6%, respectively. For the APCI-LC-MS, mean inter-day precision for both 

compounds were between 1.6-3.7%. 

Using NCI-GC-MS, ketamine was detected in urine of four persons up to 1 day and in one up to 2 days 

after drug administration. Its concentrations ranged from 58 to 1181 ngimL. Norketamine (measured in 

concentrations of 1.18 !J.glmL-50 pglmL) was detected up to 14 days (average 7 days). Using the APCI 

LC-MS method, ketamine was detected in two persons up to 2 days, in one up to 4 days, in one up to 11 

days and in one only up to one day at concentrations of 2-813 ngimL. Norketamine (at concentrations of 

1276-2 nglmL) was detected up to 3, 4, 5 and 6 days after drug administration. In one person, ketamine 

was not detected through the entire 16-day period using both methods. 

Detection window of the analytes is highly dependent on the method used for determination and 

interindividual variability. 

Keywords: ketamine, norketamine, detection window, drug facilitated sexual assault 

Page 250

C33 

NOVEL ASPECTS OF IN VITRO METABOLISM OF BUPRENORPHINE 

Van Chang* and David E. Moody 

Center for Human Toxicology, University of Utah, Salt Lake City, UT 

Buprenorphine is mainly metabolized by cytochrome P450 (P450) 3A4 mediated N-dealkylation to 

norbuprenorphine; both buprenorphine and norbuprenorphine conjugate with glucuronic acid. Previously, 

tentative 6-0-desmethyl norbuprenorphine in free and conjugated form and some unknown polar 

metabolites were observed in rats; while no other metabolites were observed in human. We find higher 

buprenorphine elimination compared to norbuprenorphine formation in human liver microsomes (HLM), 

which supports the hypothesis that other metabolic pathways might exist. In the current study, the phase I 

and phase II metabolism of buprenorphine was investigated in HLM and eDNA-expressed P450s. 

Metabolites were screened by LC-MS/MS, coupled with precursor-ion scan, product-ion scan and neutralloss 

scan. 

The incubation for phase I metabolism was performed in a mixture of 0.1 M phosphate buffer (pH 7.4, 1.0 

mM EDTA and 5.0 mM MgCh); the NADPH generating system (10 mM glucose-6-phosphate, 1.2 mM 

NADP, and 1.2 units gluscose-6-phosphate); and 0.5 mglml microsomal protein. The reaction was initiated 

by the addition ofNADPH generating system in a 37°C shaking water bath and continued for 30 min. The 

incubations ofbuprenorphine in cDNA- expressed P450s were performed as described above except that 25 

pmol eDNA-expressed P450s was used and the incubation time was 20 min. The incubation for phase II 

metabolism was conducted in the presence of 25 Ilg/ml alamethicin and 2 mM UDPglucuronosyltransferases. 

LC-MSIMS utilized a Finnigan TSQ 7000 equipped with a triple-quadrupole 

mass spectrometer and an electrospray ionization source. 

During phase I metabolism of buprenorphine, 5 metabolites (M 1-M5) in addition to norbuprenorphine were 

identified. MI and M2 are hydroxylations of buprenorphine at the tert-butyl group and ring moiety, 

respectively. M3-M5 are secondary hydroxylation metabolites ofnorbuprenorphine. The hydroxyl groups 

are on the tert-butyl group (M3) and ring moiety (M4 and M5), respectively. The metabolism of 

buprenorphine in eDNA-expressed P450s showed that P450 3A5 had highest catalytic activity in Ml 

formation, with the involvement of 2C8 and 3A7, but their activity is only 5.9% and 3.3% of 3A5, 

respectively; P450 3A4 has the highest catalytic activity in M3 formation, with contributions of 3A7, 3A5 

and IA2 to a lesser extent, approximately 12.5%, 2.7% and 2.4% of 3A4, respectively. No detectable 

signal was observed for M2, M4 and M5 by selected reaction monitoring on LC-MSIMS. M3 is the major 

metabolite of norbuprenorphine in HLM, which is mediated mainly by P450 3A4, with the contribution of 

3A5 and 3A 7 no more than 6% of 3A4. Norbuprenorphine metabolism suggested that secondary 

metabolite M3 of buprenorphine was formed through norbuprenorphine. The primary study on phase II 

metabolism of buprenorphine showed the presence of conjugated hydroxyl buprenorphine and hydroxyl 

norbuprenorphine, the structural identification will be processed in details in the future work. 

Keywords: Buprenorphine, Metabolism, P450 

Page 251

C34 

CHANNELING THE EMPEROR; WHAT REALLY KILLED NAPOLEON 

Francesco Mari l , Elisabetta BertolI, Vittorio Finnechi 2 " Steven B. Karch*3. 

IUniversity ofFlorence, Italy, 2University ofFoggia, Italy, Office ofthe Medical Examiner, City & County 

ofSan Francisco, CA 

Those with an opinion about the cause of Napoleon's death rely largely on the results of hair testing. Over 

30 different samples, of reasonable provenance, have been analyzed. Some samples were obtained during 

Napoleon's first exile in Elba, years before he arrived on Saint Helena. Others were obtained at autopsy. All 

studies have revealed elevated concentrations of arsenic and, in the most recent study, elevated 

concentrations of antimony as well. These observations, coupled with written accounts of the symptoms 

and signs exhibited by the Emperor, have led to near universal agreement that arsenic poisoning was the 

cause of death. For proponents of this view, the only question remaining is whether poisoning was 

accidental or intentional. Evidence for intentional poisoning comes from the Emperor's own hand. He wrote 

in his will, "I die before my time, murdered by the British oligarchy and its hired assassin." Others maintain 

that exposure was environmental. Many sources of environmental contamination have been proposed, but 

there is compelling evidence that Napoleon was poisoned by his own wallpaper; it was painted with 

"Scheele's green" pigment, a mixture of copper arsenides. It has been known for more than 100 years that 

certain molds, some likely to have been present in Napoleon's wall coverings, can volatilize arsenical salts. 

Other explanations are possible. Those favoring the poisoning theory tend to minimize the facts that 

Napoleon had a rather thorough autopsy. He had requested one because he believed that he, like his father, 

might have stomach cancer, and that his children were at risk. The autopsy was performed by one of 

Napoleon's personal physicians, a Corsican named Francesco Antommarchi. Five English physicians were 

present, but since Antommarchi was the only one actually trained in anatomic pathology, he performed the 

dissection. Though some physicians later expressed divergent opinions about the cause of death, none 

disputed his anatomic findings: an ulcerated, regionally invasive, carcinoma of the greater curvature, 

metastasizing into the regional lymph nodes. Since high levels of arsenic were clearly present at death and, 

according to the latest studies, present for many years, the question to be answered is whether Napoleon 

died "of" arsenic poisoning, or "with" arsenic poisoning. Recent developments in the treatment of 

promyelocytic leukemia (APL) may provide the answer. Treatment with arsenic trioxide is complicated by 

the occurrence of QT prolongation, torsades des pointes, and sudden death. Tissue culture studies have 

shown that at clinically relevant concentrations, arsenic blocks both I Kr and I ks channels and, at the same 

time, also activates kATP channels. The effects cancel each other out, and normal cardiac repolarization is 

maintained. The unpredictability ofQT interval prolongation, and the occurrence of ventricular arrhythmias 

during arsenic therapy in APL patients, is the result of competing effects, blocking and activating multiple 

repolarizing potassium currents. The balancing ofthese forces is clearly a delicate matter, one that could be 

disrupted by any number of extrinsic or intrinsic physiologic forces. Napoleon was given a huge dose of 

tartar emetic (potassium antimony tartrate) the night before he died. Antimony binds to potassium channels. 

It appears that the immediate cause of death was neither gastric carcinoma, nor chronic arsenic poisoning, 

but medical misadventure. Had Napoleon not been given the tartar emetic, arsenical effects on cardiac 

conduction would have remained balanced; he would have lived to die a natural death, probably from 

gastric carcinoma. 

Key Words: Arsenic, Napoleon, Channelopathy 

Page 252


Abstracts: . 

Forensic Urine 

Drug Testing & 

Adulteration 

Page 253

Fl 

COMPARISON OF SIMULATANEOUS CAMP AND BMBP MULTIPLE ANALYTE ENZYME 

IMMUNOASSA Y REAGENTS WITH ABBOTT AND SYV A EMIT SINGLE ANALYTE 

IMMUNOASSA Y REAGENT 

Michael 1. Coyer*, Jill Mahoney and Dana Robosky, Clinical Laboratories, Inc, A LabCorp Company, 

901 Keystone Industrial Park, Throop, PA 18512. 

The development of drugs of abuse testing (DOA) has seen significant changes since the principals 

competitive binding of Yalow and Solomon were adapted to DOA screening. The changes in the governing 

regulations, the instrumentation and testing requirements has led to more emphasis being placed on turn 

around times and cost of the test to the client. The many advances in antibody production have enhanced 

the selectivity and specificity of screening tools much to the advantage ofthe laboratory and the client. 

This investigator has begun the evaluation of a Multiple Analyte Enzyme Immunoassay (MAEl) products: 

specifically, 1.) Cocaine-Amphetamine-Morphine-Phencyclidine (CAMP) and 2.) Barbiturate-Methadone 

Benzodiazepine-Propoxyphene (BMBP) recently approved by the FDA for use in the U.S. The design of 

the dual reagent system CAMP and BMBP takes into account the values that are associated with the widely 

accepted NIDN SAMHSA screening cut-offs. The reagents are designed to test the four (4) drugs in each 

group in a single sampling. The resulting response of the instrumentation will be relative to a previously 

determined cut-off value. Any result being above the threshold for a positive will be subjected to individual 

analyte analysis. The samples giving no response will be considered negative and no further testing would 

be performed. The reagent design is based on certain statistical data, specifically, > 90% of all urine drug 

screens are negative. The design of the reagent system will enhance the turnaround time and costs 

associated with testing operations while still holding the same high quality standards required ofthe testing 

facilities. 

This report will focus on the comparison of the MAEI reagents with previously tested samples. The 

discussions will include comparisons ofthe MAEl with several reagent systems including, but not limited 

to, Abbott (FPIA), and SYVA EMIT. The correlations of these analysis and discussion of the reagent 

system will be presented. The preliminary testing has been completed and will be furthered after the 

submission of this abstract to include statistics, cross reactivity and overall effectiveness in the testing for 

drugs ofabuse. 

Keywords: Multiple Analyte Enzyme Immunoassay, drugs ofabuse, turnaround time 

Page 254

F2 

URINE ADUL TERA TION TRENDS FROM 2001-2003 

David J. Kuntz*, Chuck Jones, Kris Botelho, and Michael Feldman. Northwest Toxicology, a division of 

LabOne Inc., Salt Lake City, UT 84124, 

The use of urine adulterant products to defeat a urine drug test has become an increasingly important issue 

for the federal and private drug testing programs. Initially, adulteration products were common household 

chemicals. In approximately 1990, the first of designed adulterants appeared as Urine Luck containing 

glutaraldehyde. Nitrite quickly followed once the detection of glutaraldehyde was established. Since that 

time a number of products have evolved with chromium VI, iodine, iodate, and fluoride as the primary 

purchased adulterants. And another disturbing trend in the increase in the number of substituted and 

invalid specimens reported by the laboratory. These specimens indicate the increased use of excessive 

hydration to lower urine levels of drugs of abuse or in the case of invalid specimens, the use of an 

unidentified adulterant. 

~., 

The analysis of adulteration trends includes only specimens tested by Northwest Toxicology during the 

period of2001-2003. The total number ofspecimens during each year was approximately 600,000 samples 

per year. Each specimen tested by the laboratory receives an analysis for creatinine (specific gravity if 

creatinine is less than 20 mg/dL), pH, and adulterants. During 2001-2002, the adulterant test was an 

analysis for nitrite and chromium. In 2003, the nitrite and chromium tests were replaced by a general 

oxidant test. The analysis of the data for the three year period reveals several significant trends in specimen 

validity testing (SVT). The number of specimens reported as substituted or invalid has more than doubled 

from 2001 to 2003 while chromiumVI positive specimens have been reduced by 75%. Nitrite positive 

specimens have been reduced by 20% with unexpected increases in bleach and abnormal pH specimens. 

Iodine and iodate analysis began in 2002 and has quickly become the number one detected adulterant by 

the laboratory. Fluoride is frequently encountered when iodate is also present. 

2001 2002 2003 

Substituted 136 192 248 

Unsuitllnv 202 248 442 

Nitrites 98 88 77 

Chromium 241 102 58 

Bleach 5 30 20 

Soap 1 3 ] 

pH 48 72 97 

Iodinellodate NA 30 105 

Fluoride NA NA 9 

Glutaraldehyde 0 0 1 

0.12% 0.13% 0.16% 

The number of adulterated specimens clearly indicates that the routine testing for a single adulterant, such 

as nitrite, will not detect the majority ofadulterated samples and that a comprehensive SVT program must 

be developed for the identification ofthese specimens. 

Keywords: Specimen validity testing, adulteration, nitrite, chromium 

Page 255

F3 

ANALYSIS OF URINARY STEROID SULFATE METABOLITES USING ION-PAIRED 

EXTRACTION 

'12 

Ad am Cawley " Rymantas Kazlauskas 

l , Graham Trout l , Adrian George 2 

1. Australian Sports Drug Testing Laboratory, National Measurement Institute, Sydney, Australia, 2. 

School ofChemistry, University of Sydney, Sydney, Australia 

Urinary steroid metabolites may be excreted in their unchanged (free) form, and/or more likely, as 

glucuronide or sulfate conjugates. Routinely, doping control laboratories accredited by the International 

Olympic Committee (JOC) and the World Anti-Doping Agency (WADA) screen urine samples collected 

from athletes for the presence of free and glucuronide conjugated steroids by Gas Chromatography-Mass 

Spectrometry (GC-MS) following enzymatic hydrolysis and solid phase extraction. There is a clear need, 

however, for laboratories to analyse a number of steroids excreted primarily as their sulfate conjugates in 

order to further metabolic research and develop confirmation methods that are more specific to particular 

steroid administrations. Published methods utilising a range of chemical hydrolysis techniques to cleave the 

sulfate moiety from steroid metabolites and make them amenable to GC-MS analysis have been 

problematic to implement due to urinary matrix effects. To circumvent this problem, ion-pairing extraction 

with (-)-N,N-dimethylephedrinium bromide under basic conditions was optimised to obtain a purified 

sulfate fraction. Cleavage of the sulfate moiety was efficiently achieved by chemical hydrolysis under mild 

conditions using methanolic-HCI derived from trimethylchlorosilane (TMCS). The free steroids were 

extracted into hexane at pH 9.8 before reaction with N-methyl-N-(trimethylsilyl)trifluoroacetamide 

(MSTFA) to form TMS enol ether derivatives that were subsequently analysed by GC-MS. This method 

provided linear recoveries (R 2 2:0.9993, 100 to 5000 ng/mL) of greater than 90% for androsterone (5aandrostane-3a-oI-17-one) 

extracted as the sulfate conjugate. The potential of direct urinary sulfate 

metabolite analysis using Liquid Chromatography-Mass Spectrometry (LC-MS) is also discussed as a 

means to alleviate the need for chemical hydrolysis. While these methods are directed at steroid detection 

for IOCfW ADA laboratories they may be applied in a broader toxicological sense for the analysis of 

sulfated metabolites of other drugs of abuse. Novel analysis methods such as these may also allow more 

detailed metabolic studies to be carried out to determine new markers of specific drug abuse, thus 

demonstrating their potential as a valuable confirmation technique. Incorporated into the GC-MS method 

validation was an evaluation of a reasonable estimate of measurement uncertainty that is presented as an 

example to emphasise the importance ofISO 17025 compliance for forensic toxicology laboratories. 

Keywords: urinary sulfate metabolites, ion-paired extraction, measurement uncertainty 

Page 256

F5 

PAPAIN, A NOVEL URINE ADULTERANT 

David L. Burrows l ', M.S.; Andrea Nicolaides\B.S.; David A. Johnson 2 , Ph.D.; Michelle M. Duffourc\ 

Ph.D.; Kenneth E. Ferslew i , Ph.D., Section of Toxicologyl, Dept. of Phannacology',3; Dept. of 

Biochemistry and Molecular Biology2, James H. Quillen College of Medicine, East Tennessee State 

University, Johnson City, TN. 37614 

The estimated number of employees in the United States screened annually for illicit drugs is 

approximately 20 million, with marijuana being the most frequently abused drug. Urine adulterants 

provide an opportunity for illicit drug users to obtain a false negative result on commonly used primary 

drug screening methods such as the Fluorescence Polarized Immunoassay (FPIA) technique. Typical 

chemical adulterants such as nitrites are easily detected or render the urine specimen invalid as defined in 

the proposed SAMHSA guidelines for specimen validity testing based on creatinine, specific gravity and 

pH. 

Papain is a cysteine protease with intrinsic ester hydrolysis capability and several residues that serve as 

hydrophilic and hydrophobic binding sites that can act as a potential urine adulterant. These mechanisms 

would exists in a. novel class of urine adulterants and urine adulteration with hydrolytic enzymes can be 

attained with a relatively smaller quantity as compared to their typical chemical counterparts. The primary 

metabolite of the psychoactive chemical in marijuana, Il-norcarboxy-delta-9-tetrahydrocannibinol 

(I INC), was assayed by FPIA in concentrations ranging from 25 to 500 ng/mL, at pH values ranging from 

4.5 to 8, over the course of3 days with papain concentrations ranging from 0 to 10 mg/mL. FPIA analysis 

of other frequently abused drugs: amphetamines, barbiturates, benzodiazepines, cocaine, opiates, and 

phencyclidine, along with gas chromatography I mass spectrometry (GCIMS) of IINC and high 

perfonnance liquid chromatography I ultraviolet (HPLC/uV) of nordiazepam was perfonned in order to 

determine if the mechanism of urine adulteration by papain was analyte specific. Control and adulterated 

urine specimens (n=30) were assayed for creatinine, specific gravity, osmolarity and pH to detennine if 

papain rendered the specimens invalid based on the proposed SAMHSA guidelines. There was a direct 

pH, temperature, and time dependent correlate between the increase in papain concentration and the 

decrease in IINC concentration from the untreated control groups (p

---.,. 

F4 

IODINE CONTAINING ADULTERANT - 

DETECTION BY INTECT® 8 

ITS EFFECT ON RAPID DRUG SCREEN AND 

Beckie Chien*, Jan Sook, Raphael C. Wong, Branan Medical Corporation, Irvine, CA, USA 

Aims:New generation of commercial adulterants have included iodine as the main ingredient at sample 

concentration of about 1 mg per ml. The objective of this study was to evaluate iodine's effect on a certain 

rapid drug screen and its detection by an onsite adulterant dipstick, Intect®8. 

Method: The adulteration effect of iodine was studied by adding iodine to a urine sample containing three 

times the SAMHSA cut-off concentrations of five drugs including benzoyl ecgonine (COC), morphine 

(OPI), amphetamine (AMP), THC and PCP. The final concentration of iodine in the sample was 1 mg/mt. 

The adulterated sample was then tested by an onsite rapid drug screen Monitect® PCll over one hour 

period. The same sample was also tested with the new Intect 8 onsite adulteration test strip that contains g 

test pads including creatinine (CR), nitrite (NI), glutaraldehye CGL), pH, specific gravity (SG), bleach (BL), 

pyridinium chlorochromate (PCC) and iodine (I) to evaluate the adulterant's detection over time. 

Results: Negative (-) result in the drug screen indicates adulterant was able to modify the positive drug test 

result to negative result whereas (+1-) indicates a possible adulteration effect for that specific drug; normal 

(N) result in the Intect 8 pad suggests no effect of iodine on the specific test pad while abnormal (A) 

indicates detection ofthe adulterant by the specific pad. 

! Time (min.) COC OPI I AMP I THC PCP 

Drug 5 Invalid results due to no control line 

i 

Screen 10 + 

+ + I - 

+ 

Result 30 + + L + I - + 

60 + +1 I + I - + 

Time I· CR I Nl GL I ~H SG BL PCC L I 

I 

5 I N I N N N N A A I A 

Intect 8 

Result 

10 N : N N N N A A I 

I 

A 

30 N j N N N N A A I A 

60 I N 1 N N N N N N I N 

I 

Urine spiked with PCC or BL did not cause an abnormal result with the Iodine pad. 

Conclusion: Iodine containing adulterant is shown to mask the presence ofTHC. It also has some effect on 

opiate. This adulterant is detectable by an onsite adulteration test device Intect®g in a 30-minute window. 

Keywords: Adulteration, Intect, iodine 

Page 257

F6 

A COMPARATIVE EVALUATION OF THE INSTANT-VIEW 5 PANEL TEST CARD WITH 

ONTRAK TESTCUP PRO 5: COMPARISON TO GCIMS AND INSTRUMENT SCREENING 

WITH ONLINE REAGENTS 

David E. Moody'*, Wenfang B. Fangl, David M. Andrenyak ' , Kim S. Monti l , and Chuck Jones 2. 

ICenter for Human Toxicology, University of Utah, Salt Lake City, UT 84112, 2 Northwest Toxicology, 

Salt Lake City, UT 84124 

We have compared the ability of two on-site testing devices, Instant-View Test Card (Alfa Scientific 

Designs, Inc., Poway, CA) (I) and OnTrak TesTcup (Roche Diagnostics Corp., Indianapolis, IN) (T) to 

discriminate negative from positive urine samples for the following five drug groups: cannabinoids, 

cocaine metabolite, opiates, amphetamines and benzodiazepines. Samples for a precision study were 

prepared for each device separately due to cutoff and primary antigen differences. A drug-free urine pool 

was fortified in a random fashion to contain the primary antigen at seven concentrations (0, 2S, SO, 75, 12S, 

ISO and 17S% of cutoff). These stocks were than submitted for GCIMS confirmation and then 10 aJiquots 

from each where prepared and assigned random numbers of I-70 for each test device. All drug groups, 

except for cannabinoids were within 10% of target and target concentrations were used for the evaluation. 

Cannabinoids deviated more than IS% from target. The GCIMS-determined concentrations were used for 

cannabinoids; this resulted in samples at 0,62, 80, 102, 136, 158 and 170% of cutoff. Two individuals 

tested each sample. None of the °or 2S% (0 or 62% for cannabinoids) cutoffs tested positive with any 

device. The results for the rest of the samples can be summarized as follows where for each device we first 

list the % of below cutoff samples testing positive and the % of above cutoff samples testing negative: 

cannabinoids (1-3.3, 47.5; T-O, 40); benzoylecgonine (1-2S, 8.3; T-16.4, 0); morphine (I-2.5; 21.7; T -10,0); 

amphetamines (1-0, 60; T-22.S, 0) and benzodiazepines (I-8.8, \S; T-O, 21.7). Similar data were collected 

for an automated immunoassay using Online reagents; the only imprecise result was for cannabinoids at 

102% of cutoff where IS% of the samples tested negative. Samples submitted to NWT were used for the 

clinical study. Seventy-five samples that recently screened negative were rescreened for the five drug 

groups and the fifty with the lowest absorbance readings were selected as negative samples. Positive 

samples were selected from actual positives that had reached their disposal date. They were rescreened for 

all five drug groups. Those with results> the 75% control in the screening batch were then submitted to 

GCIMS confirmation for the particular drug group. Sufficient samples were screened and confirmed to get 

at least 45 samples above the device specific cutoff; with many samples near cutoff and some just below 

cutoff. As each on-site device will test for all five drug groups simultaneously, this provided::::: ISO 

additional samples that were presumptively negative per drug group. Samples were assigned random 

numbers, and two individuals tested each sample. Providing results as the percent of device positives when 

the GC/MS results (sum of analytes in many case) are

F7 

Direct Analysis of Opiates in Urine by Liquid ChromatographylMass Spectrometry! 

Mass Spectrometry 

Leslie E. Edinboro, MS, PhD, *,1,2 Ronald C. Backer, PhD. 3 & Alphonse Poklis, PhD.l 

1. Department of Pathology, School of Medicine, 2. Department of Pharmaceutics, School of Pharmacy, 

Virginia Commonwealth University, Richmond, Virginia, 3. AmeriTox Laboratory, Midland, Texas. 

A method for the direct analysis of ten opiate compounds in urine was developed using Liquid 

ChromatographylMass SpectrometrylMass Spectrometry (LCIMSIMS). Opiates included were: morphine- 

3-f3-glucuronide, morphine-6~~-glucuronide, morphine, oxymorphone, hydromorphone, norcodeine, 

codeine, oxycodone, 6-monoacetylmorphine (6MAM) and hydrocodone. Deuterated internal standards 

used were morphine-d 3 and 6MAM-~. Urine samples were prepared by addition of the internal standard 

solution, centrifugation to remove large particles and direct injection into the LCIMSIMS. Gradient reverse 

phase separation was accomplished on a phenyl column using acetonitrile and water modified with formic 

acid and ammonium formate. An electrospray ionization (ESI) interface was used to introduce the LC 

eluent into the MS. Multiple Reaction Monitoring (MRM) was utilized for detection in the MSIMS mode 

based upon pre-established precursor: product mJz transitions. Separation and detection of all compounds 

was accomplished within six minutes. Selectivity and possible ion suppression were evaluated using 10 

different blank urines. Linearity was established for all opiates except 6MAM from 50 nglmL to 10,000 

nglmL; 6MAM from 0.25 ng/mL to 50 ngimL. Correlation coefficients (r) for all opiates was > 0.99. Interrun 

precision (%CV) ranged from 1.1 % to 16.7%. Intra-run precision ranged from 1.3% to 16.3%. 

Accuracy (% Bias) ranged from -7.3% to 13.6% and -8.5% to 11.8 for inter and intra-run respectively. 89 

urine samples previously analyzed by GCIMS were re-analyzed by the LCIMSIMS method. The qualitative 

results found an 88% agreement for negative samples between the two methods and 94% for positive 

samples. The LCIMSIMS method identified 19 samples with additional opiates in the positive samples. 

Ten samples were oxymorphone positive that the GCIMS method was not setup to detect. Of these, 90% 

were associated with GCIMS positiYe oxycodone samples, which metabolically are justified. Additionally 

three samples were· hydromorphone positive for GCIMS hydrocodone positive samples, which also is 

likely. The remaining positive samples were oxymorphone, oxycodone, hydrocodone and hydromorphone. 

Quantitatively it was difficult to compare the two methods as the GCIMS method utilized a glucuronidase 

step that was not included in the LCIMSIMS method. Therefore only the morphine results could be 

compared if the LCIMSLMS results for the glucuronide metabolites were summed on a molar basis with the 

morphine. The R2 was 0.78. The difference between the methods is probably due to the efficiency of the 

GCIMS glucuronidase procedure. Overall the direct injection LCIMSIMS method performed well and 

permitted the rapid analysis « 6,0 minutes) of urine samples without extensive sample preparation for 

several opiates simultaneously. Additionally the method provided a lower limit of quantitation for 6MAM 

than previously reported utilizing direct injection techniques. The method could be further improved with 

the addition of compound specific deuterated internal standards and other glucuronide metabolites, i:e., 

hydromorphone. 

Keywords: Opiates, LCIMS/MS, Urine 

Page 260

F8 

UNUSUAL DRUG TEST RESULTS FROM KNOWN HEROIN USERS 

Neil Fortner*l , Doug Crook l , Dr. Robert Turk 2 , Andrew Waszyn 3 

iPharmChem Inc., Haltom City, TX, USA, 2Center for Toxicology Services, Inc, Humble, TX, USA 

,3United States Probation, Western District ofPennsylvania 

The use of total morphine levels in urine has traditionally been one approach to predicting the use of 

heroin. Recently we have encountered several situations that would suggest this practice should be revisited. 

Five individuals that were either on supervised release or on parole were tested for illicit drugs as a 

condition of their release. These individuals were comprised of two black males, one black female, one 

white male and one white female. The ages of these individuals ranged from 29 to 70 years, with an 

average age of 51 years. All individuals were experienced with heroin (either use or possession). Based on 

their history of drug use, these individuals were subject to periodic urine drug testing. These samples were 

screened for illicit drugs, including opiates, using reagents manufactured by Diagnostic Reagents, Inc. The 

screening cutoff level for opiates was 300 ng/ml. All samples screened negative for opiates. Due to their 

past experience with heroin, a special test for 6-AM was requested on each sample. All samples were 

positive for 6-AM with the concentrations ranging from 10 to 144 nglml. To investigate this apparent false 

negative immunoassay screen, all samples were analyzed for the presence of codeine, total morphine and 

heroin. All samples were negative for both codeine and heroin. All total morphine levels were less than 300 

nglml, which substantiated the negative immunoassay results. Total morphine levels ranged from 18 to 245 

nglmL These results suggest that the traditional approach for predicting heroin use based on total morphine 

levels need to be looked at more closely, as current practice may in fact underestimate instances of heroin 

use. 

Keywords: heroin, 6-acetylmorphine, morphine 

Page 261

F9 

ESTIMA TE OF DETECTION PERIOD IN URINE FOR MARKERS OF STREET HEROIN 

Sue Paterson*, Rosa Cordero, Simon Burlinson 

Toxicology Unit, Imperial College London, St Dunstan's Road, London W6 8RP, UK 

Nick Lintzeris 

National Addiction Centre, 4 Windsor Walk, London SE5, UK 

There is growing international interest in prescribed pharmaceutical heroin as treatment for individuals who 

are unable to stop injecting opiate drugs. The rationale for this treatment is one of "harm minimisation". In 

a previous paper (1) co-workers reported on the GC-MS analysis of over 1000 urine samples from patients 

attending a substance misuse service during which metabolites of various contaminants of street heroin 

were detected. They concluded that the detection of papaverine and noscapine metabolites was likely to 

provide a reasonably sensitive and highly specific means of identifying the illicit use of heroin in patients 

prescribed diamorphine. We believe that these markers offer the best means of determining the use of street 

heroin in these patients. The aim of this study was to estimate the detection period for identifying markers 

in the urine of street heroin users. Over 50 urine samples were collected from patients who admitted to the 

use of street heroin within the previous 72 hours. The researcher interviewed each subject regarding all 

episodes of drug use within the preceding 72 hours, including details of amount used, route of 

administration and time of use. The time the urine was passed was also noted. These samples were analysed 

by the method currently in use for the routine analysis of urine samples from patients attending drug 

treatment centres, that is mixed-mode solid-phase extraction (SPE) followed by GC-MS (2). Urine samples 

were subjected to enzymatic hydrolysis followed by SPE using Bakerbond narc-2 columns. The eluent was 

selectively derivatised with N-methyl-bis-tritluoroacetamide and N-methyl-N-trimethylsilyltrifluoroacetamide 

+ 1% trimethylchlorosilane. Analysis was performed using a GC-MS system operating in full 

scan mode. The study samples were analysed for the commonly abused drugs plus a range of other drugs 

including antidepressants and certain anti psychotics that are prescribed to these patients. In addition, the 

metabolites of papaverine, hydroxy and dihydroxypapaverine, and the metabolites of noscapine, meconine 

and hydroxymeconine, were looked for in each sample. Other contaminants of street heroin including 

papaverine, noscapine, thebaine, and acetylcodeine were also monitored. Using this assay the limit of 

detection for morphine was 0.05 ugimL. The limit of detection for hydroxy and dihydroxypapaverine could 

not be determined as pure standards were not commercially available. A characteristic peak pattern in the 

chromatogram was found to be indicative of the presence of the papaverine metabolites, which were then 

matched by retention time and spectra obtained from the elucidation of these metabolite derivatives as 

reported previously (1). Initial analysis of the data shows that hydroxy and dihydroxpapaverine were 

detected in urine for approximately the same period as morphine. This data supports the findings of 

McLachlan-Troup et al that hydroxy and dihydroxypapaverine can be used to determine if patients 

prescribed injectable diamorphine are "topping-up" with street heroin. Papaverine, noscapine, thebaine and 

acetyl codeine were not detected in any of the study samples. 

l. N, McLachlan-Troup, G.W. Taylor, B.c. Trathen. Diamorphine treatment for opiate dependence: 

putative markers of concomitant heroin misuse. Addiction Biology, 2001; 6: 223-231 

2. S Paterson, R Cordero, S McCulloch, P Houldsworth. Analysis of urine for drugs of abuse using mixedmode 

solid-phase extraction and gas chromatography/mass spectrometry. Ann. Ojefin. Biochem. 2000; 37: 

690-700. 

Keywords: Street heroin markers, papaverine metabolites, noscapine metabolites 

Page 262

FlO 

EFFECTIVENESS OF FREE AND TOTAL MORPHINE CONCENTRATION AS CRITERIA FOR 

SELECTING URINE SPECIMENS FOR TESTING 6-ACETYLMORPHINE 

3 4 

Sheng-Meng Wan~I.2, John W. Soper',2, Dennis Canfield 2 , and Ray H. Liu 2 . • , ICentral Police University, 

Taoyuan, Taiwan. Toxicology and Accident Research Laboratory, FAA Civil Aerospace Medical Institute, 

Oklahoma City, OK, U.S.A. 3Department of Medical Technology, Fooyin University, Kaohsiung Hsien, 

Taiwan. 4Department of Justice Sciences, University of Alabama at Birmingham, Birmingham, AL, 

U.S.A. *Presenting Author 

The U.S. Department of Health and Human Services' workplace urine drug testing program has adopted a 

2000 ng/mL total morphine concentration as the criterion for testing 6-acetylmorphine (6-AM). We were 

interested in evaluating whether such a criteria can be applied to specimens collected from workers in 

Taiwan, a genetically different group than the typical US worker. Two hundred and thirteen workplace 

specimens, testing positive for opiates by immunoassay (cutoff 300 ng/mL), were analyzed for 6-AM, and 

for free and total morphine, by GC-MS methods. Recovery efficiencies of extraction and hydrolysis 

protocols (for total morphine determination) were evaluated and used to derive analyte concentrations in 

test specimens. In reference to an earlier report by Paul et al [1], data hereby obtained were used to 

determine whether free or total morphine concentration is more effective in predicting the presence of 6 

AM in 2 different concentration ranges: >10 ng/mL and 5-10 ng/mL. As shown in Table 1,4 specimens 

were found to contain from 5-10 ng/mL of 6-AM, while 55 specimens were ~10 ng/mL. This total of 59 

specimens showed the presence of 6-AM at >5ng/ml, regardless of whether total morphine (2000 ng/mL) 

or free morphine (50,100, or 200 ng/mL) concentration was used as the cutoff for selecting specimens for 

6-AM determination. 

Table 1. Effectiveness of free and total morphine in predicting the presence of 6-AM 

,"~, 

Analyte concn No. of No. of specimen with 6-AM concn (ng/mL) 

(ng/mL) specimen >10 5.0-10 1.0-4.9 Negative" 

Total morphine 

~300 213 55 (25.8%)t 4 14 140 

~2000 162 55 (34.0%) 4 12 91 

~4000 142 55 (38.7%) 3 9 75 

Free morphine 

~50 181 55 (30.4%) 4 12 110 

~100 168 55 (32.7%) 4 12 97 

~200 154 55 (35.7%) 4 11 84 

" Limit of detection: 1 ng/mL. 

t Numbers inside parentheses are the percentages ofthe morphine positive (using the cutoff listed in the 

first column in respective rows) specimens that were found to contain ~1 0 ng/mL 6-AM. 

Data resulting from this study indicate comparable effectiveness in using either 2000 ng/mL total morphine 

or 100 ng/mL free morphine as the criterion for selecting specimens to further test for the presence of 6 

AM at the 10 or 5 ng/mL level. The free morphine option is less costly, avoids uncertainty associated with 

the hydrolysis process, and may produce more nearly accurate results. This study provides an addition to 

the free morphine-related database and adds further confidence to the use of free morphine data, which is 

not readily available. 

References: 

1. B.D. Paul, E.T. Shimomura, and M.L. Smith. A practical approach to determine cutoff concentrations 

for opiate testing with simultaneous detection of codeine, morphine, and 6-acetylmorphine in urine. 

C/in. Chern. 45: 510-519 (1999). 

Keywords: Heroin, free-Morphine, 6-mono-acetyl Morphine 

Page 263 

1

Fll 

REVIEW OF AN UNUSUAL MULTI-DRUG POSITIVE USAF MEMBER CASE 

Vincent M.Papa l *, Sarah Ozmene, Timothy Cox 2 , Terence FarreU 2 , Aaron Jacobs 3 

I Air Force Drug Testing Laboratory Brooks City-Base, Texas 78235; 2 1 FW/JA LangeJy AFB, Virginia 

23655; 3 Division of Forensic Toxicology, Armed Forces Institute of Pathology, Division of Forensic 

Toxicology, Rockville, MD 20850 

The Air Force Drug Testing Laboratory (AFDTL) tests approximately 400,000 military specimens/year for 

THC, cocaine, amphetamines, PCP, opiates, barbiturates and LSD. Only 88-90 member specimens are 

positive for multiple drugs. A case report on specimens positive for mUltiple drugs may have a complex 

interpretation. The member underwent on consent urinalysis on late Friday evening/Saturday morning. The 

week preceding this positive result, the individual member had an occupational accident where the cleaner 

" Googone " was sprayed into his eyes. He was prescribed Tylox for pain resulting from ocular damage. 

On the night preceding the drug test, a call was made to 911 after his girlfriend found him unresponsive. 

EMTs noted dilated pupils and that he was not cooperative. He was transported exhibiting combative and 

belligerent behavior to a local hospital. At the hospital, the member responded to questions but had lapses 

in memory. A drug and alcohol screen was performed at the hospital. The alcohol screen was negative but 

the drug screen was positive for cocaine and THC as benzoylecognine and I I-nor carboxy THC. A consent 

urinalysis was obtained and analyzed at the Armed Forces Institute of Pathology (AFIP). Analysis for THC, 

cocaine, amphetamines, PCP, opiates and barbiturates were performed by Axsym screen and GCMS 

confirmation. Medical history indicated that he had ingested psilocin mushrooms so an analysis for 

psilocyn was accomplished using a gas chromatography (GC) screen and GCMS confirmation. The 

following drugs were detected and quantified: 

1) Benzoylecognine 3148 ng/ml 

2)MDMA 

5947 ng/ml 

3) Psilocin 6450 ng/ml 

4) 11- nor carboxy THC 135 ng/ml 

The defense asserted that the individual was not capable of giving consent due to the number of drugs 

detected and the fact that he was taking Tylox and drinking heaVily. There were 15 pills missing. His 

girlfriend reported that he was dinking beer and mixed drinks. Additional analysis at AFIP revealed the 

presence of 165 ng/ml urinary oxycodone. Psychiatric analysis was performed for competency to consent. 

At trial, the military judge ruled the member mentally capable. Investigation revealed that the member the 

evening of the hospital admission consumed 4-5 caps of psilocyn mushrooms, one MDMA tablet and 

smoked a marijuana blunt laced with cocaine. In this case, psilocyn results were approximately 6X higher 

than the levels reported by Sticht et.aI.(I). Furthermore, there are reports of smoking marijuana blunts that 

have elevated THC percentages. Aggressive and belligerent behavior exhibited in the above report is 

consistent with synergistic effects of multiple drug consumptions ofhallucinogens and stimulants. 

l.G.Sticht, H.Kaferstein" Detection ofpsilocin in body fluids, Forensic Science International 113 (2000) 

403-407. 

Keywords: psilocin, marijuana, cocaine, mdma 

Page 264

F12 

TITLE: AMPHETAMINE CONCENTRATIONS IN URINE AFTER THE USE OF DEXEDRINE 

"GO-PILLS": COMPARISON OF SINGLE AND DOUBLE DOSES OF D-AMPHETAMINE. 

P.L. Mobley*, S.H. Constable, R.D. Vanderbeek, T.A. Frazier, C.S. Ramsey, D. Wheeler 

Presenting Author: Philip L. Mobley, Ph.D. Business Address: Air Force Drug Testing Laboratory, 

AFIOH/SDT, 2730 Louis Bauer Drive, Brooks AFB, TX 78235 

The Air Force, under the close supervision of flight surgeons, approves the use of amphetamine for both 

active duty and reserve Air Force aviators to counter performance problems during long military missions 

associated with fatigue and sleep deprivation. These pilots may also be subjected to urine drug tests, which 

include testing for amphetamines by DoD. Studies were conducted to determine the window of detection 

for amphetamine after the administration of Dexedrine ("Go-Pills") at two approved doses. Volunteers 

(22-58 years, mean=38.7 years) were administered a single IOmg dose of d-amphetamine (N=31), or two 

10mg doses four hours apart (N=31). A urine specimen was collected from each subject just prior to drug 

administration and collected ad libitum thereafter for several days. Urine was screened using the KIMS 

immunoassay. GCIMS was performed on specimens from each subject until the amphetamine 

concentration fell consistently below 350ng/ml in the screen test. Based on GC/MS analysis, peak urine 

concentrations of amphetamine ranged from 602 ng/ml to 12,191 ng/ml with a mean of 3345 ng/ml in the 

single-dose study, and from 1,339 ng/mJ to 15,359 ng/ml with a mean of 6076 ng/ml in the two-dose study. 

The time at which the highest (peak) concentration was observed ranged from 2.6 to 29.6 hours with a 

mean of 12.3 hours (sd=6.3) in the single dose study, and from 4.0 to 26.5 hours with a mean of 13.8 hours 

(sd=6.7) in the two-dose study. The time to the last identified positive (>500 ng/ml) in each subject ranged 

from 2.6 to 46.7 hours with a mean of 31.3 hours (sd=9.7) in the single-dose study, and from 29.1 to 71.5 

hours with a mean of 44.8 hours (sd=9.3) in the two-dose study. The excretion rate half-life of 

amphetamine in the urine was approximately 12 to 13 hours in both test groups. These studies indicate that 

most pilots should be below the DoD cutoff of 500 ng/mJ within 48 hours after a single 10 mg dose, and 

within 72 hours after the 2xlO mg dose. 

Key Words: Amphetamine, Urine, Go-Pills 

Page 265

F13 

WORKPLACE URINE OPIATE TESTING: A CASE OF SCIENTIFIC INJUSTICE IN THE U.K. 

William Westenbrink*, Forensic Alliance Limited, Abingdon, Oxon, U.K. 

A successful professional accepts a supervisory nursing position, provisionally, pending a negative urine 

drug test. She advises that she may have taken paracetamol within two weeks prior to the testing. 

Unexpectedly, the subject's urine, Sample A, is found to contain a 'total' morphine concentration of 403 

ngimL. The employer, based on this 'positive' test, refuses her the position, and the company's medical 

review officer will hear no explanation whatsoever. Five months later the certificate with the test result of 

Sample A and the second urine sample, Sample B, are released to the donor. Independent analyses indicate 

'free' morphine of 27 nglmL, 'total' of 687 ng/mL and the presence of thebaine in this second sample. No 

codeine is detected. The total morphine values exceed the U.K. morphine limit of 300 ng/mL, yet both 

sample results are below the U.S. and U.S. Military limits of 2,000 ng/mL and 4,000 ng/mL respectively. 

As will be discussed, selected requirements within the U.K. Guidelines for Legally Determined Workplace 

Drug Testing were not followed. Violations include: the lack oftemperature measurements, witnessing the 

original sealing of the samples followed by the samples being subsequently unsealed, repackaged, and 

resealed with the original seals, no hand washing between various samples being handled, no disclosure of 

original results, no timed release of Sample B, nor the availability of declaring a 'dietary source' on the 

disclosure forms, amongst others. No interpretations were carried out with consultation between the 

medical review officer with the original toxicologist and the general medical practitioner of the donor, 

concerning the 'positive' result, as required. In fact, the donor was originally verbally told that 'codeine' 

was present in her urine via telephone. 

The source of morphine was the consumption of 'Warburton's Seeded Batch Bread' containing 

approximately 0.5 grams of poppy seeds per slice. The donor had eaten this bread during the four days 

prior to the drug testing date and on many other previous occasions. Having provided the employer with 

this information, the company refuses to overturn its original decisions: that the donor was positive for 

morphine without reasonable excuse nor place her in the position for which she had been successful, 

despite having dismissed the original medical review officer. The donor remains unable to secure suitable 

professional employment in her field because on all subsequent employment applications she must indicate 

that she has been refused employment as a result of a positive urine drug test. Each time such violations 

occur in drug testing at the workplace programs, insurmountable injustices are placed on the victims, as this 

cases demonstrates. 

Keywords: workplace, drug-testing, morphine 

Page 266

F14 

AUTOMATED APPROACH TO NON-NEGATIVE SPECIMEN LIST (NNSL) PRODUCTION 

AND APPLICATION FOR SAMHSA-CERTIFIED LABORATORIES. 

William N. Bennett and Anthony Wu* - ACL Laboratories, West Allis, WI. 

SAMHSA-certified laboratories are required to produce a Non-Negative Specimen List (NNSL) for the 

National Laboratory Certification Program (NLCP) on a semi-annual basis. This list must be submitted to 

the NLCP staff Research Triangle Institute (RTI) on a specifically formatted Excel spreadsheet. From the 

list, NLCP staff select those specimens whose documentation are to be reviewed by inspectors at the next 

scheduled on-site inspection. For ease of review, as mandated by the inspection guidelines, these 

documents must be organized in a manner that reflects the selected NNSL. To decrease the time needed to 

compile this information, we have designed an approach to automate this organization process. Our 

application of this approach uses SQL and Turbo Pascal computer languages and the spreadsheet Excel, 

with Commercial Lab as the Laboratory Information Management System (LIMS). First, SQL queries are 

run which produce text files from resident databases on the LIMS. Second, the text files are moved to a PC 

and imported into pre-formatted Excel worksheets, which are then forwarded to RTI. Third, the files are 

refined using a Turbo Pascal program and imported into Excel calendar worksheets. Calendar entries 

include: Access number, Batch number, CCF number and the Non-negative test for which the sample was 

targeted. The listings on the calendar (placed on boxes containing the sample documentation), in 

conjunction with the selected NNSL, organize the specimen documentation for easy retrieval and review 

by the inspectors. Inspectors to our laboratory have praised this organizational scheme. As pre-inspection 

guidelines become increasingly time consuming, this approach may be useful for other SAMSHA-certified 

laboratories. 

Keywords: NNSL, SAMHSA, Non-negative 

Page 267

F15 

METHOD VALIDATION FOR THE ANALYSIS OF AMPHETAMINE, METHAMPHETAMINE, 

MDAAND MDMA IN URINE 

Mee-lung Park, Sang-Kit Choi, Eun-Mi Kim, Mi-Ae Lim*, Myungyoon PyOI), Hee-Sun Chung 

Division ofNarcotic Analysis, National Institute of Scientific Investigation, Seoul, Korea 

I)The school of Pharmacy, Sookmyung Women's University, Seoul, Korea 

The purpose of this study is to provide the standard method for the. assay of stimulants in urine, especially 

ATS (amphetamine type stimulants; amphetamine (AM), methamphetamine (MA), 

methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA». We performed 

method validation for these drugs in urine according to EURACHEM Guide (A laboratory guide to method 

validation). 

Analytical method for AM, MA, MDA and MDMA in urine was as follows. For the analysis of 

amphetamines in urine. immunoassay (fluorescence polarization immunoassay, TDxFLx) was used for 

screening test and confirmation test was performed with GC/MS. After the analysis by immunoassay, the 

positive specimens were alkalized with 6 M-NaOH and the analytes were extracted with ethyl acetate. After 

centrifugation of specimens, the supernatants were evaporated to dryness under the nitrogen stream at 450 

with vacuum. The residues were derivatized with pentafluoropropionic anhydride (PFPA). They were 

analyzed by gas chromatography/mass spectrometry (GC/MS). Ds-deuterated amphetamines were used as 

internal standards. To validate this method, selectivity, linearity of calibration, within- and between-run 

reproducibility (precision), accuracy, limit ofdetection and quantification were studied. 

As a result, the calibration curves were ranged from 100 to 8,000 ng/ml with the correlation coefficients of 

greater than 0.9. Within- and between-run precisions were measured in three different concentrations (250 

ng/ml, 500 ng/ml, 1,000 ng/ml). Coefficients ofvariance (CV %) of their precisions were under 10 % at all 

concentrations. Those accuracies (% bias) were also under 10 %. The limit ofdetection (LOD) and limit of 

quantification (LOQ) for all analytes studied were 20 ng and 100 ng, respectively. The selectivity of 

amphetamines in urine was observed by spiking high concentrations of those, which have similar chemical 

structures such as ephedrine, pseudoephedrine and norephedrine into low QC urine samples (500 ng). 

Those chemicals did not show any inferences in the analysis ofATS in urine by this method. 

It was concluded that validation data by Eurachem guide was proved to be adequate for the analysis of 

amphetamine, methamphetamine, MDA and MDMA in urine Samples .. 

Keywords; method validation, ATS (amphetamine type stimulants), urinalysis 

Page 268

F16 

URINARY EXCRETION OF MORPHINE AND CODEINE FOLLOWING THE 

ADMINISTRA TION OF SINGLE- AND MULTIPLE-DOSE OF BROWN MIXTURE 

Dong-Liang Lin,I,2, Hsiu-Chuan LiU*,1,2, Hsiu-O H02, Chung-Yi Wang l and Ray H, Liu 1 ,3,4, IInstitute of 

Forensic Medicine, Ministry ofJustice, Taipei, Taiwan. 2Taipei Medical University, Taipei, Taiwan. 3Fooyin 

University, Kaohsiung Hsien, Taiwan. 4Graduate Program in Forensic Science, University of Alabama at 

Birmingham, Birmingham, Alabama 

Parallel to the "poppy-seed defense" strategy commonly reported in the United States, donors of urine 

samples tested positive for opiates in Taiwan often claimed the consumption of Brown Mixture (BM) as the 

source of the observed morphine and codeine. Since BM contains opium powder (10-10.5% morphine), 

opium tincture (0.9-1.1% morphine), or camphorated opium tincture (0,045-0.055% morphine) and is a 

popular remedy, while heroin use is considered a serious criminal act, the claim of BM use has to be 

adequately addressed. 

In this study, BM from 7 different manufacturers (5 tablets and 2 solutions) and urine samples from patient 

and alleged heroin users were analyzed for their morphine and codeine contents. The analytical procedure 

included hydrolysis, trimethylsilylation, and monitoring of the following ions designated for 

TMS-derivatized codeine, morphine, and nalorphine (internal standard): mh 371,356,343; 429, 414, 401; 

455,440,414, respectively. The first ion listed for each compound was used for quantitation using a six-point 

calibration protoco I (50-2000 ng/mL). 

The contents of morphine and codeine and their ratios ([M]/[C]) in: (a) BM tablets (n =5), and BM solution 

(n = 2) available in Taiwan and (b) urine specimens collected from alleged heroin users and patients (n 7) 

ingesting 1-6 tablets (or 5-20 mL solution) -- one-time single dose or three times per day for two days -- are 

summarized in Table I. The contents of morphine and codeine in the tablets are very consistent, but vary 

considerably in the 2 BM solution. Morphine concentrations found in urine specimens collected from 

patients ingesting BM tablets or solution, are always 5.0) for patients ingesting BM 

tablets and alleged heroin users. It appears that (a) BM ingestion (tablet or solution) is unlikely to result in 

morphine concentration >4000 ng/mL; and (b) [M]/[C] ratio may not be an effective parameter for the 

differentiation ofBM tablet ingestion and heroin use. 

Table 1. Morphine and codeine contents in Brown Mixture tablet, Brown Mixture solution, and urine 

specimens collected from alleged heroin users and patients ingesting Brown Mixture 

Brown Mixture 

Urine specimen o collected users of 

[M]/[C] Tablet Solution Tablet Solution Heroin h 

N 25 10 56 68 90 

Range 8.46-9.18 2.56-2.93 4.88-14.8 1.31-9.09 3.15-40.8 

Mean 8.77 2.74 7.76 2.62 8.52 

Std dev. 0.19 0.15 1.92 0.87 4.03 

°Only specimens with morphine concentration higher than 300 nglmL are included. 

b Alleged heroin users. 

Key words: Brown mixture, morphine, codeine 

Page 269

F17 

A MODIFIED METHOD FOR THE LIQUID-LIQUID EXTRACTION AND GCIMS ANALYSIS 

OF METHADONE FROM HUMAN URINE IN A CAP-FUDT CERTIFIED DRUG TESTING 

LABORATORY 

Jeffrey Lavelle, Brian Brunelli, Edward A'Zary*, and Judy Keller. Quest Diagnostics Incorporated, 

3175 Presidential Drive, Atlanta, Georgia 30340 

In order to produce forensically accurate and reproducible results in a competitive, cost-effective, and time 

conserving manner, we took an existing method (1,2) for the extraction of methadone from urine and made 

several significant modifications. The internal standard was changed from phenyltoloxamine to methadoned9. 

The extraction method was changed from a solid phaselback extraction to a liquid/liquid extraction. 

Four organic solvents were tested: 

Solvent A comprised of(35:15:40: 1 0) toluene:ethyl acetate:hexane:methanol. 

Solvent B comprised of(25:25:50) toluene:ethyl acetate:hexane. 

Solvent C comprised of(10:90) toluene:hexane. 

Solvent D comprised of (5:95) isoamyl alcohol:hexane. 

All solvents were evaluated in a liquid-liquid extraction procedure for quantitation ion areas 72 m/z and}8 

mlz with calibrators spiked at 80 nglmL,. 200 ng/mL, and 2000 nglmL. Area recovery studies showed tnat 

solvent B was the most desirable of the four extraction solvents. Solvent A was eliminated because it had 

the second lowest correlation coefficient (r2 ==0.999985), and because it contained methanol, which could 

cause emulsions to form. Solvent C was eliminated because it had the lowest recovery of internal standard 

and the largest variation in internal standard recovery. Finally, solvent D was eliminated because it had the 

lowest correlation coefficient (r=0.999929) and the second largest variation in internal standard recovery. 

Linearity studies with our previous extraction method produced an LOD (Limit of Detection) of 50ng/mL, 

LOQ (Limit of Quantitation) of lOOng/mL and an ULOL (Upper Limit of Linearity) of2,OOOnglmL. The 

new extraction method allowed this laboratory to expand our ULOL from 2,000ng/mL to 10,OOOnglmL 

allowing for an accurate quantitation of high concentrated samples on a first time run without prior dilution. 

This method modification reduced the rescheduled rate from 27% to 9%. 

1. Clean Screen Basic Drug Procedure, Worldwide Monitoring 

2. Taylor, RS,Greutink, C. and Jain, NC "Identification of Underivatized Basic Drugs in Urine by Capillary 

Column Gas Chromatography". Journal ofAnalytical Toxicology, 10:205-208 (1986). 

Keywords: Methadone, LiquidILiquid, GCIMS 

Page 270

FI8 

A RAPID LCIMS METHOD FOR THE DETERMINATION OF 

METHAMPHETAMINEIDIMETHYLAMPHETAMINE AND THEIR METABOLITES IN URINE 

A STUDY OFTHE CURRENT SITUATION IN HONG KONG 

WC Cheng*, Vincent KK MOK, Forensic Science Division, Government Laboratory, Homantin 

Government Offices, Hong Kong Special Administrative Region, People's Republic of China 

Methamphetamine (MA) abuse, both in high purity "Ice" form or in tablets, continues to be one the popular 

drugs of abuse found in Hong Kong. Recently, some of drug seizures in crystalline or tablets received by our 

laboratory were found to contain N,N-dimethylamphetamine (DMA), a MA analog. The crystalline samples 

with DMA found were either in its pure form or mixed with MA. For tablets containing DMA, they were 

physically similar to those Ecstasy tablets, and the compositions were complex and usually mixed with MA, 

MDMA, ketamine and/or other drugs. As it is difficult to distinguish physically between DMA and MA, 

DMA can be mistakenly sold as MA. 

lt has been reported that DMA, N,N-dimethylamphetamine N-oxide (DMANO), MA, and amphetamine (A) 

were excreted in human urine after administration of DMA. As DMANO, one of the main and specific 

metabolite ofDMA, is thermally labile; the use of LCIMS would allow the simultaneous detection ofall four 

analytes without derivatization possible. In order to have a clear picture of the current state of MAIDMA 

abuse in Hong Kong, a rapid and sensitive LCIMS method has been successfully developed for the 

urinalysis of suspected drug abusers under the drug use surveillance program. LC/MS analysis was 

performed on a Finnigan Surveyor LC connected to a Finnigan LCQ Advantage ion trap mass spectrometer. 

Using solid phase extraction of urine with Alltech CI8 columns followed by LCIMS analysis fitted with an 

Alltech Rocket Platinum EPS C-18 column and using a mobile phase (O.OIM ammonium formate (pH=3): 

acetonitrile =77:23) at a flow rate of 0.2 ml/min, the simultaneous identification and quantitation of MA, 

DMA and their metabolites could be achieved within 5 min. Based on the urinalysis results, an overview of 

current MAIDMA abuse will be described. 

Keywords: N,N-dimethylamphetamine, LCIMS, urinalysis 

Page 271

F19 

CONFIRMATION RATES OF INITIAL DRUG ASSAYS IN A GROUP OF HHS-CERTIFIED 

LABORATORIES, JANUARY 01 THROUGH DECEMBER 31, 2003 

I: FEDERALLY REGULATED SPECIMENS 

Donna M. Bush '*, Michael R. Baylor 2, John Irving2, John M. Mitchelf, Craig A. Sutheime~: lDivision of 

Workplace Programs, CSAP, SAMHSA, Rockville, MD, USA; 2RTI International, Research Triangle Park, 

NC, USA 

As the U.S. Department ofHealth and Human Services (HHS) moved to expand the analytical methods, the 

approaches to drug detection, and the biological matrices allowed as specimens in the workplace drug 

testing program for Federal employees, an in-depth analysis ofcurrent practices was initiated. Of particular 

interest was the specificity and cross-reactivity of the immunoassays currently found in HHS-certified 

laboratories. The specificity of the immunoassays associated with urine drug testing has long been a 

subject of discussion among forensic toxicologists. While it has been known that some drug class 

immunoassays have very high rates for the confirmation of presumptive positives, it is also recognized that 

other drug class immunoassays produce a significant number of presumptive positives that fail to confirm 

when subjected to confirmatory testing by GCIMS. These observations led to an examination of the 

immunoassays currently in use with the goal of documenting the possible differences in specificities and 

cross-reactivities of the technologies . 

. The study included data from 11 HHS-certified laboratories encompassing nearly 4 million specimens 

tested under Federal mandate during 2003. These specimens represented between 55 to 60% ofall federally 

regulated specimens tested in accordance with the Mandatory Guidelines for Federal Workplace Drug 

Testing Programs (59 Fed. Reg. 29908-29931, June 9, 1994 and 63 Fed. Reg. 63483-63484, November 13, 

1998) during 2003. The data were obtained from laboratories that used CEDIA, EIA and KIMS 

technologies as a primary initial test. Some laboratories conducted additional screening of presumptive 

positives with FPIA as a second initial test. Summaries of specimen testing and confirmation rates are 

presented in the tables below. The confirmation rates are expressed as percent ofthe presumptive positives 

confirmed by GCIMS for each drug class. The mean, lowest and highest laboratory confirmation rate for 

each drug class are also provided. 

I Amphetamines BZE Opiates PCP THC-COOH 

I Specimens Tested 3,939,614 3,946,445 3,937,611 3,937,611 3,946,445 

i Presumptive Positives 21,577 23,570 21,586 1,772 54,578 

I Confirmed Positives 11,715 22,920 6,550 1,229 48,458 

IInitial Test Assay 

• Confirmation Rates 

Amphetamines 

{lstl2 nd Test) 

BZE Opiates PCP THC-COOH. 

: Mean Rate 51.9%/82.8% 98.1% 30.2% 69.7% 91.0% 

I Lowest Rate 37.40/0/81.3% 91.1% 17.3% 51.6% 73.0% 

I Highest Rate 77.8%/84.3% 99.9% 55.9% 91.0% 98.8% 

This study evaluated the presumptive positive rates and the confirmation rates for primary initial tests by 

immunoassay method as well as paired immunoassay methods (primary initial test plus second initial test). 

The results were examined with consideration of assay cross-reactivity and specificity. As expected, some 

assays and technologies appear to better identify specimens containing analytes of interest at or above the 

administrative cutoffs required by the Mandatory Guidelines for Federal Workplace Drug Testing 

Programs. While the study assesses current capabilities ofexisting technologies from a large population of 

"real" federally regulated workplace specimens, it also provides information that may be useful in 

formulating future guidelines by which newer technologies and approaches may be evaluated. 

Keywords: HHS-certified laboratories, Immunoassay confirmation rates, calendar year 2003 

Page 272

F20 

CONFIRMATION RATES OF INITIAL DRUG ASSAYS IN A GROUP OF HHS-CERTIFIED 

LABORATORIES, JANUARY 01 THROUGH DECEMBER 31, 2003 

II: NON-REGULATED SPECIMENS 

Craig A. Sutheimer l *, Michael R. Baylor I, John Irvingl, John M. Mitchell\ Donna M. Bush 2 : IRTI 

International, Research Triangle Park, NC, USA; 2DWP, SAMHSA, Rockville, MD, USA 

The specificity of immunoassays associated with urine drug testing has long been a subject of discussion among 

forensic toxicologists. While it has been known that some drug class immunoassays have very high rates for the 

confirmation of presumptive positives, it is also recognized that other drug class immunoassays produce a 

significant number of presumptive positives that fail to confirm when subjected to confirmatory testing by 

GCJMS. These observations are further confounded as initial and confirmatory drug test cutoff concentrations 

change and as the number of drug analytes in confirmatory panels are broadened. These observations led to an 

examination of the immunoassays currently in use at drug testing laboratories. The goal of this study was to 

document the possible differences in specificities and cross-reactivities of the technologies with multiple cutoffs 

in both the initial and confirmation testing procedures in addition to possible variability in the analytes defined in 

the confirmatory panels. 

The study included data from 10 laboratories certified by the U. S. Department of Health and Human Services 

(HHS) encompassing over 10 million specimens tested during 2003. These specimens were not subject to the 

criteria of the Mandatory Guidelines for Federal Workplace Drug Testing Programs. The data was obtained from 

laboratories that used CEDI A, EIA and KIMS technologies as a primary initial test, with varying initial and 

confirmatory test cutoffs. Some laboratories conducted additional screening of presumptive positives with a 

second initial test. Summaries of specimen testing and confirmation rates are presented in the tables below. The 

confirmation rates are expressed as percent of the presumptive positives confirmed by GC/MS for each drug 

class. The lowest and highest confirmation rate for each "initial/confirmatory testing cutoff pair" identified is 

provided. 

Amohetamines BZE 

Specimens Tested 10 142,363 10,136424 

Presumptive Positives 101,653 97,891 

Confirmed Positives I 57,283 

96,283 

Opiates PCP 

10,117,626 10 006,492 

103,081 5,838 

36,821 3,389 

I 

THC-COOH 

10,188,976 i 

347,905 I 

326,039 1 

T Barbs I Benzos Methadone MQL Propoxyphene 

. Specimens Tested T 4,585,890 I 4,271,398 3,943430 2,504,097 3,852,794 

Presumptive Positives I 15,281 47,639 10,305 56 28,188 

Confirmed Positives T 13,729 L 28,260 9,248 4 26,045 

I Initial Test Assay 

: Confirmation Rates 

Amphetamines BZE Opiates PCP I THC-COOH 

i Lowest Rate 3.4% 80.5% 25.0% 59.0% I 60.8% 

I Highest Rate 97.2% 99.3% 71.8% 96.4% l 100.0% 

I 

Initial Test Assay Barbs Benzos Methadone MQL ! Propoxyphene 

I Confirmation Rates 

- 

i Lowest Rate 76.1% 45.9% 69.8% NA 22.7% 

I 

I Highest Rate i 100.0% 83.7% 100.0% 8.3% 100.0~ 

This study evaluated the presumptive positive rates and the confirmation rates for primary initial tests by 

immunoassay method. The results w~re examined with consideration of assay cross-reactivity and specificity. 

As expected, some assays and technologies appear to better identifY specimens containing analytes of interest 

above the defined cutoffs. While the study assesses current capabilities of existing technologies from a large 

population of "real" specimens, it also provides information that may be useful in formulating future guidelines 

by which newer technologies, additional drug classes and additional analytes may be evaluated. 

Keywords: non-Regulated testing, HHS-certified laboratories, Immunoassay confirmation rates, Calendar Year 

2003 

Page 273

F21 

DETERMINA TION OF BENZODIAZEPINES IN HUMAN URINE USING SOLID-PHASE 

EXTRACTION AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

ELECTROSPRA Y IONISATION TANDEM MASS SPECTROMETRY 

S Hegstad*, E L 0iestad, U Johansen and A S Christophersen 

Division of Forensic Toxicology and Drug abuse, Norwegian Institute of Public Health, P.O. Box 4404 

Nydalen, NO-0403 Oslo. 

Aims: A liquid chromatography-tandem mass spectrometry (LCIMSIMS) method has been developed and 

validated for the determination of benzodiazepines registered in Norway and/or their metabolites in human 

urine. These compounds are frequently associated with misuse. The following compounds are included: 7 

aminonitrazepam, 7-aminoc)onazepam, 7-aminoflunitrazepam, alprazol am, alphahydroxyalprazolam, 

oxazepam, 3-0H-diazepam and N-desmethyldiazepam. The method was evaluated by analysing urine 

specimens from prison inmates, suspected of drug abuse. 

Methods: Urine samples (0.5 ml) were hydrolysed with l3-glucuronidase (from Patella vulgata) at 60°C for 

2 hours before solid-phase extraction with a polymer-based mixed-mode column (Oasis MCX). 

Chromatographic separation of extracts was achieved using a Waters Symmetry CI8 (2.IXlOO mm, 3.5 

/-1m) column with a flow rate at 0.3 mllmin with gradient elution. The analyses were performed on a Waters 

Alliance 2695 system in combination with a Waters Quattro Ultima Pt tandem-quadropole mass 

spectrometer equipped with a Z-spray electrospray interface. Positive ionization was performed in the 

MRM (multiple reaction monitoring) mode. Two transitions were monitored for the analytes, and one for 

the internal standards. Deuterated analogues were used as internal standard for all analytes except for 7 

aminonitrazepam and alphahydroxyalprazolam, which were quantified using 7-aminoclonazepam-d4 and 

alprazolam-d5, respectively. 

Results: The concentration range was 0.1-8.0 /-1M for 7-aminonitrazepam, 7-aminoclonazepam, 7 

aminoflunitrazepam, alprazolam, alphahydroxyalprazolam and 0.5-40 /-1M for the other compounds. The 

average recovery of the analytes ranged from 56-83 %. The between-day relative standard derivation of the 

method ranged from 3 -12 %. The limits ofquantification were found to be between 0.002 and 0.01 /-1M. 

Conclusion: The LC/MSIMS method proved to be robust and specific for the determination of 

benzodiazepines in urine. The method developed offers significant efficiency advantages in our routine 

laboratory replacing two chromatographic methods, which involved time-consuming derivatisation 

techniques. (HPLC-fluorescence, GC-MS). In addition, inclusion of new benzodiazepines is more 

convenient due to high specificity and flexibility ofthe new method. 

Keywords: Benzodiazepines, urine, LC/MS/MS 

Page 274

F22 

USE OF COMPOUNDS ALTERING VIGILANCE PERFORMANCE: PRELIMINARY RESULTS 

OF PREVALENCE IN HAULAGE DRIVERS IN THE NORD-PAS-DE-CALAIS REGION 

(FRANCE) 

L. Labatl, B. Dehon\ M. Lhermitte l ' and regional group of «Toxicomanie et Travail »2 

I Laboratoire de Biochimie et Biologie Moleculaire - H6pital Calmette - Avenue du Pr Leclercq - 59037 

Lille cedex - France; 2 Institut de Sante au Travail du Nord de la France A venue Oscar Lambret - 59037 

Lille cedex - France. 

Introduction: In 1995, a French study showed that the number of users of compounds that could alter 

vigilance performance was significantly more important in a population of workers in security posts than in 

the general working population (1). Moreover, even if the number of road accidents involving haulage 

drivers has decreased since 1985, their severity is greater. A multicentric study was initiated by a local 

group «Toxicomanie et Travail }) on haulage drivers from different cities in the Nord-Pas-de-Calais region 

(France). The objectives of this study are the standardisation of occupational medicine practices in order to 

define a harmonized policy for prevention and screening, to validate analytical screening methods, as well 

as the course of action in the case of positive detection. 

Methods: 1000 haulage drivers were included in the study. Occupation was the only criterion of selection. 

Urine samples were collected anonymously. Information collected included: haulage area, age, gender, 

nature of appointment with occupational practitioners and day of the sampling. A urine screen (test 

TriageS, BMD) was performed for methadone (cut off 300 ng/mL), benzodiazepines (300 ng/mL), 

cocaine (300 ng/mL), amphetamine derivatives (1000 ng/mL), opioides (300 ng/mL) and cannabis (50 

ng/mL). Buprenorphine detection was achieved with Elisa method (Microgenics). All positive results were 

confirmed by GC-MS or HPLC-MS. Alcohol levels were determined by an enzymatic method (Dade 

Behring). 

Results: Up to February 2004, 707 samples were collected. 99% are men and participants were on average 

36.4 +/- 9.9 years old. Results expressed as percentage of positives were: opioides (4.SI%), cannabis 

(9.05%), cocaine (0.14%), amphetamine derivatives (0%), buprenorphine (1.98%), methadone (0.56%), 

benzodiazepines (0.42%), alcohol (5.51%). The 34 positive cases for opioides were distributed as follows: 

6-MAM 0; morphine 10; codeine 11 ; pholcodine 20. THC-COOH was the only compound identified in 

urine samples from individuals tested positive for cannabis. 

Conclusion: Only legal opioid drugs were identified. They correspond to antitussive medications that are 

broadly prescribed during the winter period (time of the urine collection). The lack of sensitivity and 

specificity of the immunologic test could explain the low percentage observed for benzodiazepine positive 

cases. Our results are in good agreement with the results ofthe previously mentioned study of Haguenoer et 

aI., (1995) in the North of France, for the use of cannabis and alcohol by workers in security posts. In 

contrast, the number of positive cases for amphetamine derivatives, opioides and benzodiazepines was less. 

Other molecules were not investigated in the previous study of Haguenoer et at.. Considering these 

preliminary results, it would be of great interest to extend the screening to other psychoactive molecules by 

using a more efficient screening method such as HPLC-DAD. 

References: 1. Haguenoer lM. et coil. Prevalence des conduites toxicophiIes en milieu professionnel : une 

etude dans la region du Nord Pas de Calais. Bulletin du Conseil departemental du Nord, Ordre des 

Medecins, 1997; SO : 11-15. 

Keywords: vigilance, haulage drivers, illicit substances 

Page 275

F22 

USE OF COMPOUNDS ALTERING VIGILANCE PERFORMANCE: PRELIMINARY RESULTS 

OF PREVALENCE IN HAULAGE DRIVERS IN THE NORD-PAS-DE-CALAIS REGION 

(FRANCE) 

L. Labat l , B. Dehon l , M. Lhermitte l • and regional group of ({ Toxicomanie et Travail »2 

1 Laboratoire de Biochimie et Biologie Moleculaire - Hopital Calmette - Avenue du Pr Leclercq - 59037 

Lille cedex - France; 2 Institut de Sante au Travail du Nord de la France A venue Oscar Lambret - 59037 

Lille cedex - France. 

Introduction: In 1995, a French study showed that the number of users of compounds that could alter 

vigilance performance was significantly more important in a population of workers in security posts than in 

the general working population (1). Moreover, even if the number of road accidents involving haulage 

drivers has decreased since 1985, their severity is greater. A multicentric study was initiated by a local 

group « Toxicomanie et Travail» on haulage drivers from different cities in the Nord-Pas-de-Calais region 

(France). The objectives of this study are the standardisation of occupational medicine practices in order to 

define a harmonized policy for prevention and screening, to validate analytical screening methods, as well 

as the course of action in the case of positive detection. 

Methods: 1000 haulage drivers were included in the study. Occupation was the only criterion of selection. 

Urine samples were collected anonymously. Information collected included: haulage area, age, gender, 

nature of appointment with occupational practitioners and day of the sampling. A urine screen (test 

Triage8, BMD) was performed for methadone (cut off = 300 nglmL), benzodiazepines (300 nglmL), 

cocaine (300 ng/mL), amphetamine derivatives (1000 ng/mL), opioides (300 ng/mL) and cannabis (50 

ng/mL). Buprenorphine detection was achieved with Elisa method (Microgenics). All positive results were 

confirmed by GC-MS or HPLC-MS. Alcohol levels were determined by an enzymatic method (Dade 

Behring). 

Results: Up to February 2004, 707 samples were collected. 99% are men and participants were on average 

36.4 +/- 9.9 years old. Results expressed as percentage of positives were: opioides (4.81%), cannabis 

(9.05%), cocaine (0.14%), amphetamine derivatives (0%), buprenorphine (1.98%), methadone (0.56%), 

benzodiazepines (0,42%), alcohol (5.51 %). The 34 positive cases for opioides were distributed as follows: 

6-MAM 0 ; morphine 10 ; codeine II ; pholcodine 20. THC-COOH was the only compound identified in 

urine samples from individuals tested positive for cannabis. 

Conclusion: Only legal opioid drugs were identified. They correspond to antitussive medications that are 

broadly prescribed during the winter period (time of the urine collection). The lack of sensitivity and 

specificity of the immunologic test could explain the low percentage observed for benzodiazepine positive 

cases. Our results are in good agreement with the results ofthe previously mentioned study of Haguenoer et 

a!., (1995) in the North of France, for the use of cannabis and alcohol by workers in security posts. In 

contrast, the number of positive cases for amphetamine derivatives, opioides and benzodiazepines was less. 

Other molecules were not investigated in the previous study of Haguenoer et al.. Considering these 

preliminary results, it would be of great interest to extend the screening to other psychoactive molecules by 

using a more efficient screening method such as HPLC-DAD. 

References: ·1. Haguenoer 1.M. et colI. Prevalence des conduites toxicophiles en milieu professionnel : une 

etude dans la region du Nord Pas de Calais. Bulletin du Conseil departemental du Nord, Ordre des 

Medecins, 1997; 80: 11-15. 

Keywords: vigilance, haulage drivers, illicit substances 

Page 276

F23 

SCREENING OF BUPRENORPHINE IN URINE OF SUSPECTED ABUSERS BY ELISA 

Asimah Hamzah*, P.C. Peh, C.M. Lim, C.P.Lui, PhD, Narcotics II Laboratory, Centre for Forensic 

Science, Health Sciences Authority, 11 Outram Road, Singapore 169078 

Introduction: Buprenorphine is a synthetic thebaine derivative that has both analgesic and opioid antagonist 

properties. As an analgesic, it is 25 to 40 times more potent than morphine, and has a slower onset of pain 

relief and longer duration of action. Hence it is used for the treatment of chronic pain, and in treatment of 

heroin addiction as an alternative to methadone. In human, buprenorphine is metabolised primarily by N 

dealkylation to norbuprenorphine. Both the metabolite and the parent drug undergo extensive conjugation 

to glucuronides that are excreted in urine. It is known that buprenorphine has been abused by heroin addict; 

therefore there is a need for the laboratory to develop a screening method for the suspected abusers. 

Aim: The purpose of this study is to evaluate the use of an Enzyme Linked Immunosorbent Assay (ELISA) 

test kit in the screening of buprenorphine in urine to detect suspected abusers. 

Method: The ELISA test kits were purchased from Neogen Corp. USA. The assay principle is based on the 

competitive binding between the free drugs in the urine and the drug enzyme conjugates for antibodies, 

which are coated onto the 96-well microplate. After incubating the urine and drug enzyme conjugate in the 

wells for 45 min at room temperature, the wells were washed with buffer, and K·blue substrate was added 

to each well. After a further 30 min of incubation, Red Stop solution was added to each well to stop the 

reaction. Multiskan Ascent Microplate Photometer was used to measure the absorbance at wavelength of 

650 nm. The color intensity of each well is inversely proportional to the concentration of the drug in the 

urine. Confirmatory test, using Gas chromatography-mass spectrometry (GC·MS) was also carried out on 

all the urine specimens to compare with the screening results. 

Results: The linearity of the ELISA kit was found to be up to about 5 ng/ml of buprenorphine. The 

coefficients of variation (CVs) of the within-day and between-day variations for buprenorphine ranged 

from 2.0% to 6.7% and 8.3% to 19.1% respectively. Norbuprenorphine was found to cross react to certain 

extend. No cross-reactivity was found with other common drugs of abuse such as morphine, codeine, 

hydrocodone, hydromorphone, Il-nor-L'1 9 -THC-9-carboxylic acid, cocaine, benzoylecgonine, amphetamine, 

methampethamine, N,a-Dimethyl-3,4-(methylenedioxy)phenethylamine (MDMA), a-Methyl-3,4 

(methylenedioxy)phenethylamine (MDA), N-Ethyl-a,methyl·3,4-(methylenedioxy)phenethYlamine 

(MDEA), ketamine and norketamine. Out of the 3 urine specimens that were collected from suspected 

abusers, 17 were found to be screened positive, using the cut-off at 1 ng/ml. The linearity range of 

buprenorphine was found to be up to 50 ng/ml. The LOQ was found to be 0.25 nglml. The results from the 

confirmatory test are also discussed. 

Conclusions: The use of ELISA test kit was found to be a simple, rapid, specific and effective method for 

the screening of buprenorphine in urine. It helps to minimize the cost of the GC-MS test in a mass 

screening exercise. 

Keywords: ELISA, Screening, Buprenorphine 

Page 277

F24 

A COMPARISON OF THE MICROGENICS DRI® OPIATE AND MICROGENICS DRI® 

OXYCODONE IMMUNOASSA YS WITH GAS CHROMATOGRAPHY IMASS SPECTROMETRY 

FOR THE DETECTION OF OPIATES AND OXYCODONE IN URINE 

Joseph R. Monforte, Ph.D., DABFT *, Ronald Backer, Ph.D., DABFT and Alphonse PokJis, Ph.D., 

DABFT 

Oxycodone is a semi-synthetic opioid prescribed for pain management of moderate to severe pain. It is 

considered to have a high abuse potential and is prescribed as oxycodone HCl in preparations such as 

Oxycontin® (a controlled- release preparation), Percodan® (with aspirin) and Percocet® (with 

acetaminophen). Oxymorphone and noroxycodone are metabolites of oxycodone. Oxymorphone is a 

potent analgesic while noroxycodone is relatively inactive. 

Various immunoassays are available for the detection of opiates in urine. These assays generally have 

lower sensitivities to oxycodone and oxymorphone. Consequently, oxycodone may not be detected 

employing available opiate assays, even when taken under abused conditions. The Microgenics DRI® 

Oxycodone Assay uses specific antibodies that can detect oxycodone and oxymorphone without significant 

cross-reactivity from other opiates. The assay employs either a 100ng/mL or a 300ng/mL cutoff. 

Seventy specimens received from pain management patients were tested for opiates with the Microgenics 

DRI® Opiate and Microgenics DRI® Oxycodone Immunoassays, and gas chromatography/mass 

spectrometry. Immunoassay reagents were employed as directed by the manufacturers. The cutoff for the 

Microgenics DRI® Opiate Assay was 300 ng/mL, and the cutoff for the Microgenics DRI® Oxycodone 

Assay was 

100 ng/mL. The GC/MS SIM procedure employed hydrolysis of the specimen, treating the urine with 

hydroxylamine, a solid phase column extraction, and formation of the BSTF A derivative. The method' 

detects oxycodone, oxymorphone, hydrocodone, morphine and codeine. The limit of detection and limit of 

quantitation is 20 ng/mL for each opiate. 

Of the seventy specimens tested by the Microgenics DRI® Opiate Assay, fifty-three (53) specimens tested 

positive and seventeen specimens tested negative. All seventeen negative specimens were confirmed 

negative for the opiates included in the GC/MS SIM procedure and all seventeen specimens tested negative 

for oxycodone using theMicrogenics DRI® Oxycodone Assay. An opiate was detected by GCIMS in all of 

the specimens testing positive by the Microgenics DRI® Opiate Assay with four exceptions. 

Of the seventy specimens tested by the Microgenics DRI® Oxycodone Assay, twenty-four specimens tested 

positive for oxycodone. All twenty-four specimens tested positive for oxycodone by GCIMS, and twentytwo 

of the specimens also contained oxymorphone. One specimen, which did not contain a reportable 

oxymorphone concentration, also contained morphine (4,679 ng/mL). One specimen tested one point 

below the Microgenics DRI® Oxycodone cutoff and contained morphine (18, 522 ng/mL) but no reportable 

oxycodone. All forty-six specimens, which tested negative for oxycodone with the Microgenics DR1® 

Oxycodone Assay, also tested negative for oxycodone and oxymorphone by GC/MS. 

The results obtained with this limited number of specimens indicates that the Microgenics DRI® 

Oxycodone Assay is highly specific for oxycodone and, consequently, has the potential to be very effective 

in discriminating oxycodone from other opiates which may be present in a urine specimen. 

Keywords: Oxycodone, Immunoassay, Gas ChromatographylMass Spectrometry 

Page 278

F25 

IDENTIFICATION OF CHLORINATED 3,4-METHYLENEDIOXYMETHAMPHETAMINE 

IN ILLICIT DRUG ABUSER URINE 

4 

Vera Maresova 1 ', Jan Hamplt, Zdenek Chundela 2 , Frantisek Zrcek 3 , Miroslav Pohgek , Jiff Chadt' 

'Institute of Forensic Medicine and Toxicology, 1 st Medical Faculty and Hospital, Charles University of 

Prague; 2 Department of Doping Control, General Faculty Hospital, Prague, 3 Institute of Criminalistics, 

Prague, 4 J. Heyrovsky Institute of Physical Chemistry, Prague, Czech Republic 

Aim: The aim of this study was to identify 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 

chlorinated 3,4-methylenedioxymethamphetamine (CI-MDMA) using combined thin layer chromatography 

(TLC) and gas chromatography-mass spectrometry (GC-MS). The compound was identified jointly with MDMA 

in illicit drug abuser urine. The urine specimen has yielded positive EMIT immunoassay screenings for 

cannabinoides, amphetamines and cocaine. GC-MS confirmation of amphetamines has revealed the presence of 

amphetamine, methamphetamine, ephedrine, norephedrine, 3,4-methylenedioxymethamphetamine and 3,4 

methylenedioxyamphetamine (MDA). 

Methods: Thin layer chromatography: In the basic extract a routine analysis of urine by TLC for amphetamines 

using fractional diethylether liquid/liquid extraction identified ephedrine, the major component MDMA and an 

unknown compound. In the TLC system our unknown compound behaved differently than the MDMA. The TLC 

plate with silica stationary phase was used (Merck), eluent ethylacetate ethanol ammonia (36:2:2). Gas 

chromatography-mass spectrometry: The unknown compound isolated by TLC technique was analysed by 

positive chemical ionization (PCI). The base peak mlz 228 (MH+) exhibited a characteristic A+2 isotopic cluster 

indicating the presence of a monochloro-substituted compound. The molecular ion of CI-MDMA was found at 

227 mlz. The MS spectrum of the unknown compound in electron ionization (EI) exhibited characteristic A+2 

chlorine isotopic cluster typical for ion at mlz 169 (mlz 135-1+35) and for molecular ion at mlz 227 (mlz 193 

1+35). To confirm CI-MDMA structure, the unknown compound isolated by TLC technique was acetylated, 

trifluoroacetylated, silylated, heptafluorobutyrylated and analyzed by GC-MS. The EI-MS spectra ofMDMA and 

CI-MDMA after derivatization exhibited structural similarities. The EI-MS spectrum of MDMA contained ions 

193, 177, 135, 77, 58 [I]. The EI-MS spectra of derivatized CI-MDMA exhibited characteristic A +2 isotopic 

cluster for ion at mlz 169 (mlz 135 -I +35) for all used derivatization methods mentioned above. Another 

isotopic A + 2 cluster was created in all molecular ions of each derivatized chlorinated MDMA: acetylated CI 

MDMA had molecular ion at mlz 269 (mlz 227 - I + 43), silylated CI-MDMA had molecular ion at mlz 299 (mlz 

227 - I +73), trifluoroacetylated CI-MDMA had molecular ion at mlz 323 (mlz 227 I + 97), 

heptafluorobutyrylated CI-MDMA had molecular ion at mlz 423 (mlz 227 - I + 197). Next joint cluster at mlz 196 

(162 - I + 35) occurred in acetylated, trifluoroacetylated and heptafluorobutyrylated derivates with the exception 

of silylated derivates. GC-MS analysis were performed using Hewlett Packard 5890 Gas Chromatograph with 

5890 autosampler coupled to a Hewlett Packard 5972 MS detector and Finnigan MAT Magnum. CI-GC-MS 

Finnigan MAT Magnum conditions were as follows: ionization gas methanol vapour, ionization energy of 

electrons 70 eV, specific conditions for ion trap CIIMCI parameters: CI maximum ionization time 1500 IlSec, CI 

maximum reaction time 100 msec, CI ionizatin storage level 82 dacs, CI reaction storage level 164 dacs, reagent 

ion ejection level 85 amu, MCI ionization time 100 Ilsec, reagent ion ejection adjust 100 %, reagent reaction time 

54001lsec. 

Conclusion: A chlorinated MDMA was identified after derivatization of unknown compound isolated by TLC 

technique. With using literature source [2] and interpretation our mass spectra of heptafluorobutyrylated CI 

MDMA we can declare that unknown compound should be 6-CI-MDMA. 6-CI-MDMA is listed in New 

synthetic drugs reported in Europe and the USA since the mid-1990s in joint action of The European Monitoring 

Centre for Drugs and Drug Addiction (EMCDDA)[3]. 

Acknowledgment: The work has been supported by the project ofMSMNo. 111100005. 

l. H. H. Maurer, A. Weber, K. Pfleger: Mass Spectra and GC data of Drugs, Poisons, Pesticides, Pollutants 

and their metabolites, 1-4, VCH Verlagsgesellschaft, Weinheim, Germany, 1992 

2. R.l Lewis, D. Reed, A. G. Service, A. M. Langford: J. Forensic. Sci. 2000; 45(5): 1119-1125 

3. L. A. King, A. J. Poortman van der-Meer: Sci. & Just. 2001, 41(3): 200-202 

Keywords: chlorinated MDMA, GC-MS analysis 

Page 279

F26 

TESTING MEDICAL PROFESSIONALS FOR AN EXPANDED MENU OF DRUGS- 

PRELIMINARY SUMMARY OF POSITIVE FINDINGS 

Matthew McMullin* and Anthony Costantino. National Medical Services, Willow Grove, PA, USA. 

A 

Testing medical professional for drugs of abuse presents a challenge because of their access to drugs of 

choice that are not included in the scope of most drug testing menus. To assist substance abuse recovery 

programs, designed to allow medical professionals to continue to practice in their field of expertise, we 

developed an expanded menu of analytes that includes many of the known abuse drugs encountered in the 

medical professional population. The positive results data was summarized in three drug groups: Group I 

includes those analytes contained in a SAMHSA regulated test menu; Group 2 includes additional opiates 

and amphetamine like compounds not covered in the SAMHSA scope, benzodiazepines, propoxyphene, 

barbiturates, and methadone; Group 3 includes (cut-off concentration in nglmL) nor-alfentanil (0.5), 

butorphanol (20), nor-fentanyl (0.5), ketamine (100), MDMA (200), nor-meperidine (200), nalbuphine (8), 

nor-sufentanil (0.5) and tramadol (500). Group 1 and 2 analytes were screened by EMIT while group 3 

analytes are screened by LCIMSIMS. All screens were confirmed by either GCIMS or an alternate 

LCIMSIMS procedure. The confirmation menus were extensive with low concentration cut-offs. Ethanol 

and ethyl glucuronide were not included in this study. 

A total of 5,817 samples from medical professionals were tested for group I and 2 drugs. The number of 

positive drugs findings for the SAMHSA drugs include in group I was 100, with amphetamine at 56 and 

opiates at 34 representing most of the positive findings. With the group 2 drugs there were an additional 

386 positive drug findings with 193 benzodiazepines findings and the expanded opiates panel (excluding 

morphine and codeine) accounting for 134 of the positive findings. A total of 4,043 samples were tested 

for group 3 analytes, and there were 186 positive drug findings. Tramadol, meperidine, and nalbuphine 

were the most frequently reported positive findings in the group 3 drugs, accounted for 120, 30 and 14, 

respectively. 

Drug Groupings 

Positive Drug Findings! 

Drug Group 

Cumulative Positive 

Drug Findings 

(Group 1) SAMHSA Drugs 100 of 5817 samples 100 

(Group 2) expanded EMIT panels 386 of 5817 samples 486 

i 

. (Group 3) LCMSMS panel 

i 

207 of4043 samples 693 

These preliminary finding support the use of expanded testing menus when testing medical professionals 

who are in drug recovery programs. Caution must be used when interpreting these findings since some 

positive findings were listed as prescribed medications. 

Keywords: medical professional, positive-rate, LCIMSIMS. 

Page 280

F27 

LINEAR RELATIONSHIPS OF 6.-9-TETRAHYDROCANNABINOL METABOLITES IN URINE 

Scott Larson· and James D. Hutchison, Jr. 

Montana State Crime Laboratory, University of Montana, Montana, USA 

Many studies have focused on the metabolic patterns of marijuana and the relationship these compounds 

may have to possible time of cannabis exposure, based on their concentrations in urine samples. The 

objective of this study was to investigate the linear relationships of a group of 6,9-tetrahydrocannabinol 

metabolites in random urine samples (n=95). In this study, gas chromatography-mass spectrometry (using 

S1M profiles) was used to monitor and quantify I1-nor-6. 9 -carboxy-9-tetrahydrocannabinol (THCCOOH), 

Il-Hydroxy-6. 9 -tetrahydrocannabinol (ll-OH-THC), 6,9-tetrahydrocannabinol (THC), cannabinol (CBN), 

and cannabidiol (CBD). Deuterated THCCOOH-d J and 1 I-OH-THC-d J were used as internal controls. The 

random urine samples were collected and submitted to the Montana State Crime Lab from adult probation 

& parole, Pre-release centers, and Youth Court cases. Since these are random urine samples, there was no 

way to definitely determine time of marijuana use. The results of this study indicate significant linear 

relationships between some of the metabolites of THC, and also the ability to detect CBN and CBD in 

random urine samples. The data presented will provide a collection ofTHCCOOH concentrations in urine 

with respective comparisons to the other metabolites studied. 

Keywords: 6.-9-Tetrahydrocannabinol metabolites, Urine, GCIMS 

Page 281



Alternative 

Matrices 

Page 283

Ml 

COMPARISON STUDY OF SPE AND HS-SPME IN THE DETERMINATION OF METHADONE 

AND ITS METABOLITES EDDP AND EMDP IN HUMAN HAIR USING GC-MS 

L O'Hanlon l *, J S Oliveri, and K S Scott 2 

IPorensic Medicine and Science, University of Glasgow, Glasgow, UK 

2Department ofForensic Science and Chemistry, Anglia Polytechnic University, Cambridge, UK 

The objective of this research was to develop and optimize a robust head-space solid phase microextraction for the 

extraction of methadone and its metabolites from hair and to compare this method to a pre-existing solid phase 

extraction method. Methadone (6-dimethylamino-4,4-diphenyl-3-heptanone hydrochloride) is a synthetic opioid 

agonist with characteristics similar to those of morphine, including actions on the central nervous system and organs 

composed ofsmooth muscle. Although structurally it has no resemblance to morphine, its analgesic and spasmolytic 

properties have allowed it to be used as a substitute for heroin in opiate addiction programmes. 

Aliquots of blank hair (IOmg) were spiked with methadone and EDDP at two different concentrations and incubated 

in sodium hydroxide solution 1 M (Iml) with anhydrous sodium chloride. This was carried out in 4 ml amber screwtop 

vials with a PTPE/siIicone septum. The vial was preheated for 20 min under agitation. The needle probe holding 

the IOOJ,lm polydimethylsiloxane (PDMS) fibre was then pushed through the septum and the fibre was immersed in 

the specimen for 20 min at a temperature of 75°C while agitated. The fibre was then transferred to the GC inlet. The 

injection port (operated in splitlsplitless mode) was set to 225°C and the purge time to 3 min. The column 

temperature was initially held at 80°C for 2 min, then increased to 280°C at lOOC/min and held for 5 min. 

This SPME method was compared to the following SPE method. Aliquots of spiked hair (lOmg) were incubated in 

methanol for ISh at 37'C. This methanol was then removed and the sample was sonicated with more methanol. The 

methanolic fractions were combined and evaporated under nitrogen and reconstituted in distilled water and 

phosphate buffer, pH 6. The samples were homogenised and applied to preconditioned Clean Screen columns. 

These were selectively washed with various solvent and aqueous mixtures; Methadone and EDDP were extracted 

using dichloromethane/isopropanol/concentrated ammonia (78:20:2). These fractions were then evaporated to 

dryness under nitrogen and reconstituted in 25 J,ll of ethyl acetate. They were then analysed using the same GC-MS 

chromatographic conditions. Relative recoveries of methadone and EDDP from hair were determined by comparing 

the peak heights obtained from spiked hair with unextracted standards and both were determined to be above 5%. 

The HS-SPME method proved to be fast and efficient and also has the advantages of being solvent free and 

producing cleaner extracts. 

This paper presents a comprehensive, comparative study of both SPE and HS-SPME of methadone and EDDP from 

human hair and will report on the advantages and disadvantages of both methods. 

Keywords: Methadone, Solid Phase Extraction, Solid Phase Microextraction 

Page 284

M2 

DRUG ANALYSIS IN HAIR: COMPARISON OF CRYOGENIC GRINDING AND CUTTING 

FOR SAMPLE PREPARATION. 

Yvonne Cruickshank i *, Fiona M. Wylie 2 and Robert A. Anderson 2 .: iForensic Science Unit, University of 

Strathclyde, Glasgow Gl lXW, United Kingdom' ; 2Forensic Medicine and Science, University of 

Glasgow, Glasgow G12 8QQ, United Kingdom. 

Sample preparation is potentially the most important step in an analytical procedure. The forensic 

toxicology laboratory at Glasgow University has established methods for the extraction of drugs from 

keratinous matrices by cryogenic grinding (nail) and by cutting with scissors (hair) (1,2). To date, only one 

report has compared drug extracts obtained from cut and ground hair. Eser et al. found that both the 

qualitative and quantitative results from ground hair were better than from cut hair (3). In this report, two 

methods of sample preparation for the analysis of drugs of abuse in hair are compared, namely cutting and 

cryogenic grinding of hair. The drugs analysed were morphine, 6-monoacetylmorphine, codeine, 

dihydrocodeine, methadone, EDDP, diazepam, cocaine and ecgonine methyl ester. 

Samples of hair from 20 drug-related deaths were used for the evaluation. These were collected at autopsy 

by plucking from the occipital region of the scalp and the root balls were removed prior to analysis. Drugfree 

hair was obtained from laboratory personnel for use as a control and for preparation of standards. Each 

sample was washed with SDS (0.1 % w/v), DI water and dichloromethane before being dried at room 

temperature overnight. Each sample was weighed and separated into two portions of equal size that were 

subsequently minced with scissors or ground, respectively. Mincing was carried out in screw-cap tubes 

with long-blade scissors for 1 minute. Grinding was carried out in liquid nitrogen using a Glen Creston 

Model 6750 freezer mill equipped with micro-inserts. The optimum grinding cycle was established as 2 x 

2 min by SEM examination of the powders obtained following various grinding periods. The ground hair 

was recovered from the grinding tube and reweighed. 

Hair standards were prepared by adding solutions containing known amounts of drugs to drug-free hair and 

allowing it to dry. After addition of deuterated internal standards, hair standards and the case hair samples 

(up to 20 mg) from both series were suspended in methanol (1 ml), sonicated (15 min) and left for 12 hours 

at 45°C. The methanol was removed from the samples to clean vials and evaporated under a flow of 

nitrogen at room temperature. The mixture was redissolved in 4 ml phosphate buffer for SPE with Bond 

Elut Certify LRC cartridges. Extracts were derivatised at 70°C for 20 min using 50 Jll BSTFA containing 

1 % TMCS and analysed by GC-MS using a Thermo-Finnigan Trace instrument fitted with a 30 m x 0.25 

mm x 0.25 Jlm HP-l column. 

The cryogenic grinding device worked effectively and produced small hair particles (19-57 Jl). However 

specimen losses (up to 50%) during the procedure were significant. The grinding method was time 

consuming and, coupled with the use of liquid nitrogen, made grinding a less cost-effective procedure. 

Comparison of the extracts from cut and ground hair by GC-MS indicated that grinding resulted in more 

matrix interferences and also that ground hair was more likely to produce a false negative result. For 

example in the case of methadone five samples were identified as containing methadone using cut hair but 

only one of these samples was identified as containing methadone using ground hair. However, ground 

hair gave higher recoveries of drugs than cut hair, after correction for sample losses, in many cases up to 

twice the amount of drug was extracted from ground hair. 

The results confirmed the findings of Eser et al. that the ground hair was best in terms of quantitative 

analysis but the findings in terms of the qualitative analysis showed clearly that cut hair is the best 

preparation technique. Further work will indicate if grinding will permit the use of a shorter extraction 

period than cut hair. 

Keywords: Hair Analysis, Cryogenic Grinding, Drugs of Abuse 

(1 ) K. Takaichi, N. P. Lemos, R. Anderson, Analysis of Opiates in Nail Clippings from Chronic Heroin 

Abusers, Presented at the 39 th Annual TIAFT Meeting, Prague, 2001. 

(2) F.M Wylie, Personal Communication. 

(3) H.P. Eser, L. Potsch, G. Skopp and M.R. Moeller, Influence of sample preparation on analytical 

results: drug analysis (GC/MS) on hair powder using various extraction techniques. For. Sci. Int., 

84,271-279 (1997). 

Page 285

M3 

AMPHETAMINE BINDING TO SYNTHETIC MELANIN AND SCATCHARD ANALYSIS OF BINDING 

DATA 

Lata Gautam*, Karen S Scott, and Michael Cole: Department of Forensic Science and Chemistry, APU, Cambridge, 

United Kingdom, CBI IPT 

The determination of drug use and drug history has historically been carried out through the analysis of biological 

fluids (blood and urine). In the past decade, hair analysis has been exceedingly used in order to determine recent past 

drug use as well as long-term drug histories through the use of segmental analysis. In order to fully understand the 

implications of a positive hair test result, it is important to understand how the drugs are incorporated into hair and how 

they bind. Keratin, melanin and lipids are possible drug binding sites in hair. Keratin and melanin have many polar 

groups which serve as attachment points for the drugs. Previous research into drug-hair binding has shown that 

different amounts of drug bind in hair of different colours. There are no structural differences in hair of different 

colours other than in the type and content of melanin present. For this reason, this investigation focuses on 

amphetamine binding to synthetic melanin in order to help to explore the possible binding mechanisms. 

The binding study was carried out at room temperature in the presence of light and oxygen using 0.1 M phosphate 

buffer at pH 7.4. Both compounds were found to be stable under these conditions. Amphetamine-d-sulphate and 

melanin, synthesized by the oxidation of tyrosine, were purchased from Sigma Chemical Company. As melanin is only 

sparingly soluble in buffer, it was solubilised in dimethyl sulphoxide (DMSO) prior to dilution with buffer. An indirect 

spectroscopic method was used by measuring the decrease in absorbance of amphetamine as the concentration of 

melanin was increased. Aliquots of amphetamine were titrated against the melanin solution. Samples were incubated 

for 45 minutes with constant shaking. Background correction was done at each step using the same amount of melanin 

as present in the sample for matrix matching. The interaction was monitored using a Genesys 6v, UVNis 

spectrophotometer in scanning mode from 250-264 nm focussing on changes in the amphetamine spectral fingerprint at 

257.2 nm. Positive controls were prepared using the same amount of amphetamine as in the sample with the addition of 

buffer instead of melanin at each step. Buffer was used for the background correction to run positive controls. 

To determine the classes of binding sites, association/dissociation constants and the binding capacity of melanin, the 

data were analysed by Scatchards' method (Scatchard et al., 1949). As the molecular weight and the structure of 

melanin is still not clear, the number of binding sites can not be calculated directly as an integer. Instead, the ratio of 

the number of pmoles of drug bound and the dry weight of melanin in pg is considered. This study has proved that 

amphetamine binds to synthetic melanin in-vitro. Data analysis from the Scatchard method has yielded a curvilinear 

plot with upward concavity. The curvilinear plot indicates multiple binding sites and the upward concavity indicates 

negative co-operativity. 

Scatchard Analysis of Melanin Binding 

Capacity for Amphetamine 

0.60,.......-----------------------, 

• 

0.10 

• 

• 

• •• • • • 

0.00 

0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 ! 

Reference: Scatchard G., (1949) The attractions of proteins for small molecules and ions, Annals 0/ the New York 

Academy o/Science. 51:660-672 

Keywords: Hair, Melanin, Scatchard Analysis 

Page 286

M4 

ANALYSIS OF TRACE METAL CONTENT IN HUMAN HAIR USING LASER ABLATION 

INDUCTIVELY COUPLED PLASMA MASS SPECTROSCOPY (LA-ICP-MS) 

Wat L. Harden I * and Robyn Hannigan 2 ; IArkansas State University, Dept. of Biology, P.O. Box 419, State 

University, Arkansas 72467, 2Arkansas State University, Dept. of Environmental Sciences, P.O. Box 419, 

State University, Arkansas 72467 

Measuring the trace element content in human hair is traditionally done by acid digestion and analysis by 

atomic absorption or Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Acid digestion of 

samples can introduce contamination, which then lowers the accuracy ofthe analytical method. In addition 

to the contamination issues, acid digestion under trace metal clean conditions can be labor intensive and 

costly. Laser ablation (LA) is a sample introduction technique that, when combined with the ICP-MS, has 

the potential to provide accurate metal concentration data. 

Here we present method development data comparing traditional acid digestion liquid-based ICP-MS to 

LA-ICP-MS on two certified hair standards (International Atomic Energy Agency Reference Material 

Human Hair IAEA-085 and The People's Republic of China Reference Material Human Hair GBW 07601) 

and laboratory internal standard (USGS basalt standard BCR-2). The two sample preparation techniques 

that were compared were acid-digestion and solid sampling via laser ablation. Internal standards were used 

to calibrate both liquid and solid analysis techniques to monitor instrument drift (liquid - 115 In; solid-total 

Si in ppm). The concentrations of Fe, Zn, Ca, Cu, Mg and Mn of each sample were compared, using the 

known concentrations from each hair standard. 

Following the analysis of the BCR-2 standards and samples, we found that LA-ICP-MS analysis is 

reproducible only within an error of 20%. This could be due to matrix interferences (organic v.s. silicate) 

which were not corrected for in this preliminary study. Overall laser ablation is an excellent tool for trace 

metal analysis providing accurate element ratio data (eg; CulCa) but not concentrations at this time. Future 

studies will focus on the development of a matrix matched solid standard mitigating matrix interferences. 

Keywords: Trace Analysis, LA-ICP-MS, Hair 

Page 287

M5 

METHOD VALIDATION AND DETERMINATION OF MDMAAND MDA IN HAIR 

Hee-Sun Chung l *, Eun-Young Hanl, Won-Kyung Yang l , Jae-Sin Lee l , Yong-Hoon Park I, Eun-Mi Kiml, 

Mi-Ae Lim l and Youngchan Y002 : lNational Institute of Scientific Investigation, Seoul; 2Korea, School of 

Pharmacy, Chungbuk Nat. University, Chungbuk, Korea 

Recently, MDMA and MDA have been used as a recreational drug among young people in Korea. MDMA 

is a substitute for methamphetamine (MA), and has recently received much attention from media and law 

enforcement as a novel and popular psychoactive agent. As a result of the recent interest in designer drug, 

the abuse of MDMA and MDA has increased and there were cases in which both MA and MDMA were 

detected in hair samples. Therefore it was necessary for us to establish the detection method ofMDMA and 

MDA in hair samples. The method validation and uncertainty measurement were also studied and 

described. The prevalence of MDMA was surveyed in hair specimens submitted for drug testing by this 

method. 

MDMA, MDA, MDMA-ds, and MDA-ds were purchased from Radian International LLC (Austin, TX, 

USA). Hair samples were collected from 791 subjects who were suspected MDMA and MDA users. After 

washing, the hair was cut into small pieces of less than 1 mm and incubated. SPME extraction was applied 

and extracts were derivatized by trifluoroacetic anhydride (TF A). Gas chromatograph-mass spectrometry 

(GCIMS) analysis was performed under selective ion monitoring (SIM) conditions for the identification 

and quantitation ofMDMA and MDA. 

Limit of detection (LOD) and limit of quantitation (LOQ) of MDA and MDMA were 0.125 ng/mg, 

0.25ng/mg respectively. The linear calibration curve ranged from 0.25 to 10 ng/mg with the coefficient 

determination of R2 >0.99 . Within- and between run precisions were measured in three concentrations 

(low 0.8 ng/mg, med 4 ng/mg, high 8 ng/mg). The specificity in blank hair and dyed artificial hair was 

studied.. The high concentration of ephedrine, norephedrine, pseudoephedrine, nicotine, caffeine, 

methoxyphenamine, phenterminein in low QC hair samples (0.8ng/mg) didn't show any interference in this 

method. To value the confidence of the method, uncertainty was performed at the cut-off level 5 ng/mg 

hair samples. The measurement in uncertainty of MDMA was ±0.22 -0.30 (95% confidence interval, k=2). 

Among 791 hair samples studied, 44 hair samples were positive for MDMA or MDA (5.6 %). The 

concentration of MDA was low in the hairs where both MDMA and MDA were detected indicating MDA 

was the metabolite of MDMA, while in 4 specimens the level of MDA was very high with no MDMA 

indicating MDA was administered. However in urine samples from same 44 subjects showed 35 were 

negative for MDMA or MDA, while 9 were positive for MDMA. The hair analysis ofMDMA showed that 

a hair sample is a good specimen for the confirmation of long abuse of MDMA and MDA in comparison 

with urine. 

Keywords: MDMA, Hair Analysis, Method Validation 

Page 288

M6 

MONITORING DRUG USE THROUGH SEGMENTAL HAIR ANALYSIS. A THREE YEAR 

CASE STUDY. 

Karen S. Scott*, Kerri-Ann Hoy: Department of Forensic Science and Chemistry, AngJia Polytechnic 

University, Cambridge 

Segmental analysis of hair for the determination of past drug use is an area which receives mixed views 

within the field of Forensic Toxicology. The aim of this study was to ascertain if an accurate pattern of 

drug use can be predicted by the segmental analysis ofa length of hair. The hair sample (33cm length) was 

obtained from a 47 year old male with a known history of use of three types of medication. Atenolol 

(IOOmg) was taken as required; diazepam (3x 5mg) was taken as required and dihydrocodeine (3 x 30 mg) 

was taken daily. All three drugs had been prescribed over a period of9 years following a serious injury. 

The hair was cut into eleven 3 cm sections each representing an approximate period of three months 

(assuming a hair growth rate of I cm/month). The segments were prepared for extraction by washing and 

cutting. Blank hair was obtained from a subject known not to have used any of the drugs under 

investigation and similarly prepared. Triplicate aliquots (20 mg) of the hair were subjected to overnight 

acidified methanolic digestion along with standards and blanks. Following digestion the samples were 

filtered, evaporated to dryness, derivatised using BSTF A 1% TMCS and analysed by GCIMS in the SIM 

mode. 

Analysis of atenolol in the hair segments showed a variable pattern of use. No accurate record of atenolol 

use was obtained other than an increased use for short periods of time 9 and 18 months prior to sampling. 

The highest concentration of atenolol in the hair segments was obtained in segment three (corresponding to 

6-9 months hair growth). A slight increase in the levels measured in segment six (corresponding to 15-18 

months) was also observed. 

The analysis of diazepam in hair showed an overall increase of drug in hair from the proximal end to the 

distal end. It is known that the subject decreased the frequency of diazepam use in the 18 months prior to 

sampling which is consistent with the decreased concentrations in these hair sections. However there is a 

threefold increase in the amount of diazepam detected between segment six (15-18 months) and segment II 

(30-33 months). No drug use history of diazepam is available for this period. 

Finally, the analysis ofdihydrocodeine for which the full drug history was known (90 mg/day for 9 years) 

yielded extremely interesting results. If no environmental issues (e.g. UV exposure, washing) affect the 

amount of drug in hair then we would expect the levels in the proximal and distal segments to give the 

same concentration. However a decrease of approximately 90% in the level of dihydrocodeine detected 

was observed (r2 0.77). 

This study demonstrates that hair analysis can be used to detect drugs which were taken several years ago. 

More work is required to determine the exact relevance of history evaluation through segmental analysis 

but the results for dihydrocodeine indicate that this is not a futile exercise. The importance of obtaining a 

full drug use history from subjects participating in studies of this type is highlighted in the analysis of both 

atenolol and diazepam where gaps in the history meant that a full interpretation of the results could not be 

carried out. 

Keywords: Segmental Analysis, Drug Use History 

Page 289

M7 

EVALUATION OF KETAMINE ABUSE USING HAIR ANALYSIS: CONCENTRATION 

TRENDS IN A SINGAPORE POPVLA TION 

H.S. Leong', N.L. Tan, C.P. Lui, and T.K. Lee: Narcotics II Laboratory, Centre for Forensic Science, 

Health Sciences Authority, 11 Outram Road, Singapore 169078. 

Ketamine has been used as an anaesthetic since its discovery in 1961. Today, the drug is used primarily in 

veterinary medicine, and in some short-term surgical procedures in humans. Ketamine also produces posthypnotic 

emergence reactions such as prolonged hallucination and delirium, which has led to its abuse. 

Due to the increasing abuse of ketamine in Singapore, ketamine, norketamine and its dehydro derivatives 

were listed as Class B Controlled Drugs under the Misuse of Drugs Act (CAP.185) in September 1999. As 

a drug of abuse, ketamine can be administered orally, snorted, or injected. In Singapore, the most common 

mode of administration is through snorting. The majority of the ketamine abusers consist of teens and 

young working adults. 

The laboratory has been analyzing ketamine in urine of suspected abusers since September 1999. In the 

period of January 2000 to April 2004, there were a total of 17,133 urine samples submitted for ketamine 

analysis, out of which 26.6 % were tested positive for ketamine during the period of January 2000 to April 

2001. The number of positive samples increased significantly to 51.2 % during the period of May 2003 to 

April 2004. Recently, the enforcement agency of Singapore has indicated interest in the detection of 

ketamine in hair. This is because many of the abusers have claimed that their drinks were spiked with 

ketamine to avoid prosecution. The detection of ketamine in hair would determine whether they were 

habitual users or it was an one-off consumption. 

This paper presents a method for the detection of ketamine in hair. Hair samples (25 mg) were washed, 

pulverized and digested in hydrochloric acid (O.5M) overnight at 45°C. The samples were extracted by an 

automated solid-phase extraction procedure and the extracts were subsequently analyzed using gas 

chromatography/mass spectrometry (GCIMS) in selected ion monitoring mode (SIM). Good linearity up to 

120 ng/mg was obtained for both ketamine and norketamine (r 0.9987 and r2 0.9985, respectively). 

Limit of detection (LOD) was found to be at 0.4 ng/mg for both drugs while the limit ofquantitation (LOQ) 

was found to be 0.6 and 0.8 ng/mg for ketamine and norketamine, respectively. Other parameters- such as 

intra- and inter-day variation were also determined. 

About 9 I hair segments from suspected ketamine abusers were analyzed. In most of the hair segments, 

ketamine was found to bethe predominant analyte (norketamine to ketamine ratio < 1). This is in contrast 

to urine where the metabolite norketamine is usually found to be the predominant analyte. The range of 

ketamine detected in hair was found to be from 0.6 ng/mg to 489.0 ng/mg (Mean = 49.0 ng/mg) whereas 

the range of norketamine detected was from 0.8 ng/mg to 196.3 ng/mg (Mean = 12.1 ng/mg). At 

concentrations higher than 120 ng/mg, about 5 mg of hair is sufficient for the re-analysis of the sample. 

Based upon the· voluntary confession of the ketamine abusers, a correlation between the amount of 

ketamine detected and the frequency of abuse was observed. For abusers who snort the drug occasionally 

(once a week), the concentration ofketamine detected in hair was in the range of 1.1 ng/mg to 42.7 ng/mg 

(Mean 9.9 ng/mg). For those who abuse the drug more frequently (twice or thrice a week), the 

concentration of ketamine detected was in the range of 13.5 ng/mg to 111.l ng/mg (Mean 37.4 ng/mg). 

For those who abuse the drug daily, the concentration of ketamine detected was above 45.1 ng/mg (Mean = 

121.3 ng/mg). Based on the results of the analysis, three types of trends for ketamine abuse were observed: 

Low (1.0 - 10.0 ng/mg), medium (11.0 50.0 ng/mg), and high (>50.0 ng/mg). Segmental analysis of the 

hair samples of the abusers was also performed and its significance is discussed in relation to the history of 

drug use. 

In conclusion, ketamine in hair can be determined by using acid digestion and subsequent GCIMS analysis. 

Based on the amount of ketamine found in hair, certain patterns of consumption among ketamine abusers 

can be demonstrated. 

Keywords: Ketamine, hair analysis, GC/MS/SIM 

Page 290

M8 

THE REQUIREMENT FOR EFFECTIVE WASH PROCEDURES FOR HAIR TESTING, AND 

THE APPLICATION OF A WASH METHOD TO SAMPLES SOAKED IN COCAINE 

SOLUTIONS OF 1 TO 50 UG/ML 

Michael Schaffer*, Virginia Hill, Thomas Cairns: Psychemedics Corporation, 5832 Uplander Way, Culver 

City, C:A 

It has been proposed that issues of external contamination of hair can be resolved by relying on the 

presence of metabolites. However, even if some metabolites such as cocaethylene are sufficiently 

definitive to indicate certainty of use, others such as benzoylecgonine also occur as contaminants. In 

addition, for phencyclidine only the parent is determined. Therefore effective washing procedures are 

important in order to rule out contamination as the source of the parent and or metabolite. The protection 

provided by cutoffs is also dependent on effective washing procedures to remove surface contamination; 

such contamination can range from very little to at least as much as 20 times the amount in the hair. In 

testing head hair samples from 75 drug rehabilitation subjects with cocaine-positive urines, cocaine 

contamination ranged from 0.7 to 8009 ngllO mg hair; the amounts in the washes ranged from 4% to over 

2000% of the drug content of the hair. The wash procedure used for these samples was as follows. First, 

dry isopropanol (2 mL) was added t6 about 12 mg of hair in 12 x 75 mm tubes; the tubes were shaken 

vigorously at 37"C for 15 minutes, after which the isopropanol was removed to a separate tube and saved 

for later analysis. Then 2 mL of 0.0 1 M phosphate buffer/O.O 1 % BS:A, pH 6, was added to the hair samples 

in the tubes and the tubes shaken vigorously for 30 minutes at 37"C, after which the buffer was removed 

and saved to a another tube for later analysis.· This 30-minute wash was repeated twice more, followed by 

two 6O-minute washes using the same conditions. :After the final (Sib) phosphate buffer wash and removal 

of the buffer, the hair sample was enzymatically digested for confirmation by LCIMSIMS. The washes are 

routinely analyzed by RIA. The wash criterion is computed by multiplying by 5 the amount of drug per mg 

hair in the last wash and subtracting the result from the amount of drug per mg hair in the hair digest. The 

Wash Criterion is actually an overestimate of the amount of drug that would be removed by 5 additional 1 

hour washes. If the result after the subtraction is less than the cutoff for the parent drug, the result is 

considered negative in indicating drug use. The parent-drug cutoff value for cocaine was 5 ng 110 mg hair ( 

500 pg/mg). 

The effectiveness of the wash procedure was challenged by contaminating negative hair (including blonde, 

auburn, brown and black samples) by soaking for one hour in 1000, 10,000, and 50,000 nglmL cocaine. 

These amounts far exceed any likely real-life contamination scenarios. :After drying, the samples were 

washed by the procedures described and digested for confirmation by LC/MSIMS The uptake of the 

cocaine was approximately linear with increasing concentrations of cocaine in the soaking solutions, and 

the amounts of cocaine in the hair and washes were correspondingly increased. Total cocaine measured in 

the wash and hair ranged from 20.6 to 66.5 ng cocainellO mg hair for those samples soaked in 1000 nglm; 

from 185.6 to 590.7 for those samples soaked in 10,000 ng/m; and 885.6 to 1916.7 for those soaked in 

50,000 ng/mL. By application of the wash criterion, all samples were clearly identified as contaminated. 

The values for hair digests after application of the wash criterion were -0.04 to 1.45 ng/IO mg hair for 

samples soaked in 1000 ng/mL; -18.28 to 2.83 for those soaked at 10,000 nglml; and -258.0 to -42.9 for 

those soaked at 50,000 ngllO mg hair. Thus the wash procedure and criterion were effective even for 

extreme conditions of contamination by soaking. 

Keywords: Hair, Contamination, Washing 

Page 291

M9 

SCREENING OF BENZODIAZEPINES AND HYPNOTICS IN HAIR BY LC-MSIMS: 

APPLICATION TO FOUR DRUG-FACILITATED CRIMES 

M. VilIain 1 *, M. Cheze 2 , B. Ludes 1 , P. Kintz!; 1 Institut de Medecine Legale, II rue Humann, 67000 

Strasbourg, France and 2 ToxLab, 7 rue Jacques Cartier, 75018 Paris, France 

The use of a drug to modifY a person's behavior is not a recent phenomenon, but reports dealing with drugfacilitated 

crimes (sexual assaults, robbery ... ) are constantly increasing. Among drugs involved in such 

cases, benzodiazepines (flunitrazepam, bromazepam, ... ) and hypnotics (zopiclone, zolpidem) are largely 

concerned. 

In most cases and due to a long delay between the event and the claim to the police, blood and even urine 

samples are of little value. This is the reason why this laboratory developed a original approach based on 

hair testing. In practice, hairs of the victim are collected about one month after the alleged offense, 

segmented and analyzed by LC-MSIMS. In positive cases, the drug is supposed to be found only in the 

segment corresponding to the period ofthe event. 

The Hair strand is twice decontaminated using methylene chloride and then segmented (3 segments of 2 

cm). Each segment is cut into small pieces «lmm). About 20 mg are overnight incubated in phosphate 

buffer at pH 8.4, in the presence of 1 ng of diazepam-ds used as internal standard (IS). After a liquid-liquid 

extraction with a mixture of methylene chlorideldiethyl ether (80/20) and evaporation to dryness, the 

residue is reconstituted in 50 J.1L of acetonitrile/water (50/50). 

Chromatography is achieved using a XTerra MS CI8 column (100 x 2.1 mm, 3.5J.1m) eluted with a 

gradient of acetonitrile and formate buffer delivered at a flow rate of 0.2 mLlmin. 

A Quattro Micro triple-quadrupole mass spectrometer is used for analyses. Ionization is achieved using 

electrospray in the positive ionization mode (ES+). For each compound, detection is related to two 

daughter ions. 

To demonstrate the applicability ofthis method, 4 expertises are describe as follows: 

1. A-21 year old girl was offered a coffee that made her unconscious. The victim's hair, collected 15 

days after the alleged offense, was tested by LC-MSIMS and the first 2 cm segment was positive 

at a concentration of4.4 pg/mg whereas the 2 other segments were zolpidem free. 

2. A 16-year old girl claimed to have been raped during an afternoon, while sedated. Hair was 

sampled 9 weeks after the alleged offense and the first 3 cm segment was positive for zopiclone at 

a concentration of 4.2 pg/mg, the second (3 to 5 cm) at 1.0 pg/mg, whereas the last segment (5 to 

7 cm) was zopiclone free. 

3. A 39-year old woman felt sleepy for 24 hours after having drunk a coffee. Hair was collected I 

month after the event and the proximal 2 cm-Iong segment was positive for bromazepam at 10.3 

pg/mg and the other segments (2-4 and 4-6 cm) remained negative. 

4. A woman was confined illegally for 12 days, repetitively raped and forced to sign .cheques, while 

being under the influence of an hypnotic. Hair was positive for 7-aminoflunitrazepam in the 

proximal segment (0 to 3 cm) at 31.7 pg/mg, in the second segment (3 to 6 cm) at 2.0 pg/mg and 

was negative in the distal one (6 to 9 cm). 

In drug-facilitated crimes, hair is a very useful alternative specimen, and the concentrations measured, in 

the pg/mg range, require tandem mass spectrometry. 

Keywords: Hair, LC-MS/MS, drug-facilitated crime 

Page 292

MIO 

QUANTIFICATION OF AMPHETAMINE AND METHAMPHETAMINE ENANTIOMERS IN 

HAIR SPECIMENS BY GC-MSINCI 

Liliane Martins!, Michel Yegles 1 , Heesun Chung 2 , Robert Wennig l *: I Laboratoire National de Sante, 

Toxicologie, CRP-Sante, Universite du Luxembourg and 2 National Institute of Scientific Investigation, 

Seoul, Korea 

Introduction: Enantioselective separation of amphetamines is of great importance in clinical and forensic 

toxicology, as the S-(+)-enantiomers of methamphetamine (MA) and amphetamine (AM) are known to be 

considerably more stimulant than the R-(-) isomers. The determination of their respective enatiomeric 

ratios allows the distinction between legal intake and illicit MA and/or AM consumption. 

A method describing the chiral separation of AM and MA after derivatisation with S-(-) 

heptafluorobutyrylprolyl chloride (S-HFBPCI) and quantification by gas chromatography coupled to mass 

spectrometry operating in the negative-ion chemical ionization mode (GC-MSINCI), has already been 

applied to serum, urine and oral fluid. In the present study the previous method was modified to be suitable 

for abusers' hair analysis. 

Material and methods: Hair specimens were obtained from I I Korean suspected MA abusers. After 

digestion with 1 M sodium hydroxide at 100°C during 30 min, followed by solid phase extraction using 

Cleanscreen ZDAU and derivisation with S-HFBPCI, the enantiomers were quantified by GC-MSINCI 

using deuterated internal standards were operated. Extraction yields varied between 88.5 and 100 %, LODs 

were 10.6 and 2.2 pglmg hair for MA and AM respectively. 

Results: Concentration ranges of 25.2 to lOlA ng/mg hair for MA and 0.69 to 4.54 nglmg hair for AM 

were observed. For 2 specimens, only the S-(+)-enantiomer was detected, for 8 specimens the RlS ratio was 

of 10.1 and 1.2 was 

determined for MA and AM respectively. 

Conclusions: Similar to serum, urine and saliva, our results point out the predominance of the S-(+) 

enantiomer in hair. In one case the R-(-)-MA concentration was higher than the S-(+)-enantiomer. This 

may be due to the consumption of prescription drugs either containing R-(-)-MA or biotransformed to R-( 

)-MA. The present study shows that hair analysis of amphetamines' enantiomers may be helpful to avoid 

misinterpretation in the discrimination between prescription drug use and illegal drug consumption. 

Keywords: Hair analysis, Enantioselective Separation, Amphetamines 

Page 293

Mll 

DIAGNOSIS OF CHRONIC ALCOHOL CONSUMPTION. HAIR ANALYSIS FOR ETHYL 

GLUCURONIDE 

C. Jurado·, T. Soriano, M.P. Gimenez, M. Menendez :Instituto Nacional de Toxicologfa. P.O. Box 863. 

41080 Sevilla. Spain 

Alcohol abuse is one ofthe most serious social problems throughout the world. Ethyl-glucuronide is minor 

metabolite of ethanol and has proved to be a good alcohol marker. In addition, hair analysis has 

demonstrated its usefulness in establishing chronic consumption of drugs of abuse. The objective of the 

present study is to develop and validate a sensitive, precise and specific analytical method for the 

determination of ethyl-glucuronide in hair samples. 

During method development the efficacy of extraction of EtG from hair was compared in four extraction 

methods: a) methanol; b) methanol:water (1:1); c) water; and d) water:trifluoroacetic acid (9:1). Water was 

found to be the best extracting solvent, since it provided the highest recoveries, with cleaner extracts. The 

final method is as follows: about 100 mg of hair are sequential\y washed with water and acetone. The 

decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d 5 ) and 2 mL 

of water are added. After sonication for two hours, the sample is maintained at room temperature overnight 

Derivatization is performed with PFPA. Derivatives are injected into a GCIMS system in the electronic 

impact mode. The method shows linearity over the range of concentrations from 0.050 ng/mg to 5 ng/mg. 

Detection and quantification limits are 0.025 ng/mg and 0.050 ng/mg, respectively. Mean recoveries for the 

three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in 

intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab 

for the diagnosis of chronic alcohol consumption. Some examples will be shown. All of them derived from 

divorce proceedings where chronic alcohol consumption was required by the Court. EtG concentrations 

ranged from 0.05 to 0.75 ng/mg of hair. 

Keywords: Ethyl-glucuronide, Alcohol Markers, Hair Analysis 

Page 294

.~. 

M12 

OPIATE CONCENTRATIONS IN HAIR FROM SUBJECTS IN A CONTROLLED HEROIN 

MAINTENANCE PROGRAM AND FROM OPIATE-ASSOCIATED FATALITIES 

MusshoffF*, Lachenmeier K., Wollersen H., Madea B.: Institut ofLegaJ Medicine, Bonn, Germany 

Introduction: The objective of this study was the determination of the concentration of opiates (heroin = 

HER, mono acetyl morphine MAM, morphine = MOR, codeine COD, acetyl codeine AC) in hair, 

and in particular an investigation of dose-concentration relationships as well as the possibility of using AC 

as a marker for consumption of illicit heroin in contrast to pharmaceutical heroin. 

Methodology: After the controlled Lv. administration of pharmaceutical heroin-HCI (l0-1000 mg/d), the 

concentrations of opiates in head hair were determined (n = 46), using a validated GC-MS method with 

LOD's between 0.02 and 0.04 ng/mg. In addition, a collective of opiate-associated fatalities was examined 

(n 24). 

Results: The concentrations obtained in the proximal segment (1 cm) of the patients were between 0.04 

and 0.21 ng/mg for HER, for MAM betv.een 0.05 and 5.64 ng/mg and for MOR between 0.03 and 8.37 

ng/mg. There was no statistically significant difference in the concentrations in comparison to the opiate 

fatalities (HER 1.55-5.20 ng/mg, MAM 0.04-30.01 ng/mg, MOR 0.03-11.87 ng/mg in the proximal 

segments). After controlled heroin administration, a correlation between the dose and the total opiate 

concentration in the hair was found (r=0.66). When considering a single analyte, the coefficient of 

correlation increased when plasma half-life increases (r=OA2, r=0.58 and r=O.69 for HER, MAM and 

MOR). COD and AC were detected in 13.0 % and 10.9 % of the samples of the heroin program, as well as 

in 33.3 % and 16.7 % in opiate-associated fatalities, respectively. 

Discussion: The results confirm the first observations of Kintz et aI., who found only limited doseconcentration 

relationships after HER abuse in hair. The correlation is influenced by the plasma half-lifes 

of analytes. The lack of differences between obtained opiate concentrations in the hair of participants in a 

controlled heroin maintenance program and of opiate-associated fatalities does not support the hypothesis 

that a lack of tolerance can be regarded as a potential cause of death. In addition, the absence of AC also in 

the majority ofthe deaths questions its suitability as a characteristic marker of a preceding consumption of 

illicit heroin. 

Keywords: Hair analysis, opiates, heroin maintenance program 

Page 295

M13 

EVIDENCE OF ADDICTION BY ANAESTHETISTS AS DOCUMENTED BY HAIR ANALYSIS 

P. Kintz*, M. Villain, B. Ludes: Institut de Mectecine Legale, 11 rue Humann, F-67000 Strasbourg, France 

Chemical dependency is a disease that can affect all professions. Among the health care professionals, 

anesthesiologists represent a specific group. Numerous factors have been proposed to explain the high 

incidence of drug abuse among anesthesiologists. These include: easy access to potent drugs, particularly 

narcotics, highly addictive potential of agents with which they are in contact, and easy diversion of these 

agents since only small doses will initially provide an effect desired by the abuser. Opioids are the drugs of 

choice for anesthesiologists, and among them fentanyl and sufentanil are the most commonly used. 

Alcohol, mostly in older anesthesiologists, propofol, ketamine, thiopental and midazolam are also abused. 

In fact, all but quaternary ammonium drugs can be observed. Symptoms ofaddiction in the hospital include 

: unusual changes in behavior, desire to work alone, refuse of lunch relief or breaks, volunteer for extra 

cases, call, come in early and leave late, frequent restroom breaks, weight loss and pale skin, malpractice, 

behind on charts, etc. Toxicological investigations are difficult, as the drugs of interest are difficult to test 

for. In most cases, half-lives of the compounds are short, and the circulating concentrations weak. It is 

therefore necessary to develop GCIMS/MS or LC/MS procedures to satisfY the criteria of identification and 

quantitation. In most cases, blood and/or urine analyses are not useful to document impairment, as these 

specimens are collected at inadequate moments. Hair analysis appears therefore as the unique choice to 

evidence chronic exposure. Depending the length of the hair shaft, it is possible to establish an historical 

record, associated to the pattern of drug use, considering a growth rate of about 1 crn/month. 

After decontamination with dichloromethane, drugs are extracted from the hair after incubation in pH 8,4 

phosphate buffer either by Iiquidlliquid extraction or headspace technology. Opiates, fentanyl derivatives, 

benzodiazepines and propofol are analyzed by GCIMS, GCIMS/MS, LCIMS/MS and headspace GCIMS, 

respectively. 

The following cases will be reviewed: 

Case 1: 50-year old anaesthetist, positive for fentanyl (644 pglmg) 

Case 2: 42-year old anaesthetist, positive for fentanyl (101 pg/mg) and sufentanil (2 pglmg) 

Case 3: 40-year old anaesthetist, positive for codeine (210 pglmg), alfentanil (30 pg/mg) and 

midazolam (160 pglmg) 

Case 4: 44-year old nurse, found dead, positive for midazolam (760 pglmg) and propofol (1390 

pglmg) 

In these cases, the combination of an alternative specimen (hair) and hyphenated analytical techniques 

(tandem mass spectrometry) appears as a pre-requisite. 

Keywords: Addiction, Anaesthetist, Hair 

Page 296

M14 

CLOZAPINE DOSE-HAIR CONCENTRATION RELATIONSHIPS: EVALUATION OF 

INPA TlENT AND OUTPA TlENT DATA 

Robert Kronstrand*, Ingrid Nystrom, Markus Roman, and Martin Josefsson: National Board of Forensic 

Medicine, Dep. Forensic Chemistry, Linkoping, Sweden 

The aim of this work was to test the hypothesis that the concentration of clozapine in hair is dependent on 

the dose administered. The hypothesis was tested both in an inpatient (N=22) and an outpatient (N=41) 

population, with and without correction for hair pigmentation. 

Samples were taken from the head and stored dark at room temperature until analyzed. Five cm of each hair 

sample was analyzed. Briefly, the hair was cut into small pieces and weighed into a 10 mt screw-capped 

tube. To approximately 25 mg of hair were added 0.5 mL of mobile phase (10:10:80 mixture of 

acetonitrile:methanol:20mM formate buffer pH 3.0) and 25 /-ll of internal standard (2.0 Ilg/ml of d3 

mianserine) and the sample was incubated in a water bath (with orbital shaking) at 37 °C during 18 hours. 

A 150-/-ll aliquot was transferred to an autosampler vial and 10 /-ll were injected into the chromatographic 

system. 

The LC-MS-MS analysis was performed on a Perkin Elmer Series 200 chromatography system consisting 

of a Series 200 pump, Series 200 auto sampler, and a SCIEX API 2000 MS-MS instrument equipped with 

an electrospray interface (Turbo Ion Spray). We used a 50x2.l mm Zorbax SB-Cyano analytical column 

with 3-fJ.m particle size. For clozapine, two transitions, 327.11270.0 and 327.1/192.2, were monitored and 

for d3-mianserine 268.31208.2 was monitored. All samples were also analyzed for melanin using 

spectrophotometry (Iambda=550 nm) as previously described (Kronstrand et al. Clinical Chemistry 1999, 

45:9, pp 1485). The daily doses ranged from 125 to 800 mgs. 

The hair melanin concentrations ranged from 5 to 49 /-lg/mg and the clozapine concentrations ranged 

between 1.1 to 37 nglmg. In the outpatient population a coefficient of determination of r=0.14 was achieved 

when correlating the dose with the hair c10zapine concentration, as compared to 0.36 for the inpatient 

population. When melanin was taken into account the coefficients were increased to 0.36 (out) and 0.62 

(in), respectively. The results indicated that melanin influenced the incorporation of c10zapine into hair and 

that the outpatients might have been less compliant than the inpatients, and thus obscuring the results. In 

summary, it seems that the more controlled the dosing is, and the more factors that influence incorporation 

are taken into consideration, the better the relationship between dose and hair concentration. Our 

hypothesis was only confirmed under the certain circumstances of controlled dosing and melanin correction 

(see figure), otherwise no correlation could be found between the dose and the hair c10zapine concentration. 

Inpatient Dose vs CLZlMEL 

1,6 

1,2 • 

r=O,62 

01 

• • • 

• • 

~ 0,8 

c 

0,4 

0,0 I 

100 300 500 700 900 

mg/day 

Page 297

M15 

DRUG DETECTION IN ORAL FLUID: IDENTIFICATION OF POLY DRUG USE BY EIA AND GC 

MS 

Joe Clarke 2 *, Lolita Tsanaclisl , and John Wicks! : !Tricho-Tech Limited, The Cardiff Medicentre, Heath Park, 

Cardiff, CF14 4UJ, United Kingdom.; 2Altrix Healthcare Pic, Birchwood Science Park, Warrington, WA3 7BP, 

United Kingdom. 

The analysis of drugs of abuse in oral fluid is becoming more widely used and accepted across a number of 

testing disciplines, particularly because of its simplicity to collect in relation to urine. Commonly abused and 

prescribed drugs are known to be detectable in oral fluid. The objective of this study was to assess the prevalence 

of a number of drugs in oral fluid with specimens collected from known and unknown drug users. The cut-off 

levels for oral fluid are much lower then those for urine. Certain analytical methods that are used with urine 

samples, including immunoassay and GC-MS, are not necessarily applicable for screening oral fluid specimens. 

This study provides a compilation of data for the confirmation by GC-MSIEI, GC-MS/CI or GC-MS/MS of oral 

fluid for a wide range of analytes. Oral fluid specimens (N=8911) were collected with prior consent from various 

establishments in the U.K including private companies, rehabilitation clinics, and criminal justice services using 

the Intercept® DOA collection device (OraSure Technologies, USA) according to manufacturer's instructions. 

Specimens were initially screened for a combination of Opiates, Cocaine metabolites, Cannabinoids, 

Amphetamines, Benzodiazepines and Methadone using separate OraSure Technologies Inc. micro-plate enzyme 

immunoassays and Buprenorphine (Diagnostix Ltd, Canada), according to manufacturer's instructions. 

For the confirmation analysis by Gas Chromatography-Mass Spectrometry (GC-MS), the samples were cleaned 

using Oasis® MCX Cartridges and derivatised with TFA or BSTFA (Sigma, Poole, UK). The derivatised 

extracts were injected onto either gas-chromatograph HP5973 for GC-MSIEI and GC-MS/CI (Agilent, Berkshire, 

UK) or Varian Inc. Saturn for GC-MS/MS (Walton-on-Thames, UK) equipped with capillary column 15mxO.25 

mm Varian, Factor Four. The dynamic range was from 2 to 2000 ng/mL. Three ions for the drugs and two ions 

for the internal standards were monitored. The confirmation rate for each drug group, relative to applied cut-off 

levels, is presented in Table I. 

.r--" 

Table 1. Total oral fluid sampl es teste db)y GC.MSt)oreachdrug ~oup and con fiIrmatlon rates. 

GCMS 

Confirmation Rate 

Drug Group N N % 

Amphetamines 337 305 (91%) 

Benzodiazepines 1027 823 (80%) 

Buprenorphine 175 104 (59%) 

Cannabis 632 316 (50%) 

Cocaine 1358 1026 (76%) 

1 

Methadone 1536 11437 (94%) 

Omates 116460 15189 (80%t I 

Percentiles: 50% (Median), 95% and maximum levels for all analytes in each drug group were calculated and 

will be presented. Detailed results for the Cocaine group of analytes are shown in Table 2. 

Table 2. Results showing Percentiles: 50% (Median), 95% and maximum levels in ng/mL for Cocaine group. 

Median 95% Maximum N 

AEME 62.0 1409.1 3458.9 75 

BenzoyJecgonine 49.1 566.0 5240.2 865 

Cocaethylene 12.3 101.4 136.7 33 

Cocaine 3004 2135.6 29613.4 843 

Oral fluid testing offers a convenient, reliable and consistent method to determine the presence of poly-drug use 

and can identifY the presence of specific drug metabolites by the GC-MS methods established. 

Keywords: Drugs of Abuse, Oral Fluid, GC·MS 

Page 298

M16 

VALIDA TION OF THE COZAR~ 

AMPHETAMINES IN HAIR 

MICROPLATE ELISA FOR THE DETECTION OF 

Gail Cooper* and Chris Hand: Cozart Bioscience Ltd, Abingdon, Oxfordshire, OXI4 4RU 

The purpose of this study was to determine the performance characteristics of the Cozart® Amphetamine 

Microplate ELISA assay for the detection of amphetamines in hair samples. Hair samples (N=50) were 

collected from volunteers with a history of drug use and from drug related deaths. The hair samples were 

sonicated in methanol and then extracted overnight at 60°C. The methanol extract was evaporated to 

dryness and reconstituted in ELISA negative calibrator. 251lL of calibrator (0, 50, 100,200,300,500 and 

1000pg/mg), control and sample were assayed in duplicate according to manufacturers instructions for the 

Cozart@ Amphetamine ELISA - Forensic Application Kit. 

For GC-MS analysis, deuterated internal standards (amphetamine-d5, methamphetamine-d5, MDA-d5, 

MDMA-d5, MDEA-d6 and MBDB-d5) mixture, p-glucuronidase and O.lM, pH 6.0 phosphate buffer were 

added to approximately 20 mg of sample, or spiked blank hair and sonicated for I hour. Isolation of 

amphetamine, methamphetamine, MDA, MDMA, MDEA and MBDB was achieved through simple solidphase 

extraction using Bond Elut certifY cartridges. 

The true positives (N=20), true negatives (N=30), false positives (N=O) and false negatives (N=O) for 

different cut-offs with the ELISA were determined by comparison of the ELISA response (normalised to 

weight of hair extracted) to the GC-MS results as the reference method. Hair concentrations ranged from O. 

- 10.1 ng/mg (amphetamine), 0 - 0.8 ng/mg (MDA) and 0 17.4 ng/mg (MDMA). 

.r-~. 

The optimum cut-off for the Cozart® Amphetamine Microplate ELISA was determined to be 500pg 

amphetamine equivalents/mg hair using a 20 mg hair sample with an overall precision of 100% vs GC-MS. 

Keywords: Amphetamines, Hair, ELISA 

Page 299

M17 

CANNABINOID ANALYSIS OF ORAL FLUIDS COLLECTED FROM CLINICAL SUBJECTS 

FOLLOWING ORAL TETRAHYDROCANNABINOL (THC) ADMINISTRATION (HEMP OIL 

AND DRONABINOL) 

William D. Darwin l *, Richard A. Gustafson 2 , Jason Sklerov 3 , Dean Fritch 4 , Aaron Jacobs\ Kristen Blum 4 , 

Sam Niedbala 4 , Eric T. Moolchan l , and Marilyn A. Huestis' : IChemistry and Drug Metabolism Section, 

IRP, NIDA, NIH, 5500 Nathan Shock Drive, Baltimore, MD 21224; 2Jacksonville Naval Air Station, 

Jacksonville, FL 32212; 3Armed Forces Institute of Pathology, Research Blvd., Rockville, MD 20850; 

40raSure Technologies, Inc., 150 Webster Street, Bethlehem, PA 18015 

Therapeutic usefulness of oral cannabinoids is being investigated to address analgesia, AIDS-wasting 

disease, counteracting spasticity of motor diseases, and emesis following chemotherapy. In addition, 

cannabis is among the most abused psychoactive substances. Following cannabis use, physiological and 

behavioral changes occur necessitating testing to detect impairment. In recent years, food products (hemp 

oil) derived from cannabis have been introduced into the U.S. Analysis of hemp oils reveals that THC is 

present in a wide concentration range. If these products produce positive drug test for urine and alternate 

matrices, screening programs to identify cannabis use may be affected. A clinical study to investigate the 

pharmacokinetics and pharmacodynamics of oral THC was performed. The randomized, double blind, 

placebo-controlled within-subject, inpatient study compared the effects of hemp oil in liquid and capsule 

form to dronabinol (synthetic THC) doses consistent with therapy for appetite-stimulation. Hemp oil 

dosing followed manufacturer's recommendations. Five dosing conditions (0.0, 0.39, 0.47, 7.5, and 14.8 

mg/day) of oil/capsules were administered three times daily with meals for five consecutive days. One 

objective was to determine if hemp oil could produce positive cannabinoid alternative matrices tests. Two 

devices were used to collect oral fluids, OraSure (OraSure Technologies) and Salivette (Sarstedt) from 90 

min after the first dose to 1 day after the last dose. Salivette samples were analyzed by LCIMS. 

LiquidlIiquid extractions isolated THCCOOH from ll-OH-THC and THC. Extraction efficiencies were 

280%. An LCIMSD (Agilent) with an Eclipse C18 column and electrospray interface with data acquired in 

the NCI mode was used for analysis. SIM ions monitored were: THCCOOH, mlz 343.2; d3-THCCOOH, 

mlz 346.2; 11-0H-THC, mlz 329.2; d3-11-0H-THC, mlz 332.2; THC, m/z 313.2; and d3-THC m/z 316.2. 

Limits of quantitation (LOQ) were 1.0, 0.5, and 0.5 ng/mL and limits of detection (LOD) 0.5, 0.2, and 0.2 

ngimL for THCCOOH, ll-OH-THC, and THC, respectively. 511 Salivettes from 7 subjects were 

analyzed. OraSure oral fluid specimens were analyzed according to manufacturer's specifications with 

Cannabinoids InterceptTM MICRO-PLATE Enzyme Immunoassay (OraSure Technologies) with a 12 

ngimL cutoff and confirmed by GC/MS/MS. After SPE, extracts were derivatized with HFPA/PFIP. 

Extraction efficiency was 50%. THC was analyzed on a Saturn Ion Trap Quadrupole (Varian) equipped 

with a 5% phenyl-methyl column. Ions monitored were: THC, mlz 492 and d3-THC, mlz 495 (parent 

ions); THC, m/z 238 and d3-THC, m/z 238 (product ions). LODILOQ for THC were 0.75 ngimL. 497 

OraSures from 8 subjects were analyzed. All Salivette and OraSure oral fluid specimens were negative at 

the specified cutoffs. In contrast, several authors reported cannabinoid positive results for Salivette and 

OraSure oral fluid samples after smoked THC (Samyn, J Forensic Sci, 2002, 47: 1380; Niedbala, JAT, 

2001,75:289). In addition, Niedbala found positive tests for OraSure oral fluid samples collected after oral 

THC (20-25 mg THC-laced brownie), but concentrations were low ('5.7 ngimL). THC is well absorbed, but 

has low bioavailability «20%) via the oral route. There is evidence that deposition ofTHC into oral fluid 

is via direct sequestering into oral mucosa during drug use with minimal contribution from blood to oral 

fluid (Hawks, The Cannabinoids, Academic Press, 1983, 8). Characterization of rates of absorption and 

elimination of orally administered THC with different vehicles (flax oil, sesame seed oil, cookies/brownies, 

and emulsions) and with direct administration and encapsulation have shown variable absorption ofTHC. 

Little data have been published about oral fluid drug levels after oral THC administration; questions remain 

including the degree of absorption inherent to the collection devices and THC contamination of the oral 

cavity immediately following hemp oil/capsule administration. The fact that all oral fluid specimens 

collected were negative with two different devices for THC-containing hemp oils and capsules suggests 

little contamination of the oral fluid following ingestion of dronabinol or hemp oils by the time the first 

specimen was collected, at least by 90 minutes after dosing. 

Keywords: OraSure, Salivette, Oral Administration ofTHC 

/--.'~ 

Page 300

MI8 

COMPARISON OF ALCOHOL CONCENTRATIONS IN POSTMORTEM BODY FLUIDS 

Bruno Spinosa De Martinis*, Adriano Braga, Henrique Turin Moreira, Carolina Melo Candido de Paula 

and Carmen Cinira Santos Martin: Department of Pathology, Center of Legal Medicine, Faculty of 

Medicine of Ribeir1io Preto, University ofS1io Paulo. Rua Tenente Camo Roxo 2418, Ribeirao Preto, Sao 

Paulo, 14051-140, Brazil 

The determination of postmortem ethanol is probably one of the most important and frequently requested 

analyses in forensic toxicology. Because of its hydrophilic characteristics, ethanol distributes with body 

water and studies have demonstrated the existence of substantial site dependence. Factors such as quality 

and sampling site; trauma to the body; length of time between death and sampling; presence of 

microorganisms in the body; diffusion of alcoholic beverages present in the stomach into the pericardial 

fluid; diffusion of ethanol in aspirated vomitus into cardiac blood; blood contamination and unavailability 

of blood samples must be considered to interpret the postmortem blood ethanol concentration. Due those 

factors, there is an enormous effort to find alternative sampling sites and correlations between blood 

alcohol concentration and other biological fluids to establish different procedures for specimens to sample 

for alcohol analysis. 

In addition, considering the medicolegal purposes, the implications of postmortem alcohol concentrations 

are extremely important, especially when they are found to be above the legal level for intoxication at one 

specimen and below that level at another. 

The use of vitreous humor as an alternative specimen for ethanol analysis in postmortem cases has 

increased, because it is stable, readily available, easily sampled and less susceptible to bacterial 

contamination. 

In this work it was determinated the ethanol concentrations in biologic fluids, collected during the autopsy 

examination from 103 cadavers victims of different cause of death, to establish a correlation between the 

concentrations of ethanol in vitreous humor with urine and blood specimens, sampled from femoral, 

subclavian and heart. 

Determinations of ethanol in the specimens were performed using capillary gas chromatographylflame 

ionization detector and headspace techniques. 

Statistical analysis ofthe results indicated that there were no significant differences among urine and blood 

samples, collected in different sites, with vitreous humor. 

Comparing the vitreous humor ethanol concentration with respect to Pearson's correlation test, it was found 

0.97 for femoral blood and urine, 0.96 for heart blood, 0.94 for subclavian blood. These results demonstrate 

that all the fluids tested with vitreous humor were significantly correlated with "p" (associated probability 

for the used correlation tests) being always lower than 0,05. It indicates that vitreous humor can be use as 

an alternative sample for urine and blood. 

Keywords: Postmortem Specimens, Multisite Sampling, Ethanol. 

Page 301

M19 

METHOD DEVELOPMENT OF MICROWAVE-ASSISTED EXTRACTION AND 

SUPERCRITICAL FLUID EXTRACTION OF SOME PERSISTENT ORGANIC POLLUTANTS 

IN HUMAN SEBUM 

Diaz-Vazquez, Liz M.*; Garcia, Oscar; Madero, Mayraliz; Velazquez, Zorangelys; and Rosario, Osvaldo, 

Department of Chemistry, University ofPuerto Rico, Rio Piedras Campus, P.O. Box 23346, San Juan, P.R., 

00931-3346 

Sebum has been used to monitor use of controlled drugs as well as exposure to dioxins. Based on this, it is 

logical to extrapolate its use to also monitor exposure to other environmental pollutants. Methodology is 

being pursued which will allow the analysis of persistent organic pollutants (POPs) through sebum testing. 

Statistical Analysis methods based on Minitab® software were applied to optimize microwave-assisted 

extraction (MAE) and supercritical fluid extraction (SFE) conditions for the analysis of some standards 

representative of POPs. For practical purpose a model matrix of synthetic sebum spiked with the following 

standards: 1,4- benzodioxan, 1,2,4, 5- tetrachlorobenzene, aldrin, dursban and pyrene was used for the 

optimization of the extraction techniques. A stock solution of the model matrix was prepared as follow: 

synthetic sebum was dissolved in dichloromethane and the standards of interest were spiked into the 

solution. An aliquot 31lL of the spiked sebum solution was taken, deposited on the sebutape® and the 

solvent allowed to evaporate. The final concentration of the standards, if a 100 % recovery was obtained, 

was 3 ppm. The Recoveries of analytes were followed using GC-MS. All compounds were successfully 

extracted from sebum with recoveries ranging from 69 for 1,4 -benzodioxan to 91 % for dursban under the 

optimum MAE conditions: 5 mL acetonitrile, 70°C extraction temperature, and 10 min microwave heating. 

The average recoveries in SFE ranging from 84 for 1,4-benzodioxan to 95 % for pyrene under the 

following conditions: 30 min of extraction at 40°C, under CO 2 at 4000psi modified with 20% (v/v) 

methanol. The addition of a small volume (10-20 %) of methanol to the extraction cell enhanced the 

recoveries of representative persistent organic pollutants. A comparison of MAE, SFE was also conducted. 

The results indicated that ~ 69 % average recoveries were obtained by both optimized techniques. Good 

precision with RSD less than 10% was attained for most of the standards with both techniques. Although 

with SFE higher percents of recoveries were obtained, it also is more efficient extracting the components of 

the sebum like saturated hydrocarbons and fatty acids than MAE. In addition to further optimizing our SFE 

approach, both MAE and SFE are now being compared with ultrasonic extraction commonly used for the 

extraction of biological matrices. Solid phase extraction, with different stationary phases, was used to 

remove sebum components from the extract. Also SPME was tested to improve the detection limits. Real 

samples of human sebum are being analyzed from an area impacted by the emission from an incinerator of 

biomedical wastes. The compounds found in these persons will be compared to compounds found in air 

samples from the same region. This will show if specific air pollutants are being incorporated by this 

population. Once completed and validated this noninvasive approach will provide a powerful method for 

monitoring exogenous compounds in human. 

Keywords: Microwave-Assisted Extraction, Supercritical Fluid Extraction, Sebum 

Page 302

M20 

DRUG-FACILITATED CRIMES - INTEREST OF TANDEM MASS SPECTROMETRY AND 

HAIR ANALYSIS - STATISTICS OF DRUGS IMPLICATED DURING A YEAR OF WORK AT 

TOXLAB 

Duffort G.*, Cheze M., Pepin G. 

TOXLAB Laboratory, 7 rue Jacques Cartier -75018 Paris - FRANCE 

AIMS: Drug-facilitated crimes cases are often difficult to resolve, from an analytical point of view, since 

the drugs administered, largely benzodiazepines or hypnotics, are generally given as one therapeutic dose, 

and most often have a short half-life elimination. In France, for these kind of aggression cases, the police 

directs the victim towards a hospital or, in big cities, to an emergency forensic service office. Ifnecessary, 

after the clinical exam, blood and sometimes urine samples are taken. Using HPLCIDAD and/or GCIMS, 

immunochemical as well as toxicological screenings are performed on these samples. The immunochemical 

techniques constantly produce negative results. HPLCIDAD and GCIMS do not permit most basic 

molecules used in drug-facilitated crimes to appear because the aggression reports, and then the samples, 

are often late and thus substances are undetectable with those techniques. This is why we have developed 

an HPLC-MSIMS method for the benzodiazepines and hypnotics determination in biological fluids and 

hair. By that way, we can reach the detection limits of the nglmL order in biological fluids and of the 

pglmg order in hair. Human hair is sampled by us, about one month after the exposure. The sequential hair 

analysis by LC-MSIMS allows to affirm that the identified molecules in blood and/or in urine were in fact 

from an isolated single dose and not from a medical treatment, so it brings evidence for drug-facilitated 

crimes. 

METHODS: The detection of main benzodiazepines in biological fluids were performed by LC-MS/MS 

after an extraction of I mL with Toxitube A ® (Varian). Hair was segmented, decontaminated, cut, then 20 

mg were extracted with deuterated standard, whether one night incubation in ImL Soerensen buffer (PH 

7.6), or whether, for aminometabolites, at 95° for 15 minutes in NaOH O,IN. A liquid-liquid extraction was 

performed with dichloromethane/ether (80/20). The organic phase was filtered before being evaporated. 

The dry extract was reconstituted by 60JlL of ACNlMeOH (50/50) and injected into the LC-MS/MS 

(ThermoElectron). The liquid chromatography was carried out on an Uptisphere ODB C18 51-! 150 x 2mm 

column (Interchim) with a gradient of acetonitrile and formate 2mM pH3 buffer. The run was 15 minutes 

long. The mass tandem TSQ Quantum detection allowed the simultaneous determination of 27 

benzodiazepines in SRM mode with two runs. 

RESULTS: The detection limits reached by these techniques, are in the order or inferior to 0.5 nglmL in 

urine and about 1 pglmg in hair for the 27 benzodiazepines studied. We present two cases of drug-facilitated 

crimes in which cIonazepam and flunitrazepam were found in blood and urine samples. In these two cases, 

a single dose of these molecules appeared on the specific segment of hair corresponding to the period of the 

offense. From June 2003 to May 2004 we treated 90 cases of presumed drug-facilitated crimes, for which 

we had urine and/or hair samples. In 35.5% ofthese cases, the benzodiazepines or hypnotics were detected. 

Among these, a single dose, taken without someone's knowledge, appeared in 65.5%. In the 90 cases, 

35.5% of the victims were vulnerable following an alcohol and/or illegal drugs consumption; in 22.2% the 

investigation showed that it was not a drug-facilitated crimes and 6.7% were not able to be solved. 

CONCLUSIONS: This technique produces an analytical response that is essential to police and justice 

authorities. It brings out criminal acts and produces the scientific proof of drug-facilitated crimes with 

benzodiazepines and hypnotics as shown by our statistics and the two specific cases cited. 

Keywords: Benzodiazepines, LC/MS/MS, Hair Analysis 

Page 303

M21 

IMPROVED GCMS ANALYSIS OF THC IN SALIVA 

Charles R. Lyle, Matthew P. Clabaugh, F. Roark Galloway*: Shimadzu Scientific Instruments, Columbia, 

MD; Nadina Giorgi: Redwood Toxicology Laboratory, Santa Rosa, CA 

One ofthe most challenging GCMS applications is the determination ofTHC in saliva. Detection for THC 

at low ppb [nglml] levels is required due to several factors including; I) rapid elimination ofTHC in saliva 

from time of exposure, 2) low amount of drug for 'active' dose and 3) small sample volume available at 

time of collection. This application demonstrates an example of how the Shimadzu GCMS QP-2010 can 

provide the instrument performance necessary to complete this challenging analysis. Samples were 

collected using the Quantisal saliva collection kit. Extractions are performed by extraction with 

hexane:ethyl acetate [9:1] with 3 % acetic acid. Derivatization is completed using 50 uL BSTFA to form 

the trimethylsilane derivative. 1.0 uL of sample extract is injected for analysis. Using the standard SPL 

2010 split/splitless injection port on the GC-2010 as a high pressure injector, the method was performed 

with a splitless injection using a Restek 3.4mm. id. SIL TEK liner. With high-pressure injection of 175 kPa, 

fast temperature ramping of35 C/min and high column flow rate, the peaks eluted from the column into the 

mass spectrometer ion source in less than 3.00 minutes. These results were obtained using a 15m X 

0.25mm X .25 urn phase DB-IMS column. Calibration from 1.0 25 ppb THC produced a linearity 

coefficient of 0.9999 by internal standard calculation using THC-D3 at 12.5 ppb. Preliminary LOQ is 

determined to be 0.25 ppb. Precision at 3.0 ppb THC is 5.97%. 

1.0 ppb THC [TMS] Standard Calibration, 1.0 - 25 ppb THC 

AnaR.tliu 

2. 

,. 

y2 = 0.99986 

Keywords: THC, Saliva, GCMS 

Page 304

M22 

SIMULTANEOUS ASSAY FOR NICOTINE AND ITS METABOLITES IN ORAL FLUID BY SPE 

AND GCIMS/EI. 

Insook Kim*, William D. Darwin, and Marilyn A. Huestis: Chemistry and Drug Metabolism Section, IRP, 

NIDA, NIH, 5500 Nathan Shock Drive, Baltimore, MD 21224 

Nicotine, the major alkaloid in tobacco, is rapidly and extensively metabolized in humans, but its 

metabolism varies among individuals. Nicotine is primarily metabolized by cytochrome P450 (CYP2A6) to 

cotinine, which is subsequently hydroxylated to trans-3'-hydroxycotinine. This method was designed to 

simultaneous quantifY nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine in human oral fluid, a 

noninvasive biological matrix. An aliquot (0.5 mL) of each oral fluid sample, quality control (QC) sample, 

or calibration standard was mixed with 2 mL of 2M sodium acetate buffer (pH 5.5). Deuterated internal 

standards (d)-nicotine, d)-cotinine, and d)- trans-3'-hydroxycotinine) were added to each sample. Solid 

phase extraction (SPE) columns (200 mg Clean Screen® ZSDAU020, United Chemical Technologies, 

Bristol, PA) were preconditioned with elution solvent, methanol, water, and buffer. Each sample was 

loaded onto the SPE column and washed with water, 0.2 N HCI, and methanol. Analytes were eluted with 

methylene chloride: 2-propanol: ammonium hydroxide (80:20:2 v/v/v), and 100 J.lL of 1% hydrochloric 

acid in methanol (v/v) was added prior to evaporation. Extracts were evaporated to dryness under a stream 

of nitrogen at 40°C using a Zymark Turbovap LV Evaporator. Extracted residues were reconstituted in 

acetonitrile, derivatized with BSTF A (with 1 % TMCS), and analyzed by GCIMSIEI in the selected ion 

monitoring (SIM) mode. GCIMS analysis was performed using an HP6890 gas chromatograph interfaced 

with HP5973 mass-selective detector, equipped with HP-5MS column (30m x 0.25mm Ld.; 0.25J.1m-fiIm 

thickness) with helium gas at 1.0 mLlmin. The instrument was operated in the splitless mode. The initial 

column temperature of 70°C was held for 1 min, followed by increases to 190°C at 30°C/min, to 230°C at 

5°C/min, to 290°C at 25°C/min. The ions for each analyte were monitored in the following elution order 

(quantitative ions are indicated in parenthesis) for the derivatized analytes: d)-nicotine, mlz (87), 165; 

nicotine, m/z (84), 162; d)-cotinine, mlz (101), 179; cotinine, m/z (98), 176; norcotinine, m/z (234), 219; 

d 3 

-trans-3'-hydroxycotinine, mlz (252), 147; and trans-3'-hydroxycotinine, mlz (249), 144. The base peak 

and molecular ions of each analyte and internal standards were evaluated by SIM analysis. However, the EI 

spectra of these drugs do not possess many fragments that can be used as confirming ions. The mid-mass 

range ions for these drugs contain non-specific interferences probably from co-extracted endogenous 

material. For this reason, only two ions, the base peak and the molecular ion, were monitored in this assay. 

Eight point calibration curves for nicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine were linear 

across a concentration range of 2.5 to 500 ng for all compounds/0.5 mL of blank oral fluid. Correlation 

coefficients of the calibration curves were >0.99. The limits of determination and quantitation are 2.5 

ng/0.5 mL (50 pg on column) for all analytes. Recovery was 90 - Il5% for nicotine, 76 - Il7% for 

cotinine, 88 -101% for norcotinine, and 66 -77% for trans-3'-hydroxycotinine at 8, 80, and 400 ng/O.5 mL 

(low, mid, and high, respectively), QC sample concentrations. Intra-assay precision (% CV) and accuracy 

(percent difference between mean and target concentrations) ranged from 1.6 5.7 and 1.6 - 17.8%, 

respectively, at low, mid, and high QC sample concentrations for all analytes. Inter-assay precision and 

accuracy for low, mid, and high QC sample concentrations ranged from 4.4 - 8.8 and 0.2 - 12.6%, 

respectively, for all analytes. Suitable precision and accuracy were achieved for the simultaneous 

determination of nicotine and three metabolites in the oral fluid of smokers. This assay is applicable to 

pharmacokinetic studies of nicotine and its metabolites in oral fluid and provides a biomarker for 

identification of smoking, which could be helpful in smoking cessation programs. 

Keywords: Nicotine, Nicotine Metabolites, Oral Fluid 

Page 305

M23 

~9-TETRAHYDROCANNABINOL (THC), 11-HYDROXy-~9-TETRAHYDROCANNABlNOL (11 

OH-THC) AND 11-NOR-9-CARBOXy-~9-TETRAHYDROCANNABINOL (THCCOOH) IN 

HUMAN PLASMA FOLLOWING ORAL ADMINISTRATION OF HEMP OIL. 

Wesenyalsh Nebro'*, Allan Barnes', Richard A. Gustafson 2 , Eric T. Moolchan l and Marilyn A. Huestis l : ' 

Chemistry and Drug Metabolism Section, IRP, NIDA, NIH, 5500 Nathan Shock Drive, Baltimore, MD 

21224; 2 Jacksonville Naval Air Station, Jacksonville, FL 32212 

Usefulness of therapeutic oral cannabinoids is being investigated for several medicinal applications. 

Cannabis is among the most abused psychoactive substances causing behavioral and physiological changes 

that necessitate testing to detect impairment. In recent years, hemp oil products derived from cannabis 

have been introduced into the U.S. Analysis of hemp oils reveals that THC is present in a range of 

concentrations. A clinical study to investigate the pharmacokinetics and pharmacodynamics of oral THC 

was performed. The randomized, double blind, placebo-controlled within-subject, inpatient study 

compared the effects of hemp oil in liquid and capsule form to dronabinol (synthetic THC) doses consistent 

with therapy for appetite-stimulation. The NIDA IRB approved this study and each participant provided 

informed consent. One aspect of this study was to determine the detection times and levels ofTHC, 11-0H 

THC and THCCOOH in plasma following oral THC administration. Hemp oil dosing followed 

manufacturer's recommendations. Five healthy volunteers with a history of marijuana abuse ingested 

commercially available hemp oils/capsules of differing THC concentrations. Five dosing conditions (0.0, 

0.39,0.47,7.5, and 14.8 mg/day) of oil/capsules were administered three times daily with meals for five 

consecutive days followed by a ten-day washout period before the next dosing session began. Plasma 

samples were collected prior to and following each of the five dosing conditions throughout the study and 

were frozen at -20°C until analysis. Plasma was extracted by SPE (200 mg Clean Screen® ZSDAU020, 

United Chemical Technologies). The extracts were derivatized with BSTFA (l%TMCS), separated and 

quantified on a GCIMS(Agilent) operated in the positive chemical ionization mode with SIM monitoring. 

The following table lists the mean minimum and maximum concentrations (ng/mL) for all plasmas 

collected from 30 minutes before the first dose to 2 days after the last dose for each session for five 

subjects: 

THC ll-OH-THC THCCOOH 

Session Dose Amount Min Max Min Max Min Max 

~gh Potency Hemp Oil 14.8 mg/day 0.0 6.5 0.0 5.6 0.0 15.2 

Dronabinol Capsules 7.5 mg/day 0.0 3.6 0.0 2.6 0.0 24.4 

~emp Oil Capsules 0.47 mg/day 0.0 0.0 0.0 0.0 0.0 2.5 

Low Potency Hemp Oil 0.39 mg/day 0.0 1.2 0.0 3.8 0.0 3.1 

Placebo__ ~_ c.JJ.0 mg/day 0.0 ~~ 0.0 0.0 0.0 0.0 

~- 

Drug levels of plasmas varied within the same dose between subjects. In general, the THC, 11-0H-THC, 

and THCCOOH levels were low and erratic across the collection timeline after all doses. The peak 

concentrations and time to peak concentrations varied, sometimes considerably, between subjects. In 

general, the maximum concentrations of 1I-0H-THC were seen in the liquid hemp oil sessions. The 

sessions in which capsules were administered had lower concentrations, less than the low dose of hemp oil 

administered, or zero concentrations. Plasma THC and 11-0H-THC were negative for all participants and 

for all doses by 16 hours after the last THC dose. Plasma THCCOOH persisted for a longer period oftime 

following the two highest doses of 7.5 mg/day dronabinol and 14.8 mg/day THC in hemp oil. Ohlsson et 

al. (Clin. Pharmacol. Ther., 1980, 28:409) reported that orally administered (20 mg cookie) THC yielded 

low and irregular plasma concentrations compared to intravenous and inhaled THC. Bioavailability of 

orally administered THC is low. This may be due to poor absorption, degradation by stomach acid, or 

biotransformation to metabolites via first pass through the liver. 

Keywords: Oral Administration ofTHC, Plasma, GCIMS. 

Page 306

M24 

A MICROTITER PLATE ELISA PLATFORM FOR SCREENING ABUSE DRUGS IN ORAL FLUID 

Thomas Kupiec*, Lacinda DeCicco, Susan Bernard, Nicole Vu' Analytical Research Laboratories, Oklahoma 

City, OK. 

The purpose of this study was to evaluate the suitability of microtiter plate ELISA assays (Neogen Corp) for 

screening of abuse drugs in oral fluid (OF), and the efficiency of drug recovery from a oral sampling device, 

Sarstedt Salivette®. The following drugs were tested at the proposed cutoff levels for the respective compound 

according to the Federal Workplace Drug Testing Program draft guidelines: cocaine, morphine, 6 

monoacetylmorphine (6-MAM), d-amphetamine, d-methamphetamine, and methylenedioxymethamphetamine 

(MDMA) and methylenedioxyamphetamine (MDA). Twenty, non-smoking, drug-free donors by testimony spit 

approximately 25 mL OF into a clean cup and the sample was then poured away from the froth and centrifuged. 

All OF samples were confirmed negative by GCIMS for all of the tested analytes. The samples were then pooled 

to create a blank OF matrix pool used in all assays. The OF pool portions were spiked with the above analytes at 

the proposed cutoff levels for the respective compound. Next, spiked or blank OF was collected from a cup 

using an oral sampling device. The collector pad was held with tweezers and immersed in the cups of OF and 

then processed according to the manufacturer's protocol. OF samples of 20~L were analyzed alongside a 

standard curve by ELISA to determine the dilution factor needed to bring the %BlBo of each drug in OF at the 

standard proposed industry cutoff levels into the optimal range for the ELISA. The calibration curve was 

established by plotting log concentration VS logit transformation of the absorbance ratio for each calibrator and 

the blank sample (%BlB o ). The concentrations of the analyte in the test samples were then determined from the 

calibration curve based on their absorbance ratios, BlB n . The percentage of drug recovery from the oral 

sampling device was calculated by comparing the concentration of the processed sample using the device to the 

non-processed sample concentration. The amount of drug detected by the ELISA when no collection device was 

utilized was determined by Observed/Experimental x 100. The intra-assay precision was evaluated based on the 

mean absorbance ratio BlBo of the 16 replicate analyses of blank samples that were used in LOD determination. 

These results are summarized in Table I. The results indicated that this ELISA platform is suitable for the 

analysis of the following drugs in OF: cocaine, morphine, 6-MAM, d-amphetamine, d-methamphetamine, and 

MDMA. 

Table 1 

I 

! 

I 

Drug 

i Dilution of 

. OF 

I 

Drug Conc. 

. 

inOF 

(ng/mL) 

I 

i 

1 

Amount Recovered 

of OF 

(ng/mL) 

No Collector 

Collector 

Intra-assay 

Precision of 

BlBo 

! 

i Cocaine I: 50 

Amphetamine I: 5 i 

5 

20 

25 

50 

i 

4.76±0.15 

24.01±1.90 

! 17.86±2.56 

! 

40.86±3.85 

2.07±0.32 

14.96±2.47 

13.84±0.55 

36.55±1.94 

3.863 

3.863 

I 

I MDA 

No dil. 

I Methamphetamine 1: 5 

MDMA I: 5 

Morphine I: 50 

I 

i 6-MAM I: 5 

I 

I 

I 

I 

I 

50 

100 

25 

50 

25 

50 

20 

40 

2 

4 

10.42±2.58 

23.58±23.52 

36.37±2.56 

56.32±3.20 

30.26±6.45 

50.07±5.78 

I9.62±5.71 

42.36±0.96 

3.13±0.77 

5.67±0.72 

56.24±8.96 

63.19±16.74 

36.30±IO.l8 

58.42±2.12 

10.581 

2.755 

22.53±7.5 

44.08±6.64 I 

8.316 

15.18±4.43 

32.79±7.43 

2.43±0.19 

5.56±L25 

I 

I 

I 3.798 i 

I 

i 

4.564 

! 

I 

Keywords: ELISA, Oral Fluid, Drugs of Abuse 

Page 307

M25 

,--- 

LIQUID CHROMATOGRAPHY -ELECTROSPRA Y IONIZATION MASS SPECTROMETRY 

FOR THE DETERMINAnON OF SELECTED BENZODIAZEPINES 

O.Quintela*, A. Cruz, A. de Castro M. Concheiro and M.L6pez-Rivadulla. 

Forensic Toxicology Service. Institute of Legal Medicine. University of Santiago de Compostela. SPAIN 

A simple, rapid, and sensitive method based on the use of liquid chromatography-eJectrospray ionizationmass 

spectrometry (LC-ESI-MS), which allows the simultaneous determination of nine Benzodiazepines 

(Midazolam, Bromazepam, Tetrazepam, Alprazolam, Lorazepam, Triazolam, Diazepam, Flunitrazepam 

and Lormetazepam) was developed. The method was applied to plasma and saliva samples. The collection 

of saliva samples were done with the Salivette®device. Lorazepam-d4, Diazepam-d5, and Alprazolam-d5 

were used as internal standards. 

The separation of the nine benzodiazepines was performed by using acetonitrile/O.l% formic acid in 

gradient mode as a mobile phase, on a XTerra RP18 5 J.lm (l50x 2.1mm I.D.) reverse phase column. The 

liquid-liquid extraction procedure of the compounds and their respective internal standards was carried out 

with diethyl ether. The quantification study was done by operating in selected ion monitoring (SIM) mode, 

and two mlz ratios for each compound were chosen. The method was validated for both biological samples. 

LOD LOQ Selected 

Plasma Saliva Plasma Saliva mlz ratios 8 

Midazolam 0.5 0.1 0.2 326.1 

291.3 

Bromazepam 0.2 5 0.5 318.0 

290.0 

Tetrazepam 0.5 0.1 0.2 289.2 

261.3 

Alprazolam 0.5 0.2 0.5 309.1 

281.2 

Lorazepam 0.5 0.2 0.5 321.0 

303.1 

Triazolam 0.5 0.1 2 0.2 343.1 

308.1 

Flunitrazepam 0.5 0.1 0.2 314.2 

268.2 

Diazepam 0.5 0.1 0.2 285.3 

257.2 

Lormetazepam 0.5 0.1 0.2 335.0 

289.0 

Lorazepam-d 4 

Alprazolam-ds 

Diazepam-ds 

N.D. b 

N.D. b 

N.D. b 

N.D. b 

N.D.b 

N.D. b 

N.D. b 

N.D.b 

N.D.b 

325.1 

N.D. b 

N.D. b 290.3 

N.D. b 314.2 

"Quantifying ions are in bold characters 

~.D.==Not Detected 

In addition, a preliminary pharmacokinetic study for two of the nine Benzodiazepines (Midazolam and 

Lormetazepam) was carried out in order to check the possible saliva and plasma levels correlations. 

Keywords: Benzodiazepines, Plasma and Saliva, LC-ESI-MS 

Page 308

M26 

DETECTION OF SMOKED COCAINE MARKER (ANHYDROECGONINE METHYLESTER) IN 

NAILS. 

Catherine Ragoucy-Sengler l * and Pascal Kintz 2 : lLaboratoire de Biochimie, Centre Hospitalier 

Universitaire BP 465 97110 Abymes, Guadeloupe (FWI) and 21nstitut de Medecine Legale, 11 rue Human 

67085 Strasbourg France 

Objective: The chronic intoxication by smoked cocaine or crack results in psychic disturbances that may 

lead to asocial and aggressive behavior. For medical, legal and epidemiologic purpose it is interesting to be 

able to detect this drug-addiction. The detection of anhydroecgonine methy ester (AEME) marker of 

coca'ine pyrolysis in hair is significant ofchronic consumption. The collection ofhair is not always possible 

among Afro-Americans, and that of the nails can represent an interesting alternative to search AEME. 

Materials and methods: Two samples of 50 mg of nails collected with informed consent from two subjects 

consuming smoked cocaine were analyzed by gas chromatography coupled to mass spectrometry detection. 

The AEME was searched in these samples. 

Prior to analysis, the samples were washed with hot water then by 5 baths of dichloromethane in order to 

eliminate any possibility of contamination by the contact with the product or the smoke before being 

analyzed. The analysis was carried out in mode SIM on a Gaz Chromatograph (6890) coupled to a mass 

selective detector (5973) Agilent Technologies, (Palo Alto, CA) (1). AEME was quantitated by comparing 

ratios of peak areas with those ofdeuterated cocaine. 

Results: The analysis of the nails samples of both subjects (1 and 2) leads to detection of cocaine, its 

metabolites and the AEME. The analysis of dichloromethane baths were negative from the 3 me bath. 

ng/mg COC BE EME CE AEME 

Subject 1 28.7 7.3 6.3

M27 

DETERMINA TION OF THC IN ORAL FLUID SAMPLES USING LC-MS 

Manuel Rivadulla*, Oscar Quintela, Marta Concheiro, Ana Rios, and Angelines Cruz.: Forensic 

Toxicology Service, Institute of Legal Medicine, University of Santiago de Compostela, Galicia, Spain, 

15782 

Analysis of THC and its metabolites in biological samples is of great relevance for forensic purposes. In the 

case of oral fluid the analysis should determine THC, whereas in urine, it detects the inactive metabolite 

THC-COOH. Most laboratories analyze THC in such samples using GC-MS methods, but these 

procedures are time-consuming and involve unavoidable previous extraction and derivatization. In this 

paper we report a validation method in which the THC is isolated from oral fluid by a simple liquid-liquid 

extraction with hexane. The separation and quantitation was done on a positive ion electrospray (BSI 

positive) high performance liquid chromatography-mass spectrometry (LC-MS) in the selected ion 

monitoring (SIR) mode. Calibration curves for ,1.9-THC were performed in oral fluid, achieving linearity 

between 2 nglmL and 100 ng/mL. The procedure was validated in terms de linearity (coefficient of 

correlation: 0,9956±0,0035), repeatability (5.33-10.02 %, n=6), reproducibility (2.58-14.7 %, n=6). The 

limit of quantification (LOQ) was defined as the lowest concentration of analyte that could be measured 

reproducibly and accurately (CV < 20% and bias> 80%), which was 2 ngimL. The LOD was estimated on 

the criteria of the lowest concentration of an analyte that the bioanalytical procedure can reliably 

differenciate from the backgroud noise, which was 1 ng/mL. The mean recovery was 86.62%. The method 

requires only 200 ilL of oral fluid, and has been used to analyze oral fluid samples collected from drivers, 

by spitting, salivette and intercept@ modes. 

Keywords: LC-MS, Oral Fluid, THC 

Page 310

M28 

DOSE-RELATED DISTRIBUTION OF CODEINE, COCAINE AND METABOLITES INTO 

HUMAN HAIR FOLLOWING CONTROLLED ORAL CODEINE AND SUBCUTANEOUS 

COCAINE ADMINISTRATION 

Karl B. Scheidweiler l *, Edward J. Cone 2 , Eric T. Moolchan l and Marilyn A. Huestis l : 1 National Institute 

on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD: 2 ConeChem Research, LLC, Severna Park, 

MD 

Ten volunteers with histories of cocaine and opiate use consented to reside on a secure clinical research 

ward at the Intramural Research Program, National Institutes on Drug Abuse, National Institutes of Health 

to participate in a ten-week controlled cocaine and opiate administration study. This Intramural Research 

Board approved study was designed to investigate excretion of opiates, cocaine and metabolites into hair 

following multiple subcutaneous (s.c.) cocaine hydrochloride and oral (p.o.) codeine sulfate doses. The 

first three weeks of the study comprised the drug washout phase. During the low dose drug administration 

week (week 4) 75 mg/ 70 kg cocaine, s.c. and 60 mg/ 70 kg codeine, p.o. doses were administered on 

alternating days for a total of three doses for each drug. The high dose drug administration week began on 

week 8 during which, subjects received three doses of 150 mg/ 70 kg cocaine, s.c. and 120 mgi 70 kg 

codeine, p.o. on alternating days. An electric razor was used to collect hair at the end of each week during 

the study. Drugs were extracted from pulverized hair via methanolic sonication and extracts were analyzed 

for cocaine, norcocaine, benzoyl ecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), codeine, 

norcodeine, morphine and 6-acetylmorphine (6-AM) via liquid chromatography-atmospheric pressure 

chemical ionization tandem mass spectrometry. Limits of quantitation (LOQ) for cocaine and metabolites 

were 17 pg/mg, except for EME (50 pg/mg). Opiate LOQs were 83 pg/mg, except for norcodeine (240 

pgimg). 

Maximum cocaine and metabolite concentrations (Cmax) were generally measured in hair samples 

collected two weeks after drug dosing (range: one to three weeks). Average cocaine and metabolite 

Cmax's (::I: standard error of the mean) measured in hair reflecting the low dose cocaine week 

administrations were 2,997 ::I: 619, 314 ::I: 51, 187 ::I: 27, 84 ::I: 21 pg/mg of cocaine, BE, EME and 

norcocaine, respectively. Mean cocaine and metabolite Cmax's measured reflecting the high dose cocaine 

week administrations were 6,419::1: 1,698,708::1: 163,425::1: 65, 218::1: 46 pg/mg of cocaine, BE, EME, and 

norcocaine, respectively. Norcocaine did not exceed the LOQ after the low cocaine dosing in one often 

individuals. In all other cases, cocaine and metabolites exceeded the LOQ after low and high cocaine 

dosings. Comparison of cocaine and metabolite hair Cmax's that occurred after low and high dose cocaine 

weeks revealed significant dose-relationships and were significantly different from hair concentrations at 

the conclusion ofthe washout phase as evaluated using Wilcoxon signed rank test (p

M29 

CODEINE AND METABOLITE DISPOSITION IN HUMAN SWEAT FOLLOWING 

CONTROLLED ORAL CODEINE ADMINISTRATION 

Eugene W. Schwilke 1 *, Sherri L. Kacinkol, Allan J. Barnesl, Jonathan M. Oyler2, Edward 1. Cone 3 , Eric T. 

Moolchan l , and Marilyn A. Huestis l : IChemistry and Drug Metabolism Section, Intramural Research 

Program, NIDA, NIH, Baltimore, MD, 2Department of the Army, US Army Research, Development and 

Engineering Command, Aberdeen Proving Ground, MD, 3ConeChem Research, LLC., Severna Park, MD 

Analysis of codeine is included in drug treatment, military, criminal justice and workplace programs 

because of its abuse potential and performance impairing effects. Interest in monitoring drug exposure with 

human sweat, as an alternative biological specimen is increasing, as sweat collections are convenient, less 

invasive and difficult to adulterate. Our objective for this study was to determine the excretion profile of 

codeine and metabolites into human sweat using the PharmChek Sweat Patch. Volunteers with a history 

of opiate use (n=9) provided informed consent to participate in this IRB-approved study and resided on the 

closed clinical research ward for 12 weeks. Low (60mg/70kg) and high (l20mg/70kg) doses of codeine 

sulphate were administered three times a week, four weeks apart. Codeine, norcodeine, morphine, 

normorphine, and 6-monoacetylmorphine were isolated by SPE and GCIEI-MS. The limit of quantitation 

(LOQ) for codeine and morphine were 2.5 ng/patch, and 5 ng/patch for metabolites. 

We present data from patches representing four categories of sweat collection. First, weekly sweat patches 

were collected from the time of entry onto the secure ward until the time of first codeine administration, 

reflecting excretion of previously self-administered opiates. No patch was positive for 6-acetylmorphine, 

codeine, morphine or metabolites at the methods' LOQs. 

Second, weekly sweat patches also were applied prior to and removed 7 days after dosing for 5 of 9 and 3 

of 6 participants for the low and high doses, respectively. 80% of the low and 100% of the high dose 

weekly patches were positive for codeine, but negative for metabolites. The mean codeine concentration for 

these patches was 65 ng/patch (median 27, SD 92, range 16-225) for the low dose and 82 ng/patch (median 

84, SD 15, range 67-96) for the high dose. 

Third, other sweat patches were worn for between 1 to 15 hours during the first 48 hours after dosing. Of 

139 patches worn during the first 48 hours following the first codeine administration, only 11 (8%) 

contained codeine levels greater than our LOQ. Codeine was the only analyte detected and could be found 

as early as 1 hour after dosing. A total of7 patches (8%) were positive for codeine following the low dose, 

and 4 patches (8%) following the high dose. The mean codeine concentration (data from 3 of9 subjects) in 

sweat was 43 ng/patch (median 28, SD 50, range 5 - 153) for the low dose and 12 ng/patch (median 11.0, 

SD 9.0, range 3-24, data from 3 of 6 subjects) for the high dose. Codeine was detected in sweat patches for 

up to 26 h after administration; patches applied after 26 hours and worn for up to 15 hours were below the 

LOQ. 

Fourth, weekly patches also were applied to 4 of 9 participants two weeks after the last codeine dose. All 

of the patches also were negative for codeine and metabolites. 

Sweat has recently been proposed by the Substance Abuse and Mental Health Services Administration 

(SAMHSA) for federally-mandated workplace drug testing. A confirmation cutoff of 25 ng/patch has been 

proposed for patches worn 7 to 14 days. Two patches for one subject and I patch for another collected 

within the first 48 hours following dosing, would be positive using these criteria. In comparison, 6 of 8 

patches worn for 7 days during the dosing period would be positive under the new SAMHSA guidelines. 

Sweat provides an adequate matrix for detection of opioid use. There was large intra- and inter-subject 

variability. No correlation of codeine concentration with dose was found for the doses investigated. 

Patches worn for 1 week were more likely to contain codeine above the LOQ than patches worn for 1 to 15 

hours. 

Keywords: Sweat, Codeine, GCMS 

Page 312

M30 

DOPING STEROID ANALYSIS IN NAIL CLIPPINGS 

Kenichi Takaichi l ., R.A. Anderson I and D. Thieme 2 : IDepartment of Forensic Medicine and Science, University of 

Glasgow, Glasgow G 12 8QQ, United Kingdom and 21nstitute of Doping Analysis/Sports Biochemistry, Dresdner Strasse 

12, D-01731 Kreischa, Germany. 

Extraction ofanalytes from fingernail is difficult due to its hard keratin composition. Our group has shown the feasibility 

of drug analysis in nail using a cryogenic grinding technique, including cannabinoids and opioids (I). In particular, 

cryogenic grinding has been shown to allow the extraction and analysis of intact esters such as diamorphine by avoiding 

the alternative alkaline hydrolysis step. To date, few papers have reported the detection ofsteroids in nail (2). By contrast, 

several papers have reported the detection and analysis of anabolic steroids in hair. 

In the present study, cryogenic grinding was used to prepare nail clippings from doping abusers for extraction of 

endogenous and anabolic steroids to show that both can be detected, as in hair. This method was compared to blood 

(plasma) and urine samples that had been analysed at the same time. 

Fingernail clippings from users of anabolic steroids (including testosterone esters, stanozolol and methenolone acetate) 

were obtained from the Institute of Doping Analysis/Sports Biochemistry, Kreischa. Blank nail samples were obtained 

from volunteers. The nail samples were first decontaminated by washing with 0.1% sodium dodecyl sulfate, water (x 3) 

and methanol (x 3). After drying, the samples were pulverised for 4 min in a liquid-nitrogen cryogenic miII (SPEX 

CertiPrep 6750 Freezer Mill). The powdered nail was then extracted with methanOl/ethyl acetate (7:3 v/v, 7 mL). 

Deuterated anabolic steroids (5a-estran-3B-oI-17-one-d 3 , testosterone-d 3 , and stanozolol-d 3 ) and medroxyprogesterone 

were used as internal standards. 

The extracts were converted to TMSi derivatives with MSTFAlNH 4 112-mercaptoethanol and analyzed by GC-MS in the 

EI + full scan and SIM modes, on a Finnigan Trace instrument equipped with an HP-5 column (30 m x 0.32 mm i.d.; film 

thickness 0.25 J.lm) with temperature programming from 180°C to 240 °C at 3 °C/min, 240°C to 300 °C at 5 °C/min, and 

final temperature held for 10 min. 

In this initial study endogenous steroids (androsterone (And), etiocholanolone (Etio), dehydroepiandrosterone (DHEA), 

epiandrosterone (epi-And), epitestosterone (epi-Test) and testosterone (Test» were identified and quantified in the nail 

samples from both steroid users and non-users. 

No. And Etio DHEA epi-And epi-Test Test 

Suspect # I (Nail) 4.1 0.0 5.6 3.3 3.0 4.3 

Suspect #2(Nail) 4.7 0.0 3.0 1.9 1.5 3.9 

Suspect #3(Nail) 9.6 0.0 25.3 14.3 11.0 10.6 

Suspect #4(Nail) 12.8 0.0 12.0 6.2 10.9 25.0 

Suspect #5(Nail) 1.7 0.0 1.8 0.8 0.6 0.6 

Healthy # 1 (Nail) 1.5 0.0 1.4 0.7 0.6 6.5 

Healthy #2(Nail) 8.4 0.0 4.5 1.7 2.6 l.l 

Healthy #1(Plasma) 30322.9 54222.5 11218.3 Trace 222.2 3583.8 

Healthy #2(Plasma) 9923.3 2966.7 100.6 Trace 200.2 196.0 

Healthy # I (Urine) 351.6 392.5 11.2 Trace 9.0 11.0 

Healthy #2(Urine) 926.8 868.7 115.8 Trace 7.4 13.6 

* Suspect #3 

Test 292 pg/mL in urine, 

Test-enanthate, decanoate 

in hair were 20 and 18 

pg/rng, respectively. 

* Suspect #4 

Test 30 pglrnL in urine, 

Test-enanthate, 

phenyl-propionate, 

decanoate, and isocaproate 

in hair were 25, 425, 220, 

and 340 pg/rng, 

respectively. 

In general, endogenous steroid concentrations in nail were low, in the pgjmg range. Also, elevated concentrations of 

testosterone in nail were positively associated with high concentrations in plasma and urine. However, although the 

analytical results provided evidence for the presence of anabolic steroids in the samples from steroid users, including 

testosterone and testosterone esters at low concentrations, it has not yet been possible to confirm this due to interference 

from other endogenous substances. Nail remains a potential, but still to be confirmed, alternative biological specimen to 

hair for the detection ofpast exposure to doping steroids. 

References: 

(1) K. Takaichi, N.P. Lemos, R.A. Anderson, Analysis of Opiates in Nail Clippings from Chronic Heroin Abusers, 

presented at the 39 th Annual TIAFT Meeting, Prague, 200 I. 

(2) M.H. Choi, Y.S. Yoo, B.C. Chung, Measurement of testosterone and pregnenolone in nails using GC-MS., 

J. Chromatogr., B 754, 495·501 (2001). 

Keywords: Nail Analysis, Doping, Anabolic Steroids 

Page 313

M31 

DETERMINATION OF A'.TETRAHYDROCANNABINOL (THC) IN ORAL FLUID USING 

SOLID PHASE EXTRACTION AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 

AND ELECTROSPRA Y IONIZATION MASS SPECTROMETRY 

H.M. Teixeira *, P. Proenyal, A. Castanheira l , M. L6pez-Rivadulla 2 , F. Corte-Reali, E.P. Marques l , D.N. 

' 

Vieira l : I National Institute of Legal Medicine, Coimbra, Portugal and 2 Legal Medicine Institute, Santiago 

de Compostela, Spain 

Aim: High-performance liquid chromatography with electro spray ionization mass spectrometry was used to 

determine /19-tetrahydrocannabinol (t/-THe) in oral fluid. 

Methods: Oral fluid samples were extracted using Bond Elut LRC-CertiiY solid-phase extraction columns 

(lOcc, 300mg), elution performed with n-hexane/ethyl acetate. The separation and quantitation was done on 

a positive ion electro spray (ESl positive) high performance liquid chromatography-mass spectrometry (LC 

MS) in the single ion recording (SIR) mode. Quantitation was achieved by the addition of the deuterated 

analogue as internal standard. The compounds were detected by single ion monitoring ofmlz 315 and mlz 

318 for the protonated molecules [THC+H+J and [d 3 -THC+H+J, respectively. In the chromatographic 

separation, a X-Terra MS CIS column (2.1x50mm, 3.5j.1m) was used and the mobile phase was 

composed of acetonitrile and ammonia 0.05%, at a OJ mLimin flow rate. 

Results: No interferences were detected in 10 blank oral fluid samples of different origin. The precision and 

accuracy were tested on spiked oral fluid samples at three different concentrations (2, 25 and 100nglmL). 

The mean recovery was 79%, coefficients of variations were between 2.9-6.9% and the limits of detection 

(LOD) and quantitation (LOQ) were 1.0 nglmL and 2.0 ng/mL, respectively. Calibration curves for /e/. 

THC were performed in methanol solutions and in oral fluid samples using seven different concentrations, 

achieving linearity between 2 nglmL and 100 ng/mL. This validated method is currently being applied to 

real cases where oral fluid specimens are collected with the Salivette® and also by spitting from marijuana 

smokers attending the world's largest music event in recent years, "Rock in Rio - Lisboa". 

Conclusions: The method is sensitive, accurate and reproducible and may be utilized in ongoing controlled 

cannabinoid administration studies and in roadside studies and thus, important for the fields of forensic 

toxicology. 

Keywords: /1'-THC, Oral Fluid, LC-MS 

Page 314

M32 

LEGAL ISSUES OF ADDICTION AND HAIR TESTING FOR DRUGS OF ABUSE 

Aristidis Tsatsakis*, Manolis Savvopoulos, Joanna Dialyna, Manolis Tzatzarakis, and Eleftherios Pallis 

Center of Toxicology Science and Research, Department ofMedicine, University of Crete, Greece. 

Aim: The purpose ofthis paper is to present legal issues of addiction and hair testing results regarding to the 

service of Law in Greece. 

Methods Legal Part: Details (such as the process, the decision and the competence of the Court, the 

police record, the indictment, the expert reports, the defendant's individuality, the crimes and the penal 

confrontal and many others) from legal cases related to addiction and its judicial verification were collected 

and analysed. 

Methods - Laboratory Part: Laboratory data of cases concerning the laboratory evaluation of addiction in 

drug users and also occasionally the legal course of cases with addict defendants are presented. In four 

representative cases segmental hair analysis proved that as long as the individuals were imprisoned findings 

with drug substances corresponding to that period were lesser or practically absent comparably with 

samples corresponding to the time out of prison, which proved increased drug abuse. 

Results: Hair analysis provides information on chronic exposure rather than acute poisoning. Its detection 

window varies from some days to months or even years and the length of the hair is the only limit. The 

procedure that the Law lays in many cases is insufficient and in most cases unable to be abided by. Details 

about the addicted person's profile are presented. Our study presents that the segmental hair analysis 

method is a subject of growing interest for drugs of abuse because it provides information on chronic 

exposure to the drug and the drug history profile ofeach subject 

Conclusions: Although hair testing for drugs of abuse is not specially referred by any law, Greek Courts 

accept the method for the final conclusion about the defendant's claim of addiction. Segmental hair 

analysis may be the only tool to prove this claim, not only when the medical examiner is not able to decide 

about defendant's addiction and it's severity but also in cases where the time period of drug abuse is to be 

defined. 

Keywords: Addiction, Legal and Judicial Issues, Segmental Hair Testing. 

Page 315

M33 

ENHANCED SENSITIVITY FOR DRUGS OF ABUSE IN ORAL FLUID USING THE 

INNOVATIVE NEW ORALSTAT TEST SYSTEM FOR ON-SITE TESTING. 

Yli Remo Vallejo*, Martin Gould, Matthew Lomonico, and John Donovan: American Bio Medica 

Corporation, 603 Heron Drive, Unit #3, Logan Township, New Jersey 08085 

The New OralStat is an innovative test system for the rapid on-site detection ofdrugs of abuse in oral fluid 

that dramatically improves the limits of detection. This test system consists of an untreated collector 

sponge with a built-in sample adequacy feature, an integrated test device containing separate receptacles for 

a sample buffer, gold conjugates, and immunochromatographic test strips. The device allows the lateral 

flow process to be separated into discrete stages by enabling the dilution of the oral fluid sample with 

buffer, the preincubation of the sample with the antibody-coated gold reagents, then allowing the sample 

mixture to chromatograph on the lateral flow test strips. The use of a buffer minimizes the problems 

associated with viscous saliva. The. preincubation of the sample with the antibody-coated gold reagent 

greatly improves sensitivity. The device can accommodate any combination of6 drug assays. 

The folfowing performance was observed in a study involving spiked oral fluid from 3 non-users: 

Drug 

Sensitivity (nglmU 

THC (parent compound) 25 

PCP 5 

Amphetamine 25 

Methamphetamine 25 

Opiates 20 

Cocaine (Benzoy I Ecgonine) 12 

Benzodiazapines 25 

Methadone 25 

The NewOralStat test system can detect PCP, Opiates, Amphetamine, Methamphetamine, and Cocaine 

metabolite at lower levels than the SAMHSA proposed cutoffs ..THC detectability at 25 ng/mL, though 

still above the SAMHSA cutoff of 4 ng/mL, is a major improvement over current POCTs, improving the 

detection of recent marijuana use by many hours. The results of a current study with samples from drug 

users will be presented. 

The New OralStat test system is visually read and requires no instrumentation to attain these levels of 

sensitivity. It is however expected that the use of a reflectance reader along with agitation during the 

preincubation period will further improve these limits of detection. Initial results show that by applying 

such measures the THC cutoff of 4 ng/mL can be achieved. 

Keywords: Oral Fluid, Drugs ofAbuse, On-Site Testing 

Page 316

M34 

COMPARISON OF ORAL FLUID WITH URINE TESTING IN DRE CERTIFICATIONS 

Michael A. Wagner!*, Colleen Scarneo l , Emily Rice l , Kris Valas l , Susan Lefebvre l and Christina Werner2 

IOepartment of Safety, State Police Forensic Toxicology Group 33, Hazen Dr., Concord, NH 03301, and 

20raSure Technologies, 150 Webster Street, Bethlehem, PA 18015 

Determination of impairment by drugs in OWl cases or in the work place often requires the analysis of biological 

specimens. The most common matrices tested are blood, urine and saliva. Each of these matrices has inherent 

advantages and disadvantages. For example, urine has advantages of providing a historical perspective to drug 

use yet does not supply impairment interpretation. Saliva has the potential for supplying interpretation with 

respect to blood concentration and impairment with similar detection widows to blood. This abstract reviewed 

61 ORE cases, July 8 1h 2002 until June 30 1h 2003, involving roadside stops for OWl and ORE certifications 

events. Twenty-six of the 61 cases involved ORE certification events such as; concerts, and evaluation nights 

arranged by various police departments were the subject of this initial study. During the certification events the 

laboratory personnel was able to attend and assist law enforcement with the collection of oral fluids and urine 

samples. Oral fluids were collected using the Intercept Collection device from OraSure Technologies Inc. The 

process entailed having the subject place an oral collection pad (attached to a handle) between the lower cheek 

and gum, rubbing back and forth until moist. Most subjects just sucked on the pad until the sampling time 

interval was completed (2 minutes). The pad was placed in the sampling tube containing 800 uls of OraSure 

buffer solution. The specimens were collected typically upon completion of the ORE evaluation. Matched urine 

and oral fluids specimens were collected. The oral fluids were tested in the on Intercept® Micro-Plate EIA, 

while the urines used the on Micro-Plate EIA serum kits optimized for urine analysis. Both analyses were 

performed on a PersonalLABTM automated analyzer. All subjects were screened for seven classes of drugs: 

Benzodiazepines, Cocaine, Cannabinoids, Opiates, Methamphetamines, Amphetamines, Barbiturates, PCP, and 

Methadone. 

Summarized in Table I are the correlated analytical results between urine and oral fluid testing as they relate to 

ORE interpretation. The positive rates with respect to the various drug categories appears to be the highest for 

Cocaines and Cannabinoids, 87% and 94% respectively, while Barbiturates had the highest percentage of oral 

fluid positive results 75% versus 25% for urine. ORE percent corroboration varied between 25% for Stimulants, 

36% for Depressants, 47% for Cannabinoids, and 100% for Narcotic Analgesics. These results seem to parallel 

with the subject's admission rate. Certainly one issue confounding the ORE corroboration was the percentage of 

positive polydrug cases. 57% of the cases had two or more drug categories test positive, 27% screened positive 

for three or more drugs, 15% of the cases screened positive for four or more drugs, and 8% of the cases screened 

positive for five or more drugs. 

The ease of administration for oral fluid testing in ORE evaluations may provide additional information with 

respect to impairment interpretation. This initial study demonstrated that Cocaine and Cannabinoids appeared to 

have a better correlation with urine as compared to other drug classes, while Barbiturates had the highest 

specificity for oral fluids. 

Table I 

! Drug Urine Oral No. Tests Urine OF ORE Subject 

Categories NH-ForenTox NH-Foren Tox "'+" "+" Opinions Admission 

Cutoff ng/mL Cutoffng/mL 

Benzodiaz 100 1.0 11 11 2 3 2 

cac 100 5.0 16 13 14 4 4 

THC 20 1.0 17 16 12 8 IO i 

Opiates 200 10 8 8 4 8 5 

Methamph 300 40 4 4 1 1 2 

Barbiturate 200 20 3 1 3 2 2 

Methadone 300 5.0 3 3 1 3 3 

PCP 25 1.0 ND - - - - 

Amphet 300 100 NO - - - - 

Key Words: DRE, Oral Fluids, Urine Drug Screen, ErA 

Page 317

M35 

DRUG DETECTION IN HAIR: ASSESSMENT OF LEVELS FROM LARGE VOLUME SCREENING AND 

CONFIRMATION TESTING 

John Wicks*, John Sullivan and Lolita Tsanaclis: Tricho-Tech Limited, The Cardiff Medicentre, Heath Park, Cardiff, 

CFI4 4UJ, United Kingdom. 

In recent years understanding of the benefits of the use of hair in drug testing has increased, particularly its advantages 

over urine, and more recently, oral fluid. The key benefit is the long time window of detection. This understanding 

together with reliable techniques has contributed to the dramatic increase in the use of hair samples in the detection of 

drug use. Amongst the wide variety of sectors that currently use hair for the detection of drugs, the largest are clinics, 

family law firms, the police and various organisations for workplace testing. The main purpose is to verifY drug use or 

monitor drug abstinence. Very often when the analysis is performed and the results issued, the most common question 

that is raised is: what do the levels detected tell me about how much drugs were used by the person being tested 

Because it is difficult to establish an accurate estimation of the amount of dose taken in relation to levels of drug in 

hair, the levels of drugs detected in hair are best used as a guide to changes in use in the individual when sectional 

analysis is performed or two different periods are compared in the same individual. 

Nonetheless, it can be very useful, as a guide when writing reports or communicating with clients, to compare results 

obtained from a hair test from an individual's hair sample with results obtained in other samples from a large group of 

people. Hair samples (N=12,218) from various sources were received by Tricho-Tech for screening for drugs over a 

two-year period and analysed using the same method. All samples were washed, extracted then screened using coatedplate 

ELISA test for each drug group (Immunalysis Corporation, Pomona, CA, USA) and processed by an automated 

analyser Triturus (Grifols, Cambridge, UK) at a cut-off of 0.5 ng/mg (Table I). Samples above the cut-off were 

submitted for confirmation analysis by Gas Chromatography-Mass Spectrometry (GC-MS or GC-MS/CI) using 

HP5973 (Agilent, Berkshire, UK) or Varian Inc. Saturn for GC-MS/MS (Walton-on-Thames, UK). The dynamic range 

was from 2 to 160 ng/mL. Three ions for the drugs and two ions for the internal standards were monitored. 

Tabl e I H' air samples analyse dbJy ELISA and GCMS WI·th confiIrmatlOn rates or eac hdruJg group. 

I iScreened GCMS Confirmation Rate 

i 

i Group .N 

N % N % 

Amphetamines 7355 4128 (56%) 1839 (45%) 

Benzodiazepines 5804 1879 (32%) 

Buprenorphine 786 

1415 (75%) 

113 (14%) 63 (56%) 

Cocaine 8919 3150 (35%) 2490 (79%) 

Methadone 6127 .1893 (31%) 

1666 (88%) 

Opiates 8970 4164 

1 

J46%) 2901 -.i70o/~ 

Percentiles 25%, 50% (Median), 75%, 95% and maximum levels for all analytes in each drug group were calculated 

and will be presented. Results for Cocaine Group are shown in Table 2. 

Percentiles and maximum levels in n m of hair for Cocaine rou 

25% 50% 75% 95% Maximum N 

0.3 0.8 1.6 5.5 34.8 305 

i""WY',,,,,,i", 

Cocaethylene 0.3 0.5 1.2 4.1 9.4 339 

0.4 1.1 3.6 16.5 163.7 1988 

Cocaine 0.9 2.9 11.9 59.5 814.2 2483 

Using Cocaine as an example of drug levels by sector, median levels were: Clinical=6.9 ng/mg; Police=5.0 ng/mg; 

Medico-Legal=2.5 ng/mg and Employment =0.4 ng/mg. A consistent pattern was seen for the other drug groups. 

The results show a wide range of levels for all analytes, frequency and distribution. Thus, this assessment of levels 

provides an important comparative aid for interpreting results of detected drugs in samples of hair. 

Keywords: Drugs, Hair, GC-MS/MS 

Page 318

M36 

EV ALUA TION OF A RAPID ORAL FLUID POINT OF CARE TEST FOR MDMA 

Lisa Wilson*, Gail Cooper, Chris Hand and Ahmed lehanli: Cozart Bioscience Ltd., 45 Milton Park, 

Abingdon, Oxfordshire, United Kingdom OX144RU 

Recreational use of 3,4-methylenedioxymethamphetamine (MDMA) either alone or in combination with 

other drugs such as alcohol and cannabis has become increasingly popular among young people throughout 

the UK, Europe and the US. The Cozart® RapiScan oral fluid drug test system has been use successfully for 

monitoring compliance within treatment centres, criminal justice settings and more recently at the roadside. 

The aim of this study was to evaluate the Cozart® RapiScan test cartridge developed for the detection of 

MDMA and methamphetamine in oral fluid. Fifty oral fluid samples were obtained from drug addicts 

attending drug dependency units and were screened initially using the Cozart® microplate ELISA for 

amphetamines. All samples were then screened using the Cozart® RapiScan test cartridge for MDMA 

followed by confirmation by gas chromatography-mass spectrometry (GC-MS). 

A total of23 samples screened positive and were confirmed for MDMA either alone or in combination with 

amphetamine. A further 15 samples were confirmed negative for all amphetamines by GC-MS and 14 of 

these samples also screened negative using the Cozart® RapiScan. The false positive sample was 

investigated further and was found to contain an extremely high concentration of pseudoephedrine. The 

remaining 12 samples were confirmed positive for amphetamine only. Six samples screened negative and 

the other six screened positive due to the extremely high concentrations of amphetamine in these samples. 

No samples tested in this study contained methamphetamine. 

The Cozart® RapiScan Drug Test System for the detection of MDMA correctly identified 100% of MDMA 

~. positive samples ranging from 53nglmL to concentrations in excess of lS0ng/mL. False positive results 

were only observed when samples contained extremely high concentrations of amphetamine or 

pseudoephedrine, which is consistent with the cross reactivity profile of the drug test. 

Keywords: MDMA, Oral fluid, Onsite Test 

Page 319

M37 

ALCOHOL TESTING BY AN ONSITE ONE-STEP ORAL FLUID ALCOHOL AND DRUG 

COMBINATION TEST DEVICE ORATECTPLUSTM 

Raphael C Wong·, Jan Sook, Richard Zoltek: Branan Medical Corporation, 10015 Muirlands Road, Suite 

F" Irving, CA 926l8, USA 

Aims: The objective of this study is to compare the alcohol detection ofOratectPlus, an onsite oral fluid 

alcohol and drug combination test device with Alco-screenTM a saliva alcohol test. 

Methods and Results: Oratect is a one-step rapid drug screen that integrates oral fluid collection, drug 

testing and confirmation sampling in one single device. OratectPlus is a new generation of Oratect that 

includes alcohol testing with drug testing and provides faster collection and test times. The new device 

consists a cap and a plastic housing containing a collection pad that is connected to an alcohol test pad on 

one side and two lateral flow immunoassay test strips that test for 6 abused drugs on another side. To run 

the test, the collection pad end ofthe device is inserted into the donor's mouth for one to two minute till red 

color fluid starts to appear on both lateral flow test strips. The color on the alcohol test pad is noted 

immediately. Negative alcohol level does not change the original tan color pad. Presence of bluish gray 

color on this pad indicates alcohol level at or exceeding 0.02% blood alcohol concentration. Drug test 

results are available from the two lateral flow strips after 3 minutes with the absence of red lines indicating 

positive results. In a field trial, forty individuals without drinking any alcohol were all tested negative with 

the OratectPlus and Alco-screen. Another forty individuals allowed to consume beer and then tested for 

alcohol within 20 minutes were all found to test positive for both OratectPlus and Aleo-screen. Nonalcoholic 

drinks including sodas, tea and coffee were found not to interfere with the test result. 

Conclusion: OratectPlus is shown to be a viable onsite device for testing alcohol use. 

Keywords: Alcohol Testing, Oral Fluid, Drugs of Abuse 

Page 320

M38 

Ll9-TETRAHYDROCANNABINOL (THC) IN SWEAT PRIOR TO AND FOLLOWING 

CONTROLLED ORAL ADMINISTRATION OF HEMP OIL AND MARINOLTM TO CANNABIS 

USERS 

Abraham T. Wtsadik 1 *, Karl Scheidweilerl,Takeshi Saito), Richard A. Gustafson 2 , Eric T. Moolchan 1 and 

Marilyn A. Huestis l : IChemistry and Drug Metabolism, IRP, NInA, NIH, 5500 Nathan Shock Drive, 

Baltimore, MD 21224, 2Jacksonville Naval Air Station, Adams Avenue, Jacksonville, FL 32212 

3Department of Forensic Medicine, Tokai University School of Medicine Bohseidai, Isehara, Kanagawa 

259-1193 Japan 

Six healthy individuals with a history of cannabis use resided on the secure clinical research unit of the 

Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health for the 10 to 

12 week study. Participants provided informed consent for this Institutional Review Board-approved oral 

cannabinoid administration protocol. This was a randomized, double blind, double dummy, placebocontrolled 

within-subject study. Sweat was collected throughout the study with PharmCheck sweat 

patches worn for 24 hours or 7 days. The initial washout phase of this study lasted from admission until 

subjects' urine cannabinoid concentrations were below 10 ng/mL by fluorescence polarization 

immunoassay. Sweat patches worn during the initial washout phase enabled investigation of excretion of 

THC resulting from previously self- administered cannabis. The dosing phase of the study followed the 

initial washout phase, during which the participants were dosed three times a day for five consecutive days 

followed by a ten-day washout period before the next dosing session. All subjects ingested commercially 

available hemp seed oil of differing THC concentrations: 0, 0.39, 0.47 (contained within capsules), and 

14.8 mg THC/day. In addition, 7.5 mg/day dronabinol or Marinol, was administered as a positive control. 

PharmChek sweat patches collected throughout the study were frozen at _20DC until analysis. Patches 

were extracted in methanol: 0.2M sodium acetate buffer, pH 5.0, 3:1 v/v, followed by solid phase 

extraction using Clean Screen, ZSTHC020, (United Chemical Technologies, Bristol, PAl. Dried extracts 

were derivatized with trifluoroacetic acid (TFAA) and analyzed using gas chromatography negative 

chemical ionization mass spectrometry (GC-NCI-MS) for THC. Daily and weekly sweat patches collected 

during the initial washout period were analyzed along with weekly patches collected during and following 

dosing. None of the patches worn during or following dosing tested positive for THC with a cutoff of 0.4 

ng/patch. Patches worn during the first week of residence on the research unit, reflecting drug excretion 

from previously self-administered cannabis tested positive for THC in two individuals. The concentrations 

of THC were 0.51 and 0.82 ng THC/patch for patches worn for the first seven days on the research unit. 

These participants self-reported cannabis smoking an average of 3.0 and 4.5 days per week and claimed to 

have last used cannabis one to two days prior to study enrollment. However, these usage histories did not 

differ significantly from those reported by the other four subjects and whose sweat patches tested negative 

for THC. In conclusion, this study demonstrates that oral administration ofTHC at, as high a dose as 14.8 

mg/day for 5 consecutive days did not produce positive sweat tests for cannabinoids with a 0.4 ng 

THC/patch cutoff in patches worn for seven days. The cannabinoid confirmation cutoff for THC in sweat 

as proposed by the Substance Abuse Mental Health Services Administration is 1 ng THC/patch. It is 

important to note that the bioavailability of oral THC has been estimated to be 6 to 20% and plasma THC 

concentrations did not exceed 6.5 ng/mL following any of the dosing regimens during this study. These 

data indicate that previously administered THC in daily cannabis smokers did not produce positive 

cannabinoid sweat tests when patches were applied at the beginning of abstinence and worn for 7 days if a 

1.0 ng THC/patch cutoff is used. In addition, no sweat patches were positive at this cutoff when up to 14.8 

mg THC/day for 5 consecutive days was ingested by cannabis users. Ingestion of commercially available 

hemp oil products and Marinol up to 7.5 mg/day did not produce positive sweat cannabinoid tests. 

Keywords: /':,.9-Tetrahydrocannabinol, Oral Cannabinoids, Sweat Patch 

Page 321

M39 

QUANTITATIVE ANALYSIS OF DEXTROMETHORPHAN, CARISOPRODOL AND THEIR 

METABOLITES IN HAIR BY GC-MS 

Wonkyung Yang l *, Eunyoung Han l , Yonghoon Park l ,Miae Lim', Myungyun Py02 and Heesun Chung I : 

INational Institute of Scientific Investigation, Seoul; 2Korea, School of Pharmacy, Sookmyung Women's 

University, Seoul, Korea 

Dextromethorphan, which is used as an antitussive agent, and carisoprodol, which is used as a muscle 

relaxant, have been widely abused among young people in Korea. Even though these are known to be very 

safe drugs for therapeutic use, people abused them for their hallucinogenic properties. Due to the serious 

abuse liability potential of these drugs, our government recently decided to control them as psychotropic 

agents. It was necessary for us to establish a detection method for dextromethorphan and carisoprodol and 

their metabolites in hair to demonstrate the abuse of these drugs. A method was established to 

simultaneous quantify dextromethorphan, dextrorphan, 3-methoxymorphinan, carisoprodol and 

meprobamate in hair samples. Analytes were extracted from fine cuttings of hair in 1 % HCI in methanol for 

16 hours. After evaporation under N2, residues were separated on a HP - 5MS column using a 16 min 

program and identified by mass spectrometry in the SIM mode (EI-GC-MS).This method was validated for 

the recovery, linearity of calibration, within-and between-day precision, accuracy, limit of detection and 

quantification. Calibration curves exhibited correlation coefficients > 0.99. Within and between-run 

precision was calculated at 8, 80 and 400 nglmg of drug or metabolite in hair with coefficients of variation 

less than 14 %, except for meprobamate (17%). Accuracy at the same concentrations was 14% oftarget for 

all analytes except meprobamate (18%). Recoveries at 10 and 100 ng of drug or metabolite in hair were 88 

to 117 %. With this method, we performed quantitative analysis of dextromethorphan and carisoprodol in 

abuser's hair. The concentrations of dextromethorphan, dextrorphan and 3-methoxymorphinan ranged from 

1.4 - 225.0, 1.3 - 44.7, and 0 - 42.7 nglmg in hair from 31 donors studied, respectively. The level of 

carisoprodol and meprobamate were 2.3 - 52.9 and 11.3 - 365.1 ng/mg respectively in 11 hair samples. We 

present a validated, sensitive and specific GC-MS method to simultaneously quantify dextromethorphan, 

carisoprodol and metabolites in hair. 

Keywords: Dextromethorphan, Carisoprodol, Hair Analysis 

Page 322

M40 

EV ALUA TION OF THE IDS ONE-STEP ELISA KIT FOR THE SCREENING OF KETAMINE IN 

HAIR 

C.Y. Yong*, H.S. Leong, and C.P. Lui: Narcotics II Laboratory, Centre for Forensic Science, Health 

Sciences Authority, 11 Outram Road, Singapore 16907S. 

An evaluation study was conducted with the commercially available ketamine microtiter plate enzymelinked 

immunoassay (ELISA) ofInternational Diagnostic System (IDS), USA, in the screening ofketamine 

in hair. The assay principle is based on the competitive binding between the ketamine-enzyme conjugate 

and the free ketamine extracted from the hair for a limited number of antibody-binding sites. Aliquots of20 

III of the sample or calibrator was added to the antibody-coated microwell followed by 100 III of diluted 

enzyme conjugate. After an incubation period of 30 min at room temperature, any unbound material was 

removed with washing. An enzyme substrate was then added and further incubated for 15 min for the 

colour development. An acid stop solution was added to stop the reaction. The absorbance is measured at 

450 nm with 650 nm as the reference filter. The absorbance obtained is inversely proportional to the 

amount ofketamine present in the sample. 

During the sample preparation, the hair specimens were washed and decontaminated before being 

pulverized in a Retch ball mill. About 25 mg of the pulverized hair was digested overnight at 45°C in 1 ml 

of 0.5 M hydrochloric acid. The samples were allowed to cool before the addition of 1 ml of 0.5 M sodium 

hydroxide and 1 ml of phosphate buffer solution (pHS). 20 fll of the clear supernatant were used for the 

immunoassay. 

The objective of this study is to determine if a commercially available ELISA system was sufficiently 

sensitive for the routine screening ofketamine in hair. We evaluate the immunoassay performance in terms 

of precision, accuracy, efficiency, sensitivity and specificity. The limit of detection (LOD) of the kit was 

found to be 0.6 ng/mg. The linearity range for ketamine was found to be up to 4.3 ng/mg using spiked hair 

samples. The intra-day % CV (within-day precision) for ketamine at concentrations of 0.6, O.S and 1 ng/mg 

was found to be from 3.73 % to 6.12 %. Interference study was evaluated by measuring the extent of cross 

reactivity of various cOmmon drugs of abuse such as opiates, 11-nor-b. 9 -THC-9-carboxylic acid, 

amphetamines and their ring analogues, cocaine and its metabolites, benzodiazepines, buprenorphine and 

its metabolites, LSD and its metabolites with ketamine. All the drugs above do not interfere with the 

detection of ketamine using ELISA. Only a low cross-reactivity was observed for low concentrations of 

norketamine. 

A total of 62 hair segments from suspected ketamine abusers were screened using the ELISA test kit. These 

hair specimens were obtained from suspected ketamine abusers. The effciency, sensitivity and specificity of 

the immunoassay were evaluated in comparison with gas chromatography/ mass spectrometry (GCIMS) 

using these hair specimens. Confirmatory cut-off for ketamine was 0.6 ng/mg. True positives, true 

negatives, false positives, and false negatives were determined using immunoassay cut-off at 0.8 ng/mg and 

1.0 ng/mg. An optimum immunoassay cut-off concentration of 1.0 ng/mg was determined for the kit. At 

this cut-off, a total of 55 hair samples were screened positive and 7 screened negative. Comparing with 

GCIMS results, 2 false negatives and no false positives were determined. The immunoassay exhibited an 

efficiency of 96.7 %, a sensitivity of96.4 % and a specificity of 100 %. 

The results collected support that the IDS One-StepTM ELISA test kit provides a suitable preliminary 

screening procedure for the detection of ketamine in hair specimens. It offers a rapid, sensitive, and 

effective method to determine the presence ofketamine in hair in suspected ketamine abusers. 

Keywords: ELISA, Ketamine, Hair Analysis 

Page 323

M41 

GAS CHROMATOGRAPHY:'HIGH-RESOLUTION MASS SPECTROMETRIC METHOD FOR 

DETERMINING METHAMPHETAMINE AND ITS MAJOR METABOLITE AMPHETAMINE 

IN HUMAN HAIR 

Jin Young Kim* and Moon Kyo In, Drug Analysis Laboratory, Forensic Science Division, Supreme Public 

Prosecutors' Office, Seoul 137-730, Korea 

Objective: The purpose of this study is I) to effectively eliminate the biological background or interference 

by cosmetic treatment and 2) to develop a suitable confirmatory procedure for analyzing methamphetamine 

(MA) and amphetamine (AMP) in human hair using a gas chromatography high-resolution mass 

spectrometric (GC-HRMS) technique. 

Nature ofthe study: GC-HRMS method development for detecting MA and its metabolite AMP containing 

an extremely low concentration in human hair, and eliminating co-eluted compounds in cosmetically 

treated hair samples. 

Material and methods: We measured the total length of hair samples, and noted special features such as 

coloring, bleaching, etc. Before we analyzed hair samples, we 1) washed them twice with 10 mL water and 

10 mL acetone, 2) then dried them under a fume hood, 3) finely cut the hair with scissors into small 

fragments below 1 mm, and 4) weighed each hair sample. We then transferred thirty milligrams of hair to a 

Teflon-faced, rubber-lined screw-cap test tube (16 x 100 mm). To extract analytes from the specimens, we 

added 2 mL of methanol-5 M hydrochloric acid (20:1, vlv) containing 100 ng of AMP-ds and MA-ds as 

internal standards into test tubes. Then, we directly extracted the hair samples under ultrasonication for lh 

and left them to stand at room temperature overnight. The hair was filtered off and filtrate was evaporated 

under a nitrogen stream in a TurboVap LV evaporator (Zymark Corp., USA). We dissolved the dried 

sample in 50 ilL of trifluoroacetic anhydride (TFAA) and 50 ilL of ethyl acetate and placed it in a dry /~, 

heating block for the derivatization at 70°C for 30 min. The mixture was allowed to cool to room 

temperature. The residue was reconstituted with 40 ilL volume of ethyl acetate. We injected an aliquot (1 

ilL) of the sample solution into GC-MS. 

Results: With the HRMS method the limits of detection (LOD) were 9 pglmg for MA and 21 pg/mg for 

AMP using a 30 mg hair sample, and the SIM responses were linear with coefficients of correlation 

ranging from 0.9998 to 0.9999. The recoveries were found to be 91.1-92.3%. By using HRMS (resolution 

of 5000), we improved the detection sensitivity due to eliminating the biological background. The LODs 

for MA and AMP were 2.4-4.4 times lower than low-resolution mass spectrometry (LRMS). We applied 

the described method to hair samples from suspected MA abusers. In nineteen of the thirty hair samples 

not detected by the LRMS SIM technique, we confirmed AMP using an accurate HRMS SIM measurement 

of diagnostic ions. In cases where there is a low concentration of AMP in the hair, high resolution SIM is 

useful to eliminate chemical background, which is present in single-quadrupole LRMS due to solvent 

and/or matrix ionization. The HRMS technique makes the detection of rather low concentrations of drugs 

in hair samples possible. In this experiment, the hair samples analyzed using GC-HRMS are not specially 

prepared and are the same as those analyzed by GC-LRMS. 

Conclusion: The proposed GC-HRMS method shows a high sensitivity and specificity. This method can 

also be used as a suitable analytical tool for identirying AMP as major metabolite of MA in human hair, 

where identirying metabolites at a low amount in hair samples is requested, and especially in cosmetically 

treated hair. 

Keywords: Hair, GC-HRMS, Methamphetamine, Amphetamine 

Page 324

M42 

A MIXED MODE SOLID-PHASE EXTRACTION METHOD FOR THE LC/MS 

DETERMINATION OF DRUGS OF ABUSE IN PRESERVED SALIVA 

Kevin M. Jenkins, Michael S. Young*: Waters Corporation, 34 Maple Street, Milford, MA 

Saliva is a less invasive alternative to urine or blood in forensics analysis for drugs of abuse. Because drug 

residues are typically less concentrated in saliva than in other biological fluids such as blood or urine, 

quantification limits for saliva samples must be considerably lower than for urine, in the low nglmL range. 

Compared with GCIMS, LCIMS is a simpler analytical approach requiring no derivatization. However, the 

saliva collection fluid contains ingredients (stabilizers and preservatives) that present some interference 

problems for LCIMS. A mixed-mode (cation-exchange) SPE cartridge, Oasis MCX, provided effective 

cleanup suitable for LCIMS determination of basic drugs of abuse in preserved saliva. The drugs studied 

were cocaine, codeine, morphine, amphetamine, methamphetamine, ecstasy (MDMA) and phencyclidine 

(PCP). MDMA-d5 was used as an internal standard. Calibration was accomplished in the range from 5 to 

250 ng/mL. Correlation coefficients (r2) for all constituents were better than 0.998. Reproducibility for all 

constituents was better than 10 % (RSD) for 6 replicate samples fortified at the 10 nglmL and 50 nglmL 

levels. Recovery was better than 85 % for all constituents. 

Keywords: Saliva, LCIMS, Drugs of Abuse 

Page 325

M43 

COCAETHYLENE: A POTENTIALLY LETHAL TOXICANT 

Danyel H. Tacker* and Anthony O. Okorodudu: Department of Pathology, University of Texas Medical 

Branch, Galveston, TX, USA 

Introduction: Cocaethylene (CE) is an active metabolite of cocaine and ethanol, produced by the liver in a 

majority of cocaine abusers who concomitantly abuse ethanol. Though the mechanism of death associated 

with cocaine overdose has historically been relative to vasospasm and cardiac/cerebral infarct and failure, 

we propose a role for CE as a toxicant to the vascular endothelium. We investigated the effects ofCE on 

microvascular endothelium to further characterize mechanisms of vasculotoxicity and associated tissue 

ischemia. 

Methods: An in vitro model of microvascular endothelial CE exposure (ImM) was used. Cytotoxicity 

assays included viability and LDH release analysis. Morphological (silver stain) and electricallresistance 

analysis was used to determine the effect of CE on monolayer permeability. Analysis of variance for n :: 3 

was significant when p

M44 

FORENSIC ENTOMOTOXICOLOGY: A STUDY IN THE DEPOSITION OF BARBITURATES 

INTO THE LARVAE OF THE BLACK BLOW FLY, PHORMIA REGINA 

Michelle R. Peace, M.F.S.* and Alphonse Poklis, Ph.D., Department of Pathology and the Forensic Science 

Program, Virginia Commonwealth University, Richmond VA 23284 

Due to events in severe decomposition, either no soft tissue remains on which to perform a toxicological 

analysis or putrefactive fluids complicate and interfere with the analysis of the soft tissues. The purpose of 

this experiment was to study the trends in the deposition of barbiturates into the larvae of the black blow 

fly, Phormia regina, in order to better understand the value of entomological evidence as toxicological 

specimens. Drug deposition was analyzed by linear regression to find a correlation between whole larvae 

drug concentration and food source drug concentration. P. regina larvae were raised at 21°C on pork 

homogenized with three concentrations of barbital (200, 400, 800mglkg), phenobarbital (20, 40, 80mglkg), 

pentobarbital (20, 40, 80mglkg), and thiopental (15.5, 31, 62mglkg). The middle dosage of each drug 

reflected the LDso of the drug in rabbits. At the end of the feeding stage, the larvae were harvested, 

washed, and frozen. Ten larvae were subsequently homogenized, diluted in 2 ml water, and subjected to a 

liquid-liquid extraction. The extracted drugs were derivatized with MethElute and analyzed by GCiMS. In 

the concentration ranges investigated, the concentrations of barbital, phenobarbital, pentobarbital, and 

thiopental found in the larvae strongly correlated with the concentration of the drug in the food source (R 2 

= 0.9976, 0.9914, 0.8938, and 0.9095, respectively). Larvae feeding on pork homogenized with 800mglkg 

of barbital accumulated 1.8 times more drug than those feeding on 200mgikg. Larvae which fed on the 

high concentrations of phenobarbital, pentobarbital, and thiopental accumulated, respectively, 3.1, 5.4, and 

68.5 times more drug than those that fed on the low concentrations of drugs. This data indicated that as the 

Iipophilicity of the drug increased, the larvae accumulated significantly more drug. 

Keywords: Entomology, Toxicology, Barbiturates 

Page 327

M45 

DETECTION OF COTININE IN EXHALED BREATH BY GAS CHROMATOGRAPHY-MASS 

SPECTROMETRY: A PRELIMINARY STUDY 

3 

Michele L. Merves J *, Chris W. Chronister l , Bruce A. Goldberger l • , Richard J. Melker and Mark S. Gold 3 • 4 : 

University of Florida College of Medicine, IDepartment of Pathology, Immunology and Laboratory Medicine; 

2Department of Anesthesiology; JDepartment of Psychiatry, Division of Addiction Medicine; 4Departrnents of 

Neuroscience, Community Health and Family Medicine; 'Gainesville, FL; 4800 S.W. 35 th Street, Gainesville, 

FL 32608. 

Exhaled breath analysis is a very useful diagnostic tool in the fields of medicine and forensic science. Exhaled 

breath is routinely used to detect markers of disease, as well as correlate ethanol concentrations with effects on 

human performance and behavior (DUI). Compared to other biological specimens, the collection of exhaled 

breath is simple and non-invasive, with minimal exposure to infectious material. Because exhaled breath is a 

physiological waste product, the quantity available exceeds those of other biological matrices such as blood and 

urine. In addition, little sample preparation is required. Finally, exhaled breath accounts for all routes of drug 

exposure including oral ingestion, injection, inhalation, and dermal absorption. 

This is a preliminary study to determine if cotinine, an oxidative metabolite of nicotine, is detectable in exhaled 

breath of humans. Cotinine was chosen as a metabolic marker of nicotine use because of its long half-life 

compared to nicotine. Exhaled breath specimens were collected onto commercially-available XAD-4 traps 

(Supelco) and analyzed by gas chromatography-mass spectrometry (GC-MS). The resin, XAD-4, is a polymeric 

adsorbent available as beads within a small glass tube (70 mm x 6 mm). Upon collection of the exhaled breath, 

the resin was removed, fortified with drcotinine (Cerilliant Corporation), and sonicated in methanol for 30 

minutes. The organic layer was separated from the resin and dried under a gentle stream of nitrogen at 40°C. The 

residue was reconstituted in phosphate buffer (PH 6). The cotinine was isolated from the buffer utilizing 

CleanScreen® solid-phase· extraction (SPE) cartridges (ZSDAU020) manufactured by United Chemical 

Technologies, Inc. Following elution from the SPE cartridges using a solution ofmethylene chloride, isopropanol, 

and ammonium hydroxide, the extracts were dried under a gentle stream of nitrogen at 40°C and reconstituted in 

methanol. The extracts were subjected to GC-MS analysis using an Agilent Technologies 6890N series gas 

chromatograph equipped with a Agilent Technologies 5973 network mass spectrometer operated in electron 

ionization and selected ion monitoring (SIM) modes. Helium was used as the carrier gas at a constant flow rate of 

1.0 mLimin. Automated injections (2-3 ).iL) were made onto a capillary GC column. The ions (mlz) monitored 

for cotinine were 98.1, 175.1 and 176.1, and the ions (mlz) monitored for d 3 -cotinine were lOLl and 179.1. 

Initial studies have demonstrated cotinine in the exhaled breath of cigarette smokers, while cotinine was not 

detected in control (non-smokers) exhaled breath. These data will serve as a model for detection of other 

compounds (drugs and their metabolites) present in exhaled breath which may be beneficial for many other 

medical, forensic, and toxicological applications. 

Keywords: Cotinine, Exhaled Breath, GC-MS 

Page 328

M46 

"THE MECONIUM PROJECT": AN ITALIAN-SPANISH JOINT STUDY TO ASSESS 

EXPOSURE TO ILLICIT DRUGS DURING PREGNANCY AND BIRTH OUTCOMES 

S. Pichini*, P. Zuccaro, E. Marchei, M. Pellegrini, J Murillo, C. Puig, O. Vail, R. Pacifici, 6 Garcia-Algar: 

Istituto Superiore di Sanita, Rome Italy, V. Le Regina Elena 299, Rome, RM 00161, Italy 

Servicio de Pediatria, Hospital del Mar, Barcelona, Spain 

The accurate assessment of fetal exposure to drugs of abuse through the objective measure of biomarkers 

could provide the basis for appropriate treatment and follow-up of new-borns, which can present symptoms 

of drug withdrawal. Furthermore, information regarding the real prevalence of drugs of abuse use during 

pregnancy could also be disclosed. 

For the first time in Europe the "Meconium Project" aimed to estimate the prevalence of drug use by 

pregnant women and the effects of exposure to illicit drugs during pregnancy on the mother, fetus, and 

infant. 

Between September 2002 and February 2004, among the mother-infant dyads from the Hospital del Mar, 

the fourth biggest hospital of the city, 1439 dyads met eligibility criteria and 1151 (79%) of those eligible 

agreed to participate in the study. Exposure was defined as an admission of use of cocaine, opiates or 

amphetamines by structured interview or the presence of drugs and their metabolites in 24 and 48 h 

meconium as determined by use of liquid chromatography-mass spectroscopy assay. Nonexposure was 

defined as a negative drug use history by interview and a negative analytical result. When exposure could 

not be confirmed, such as when meconium was not obtained or was inadequate for confirmatory analysis, 

the mother-infant dyad was excluded (n =288). 

Preliminary results on the analyzed 497 mother-infant dyads showed that structured interview disclosed a 

0.8, 1.8 and 2.2% mothers exposed to opiates, cocaine and both drugs while only one mother declared 

ecstasy consumption. Meconium analysis showed that prevalence of opiates, cocaine and combined drugs 

exposure was 9.1,3.8 and 10.7%, respectively and confirmed the case of ecstasy use. Arecoline, the main 

Areca nut alkaloid, was found in meconium specimens from 4 out of 10 Asiatic newborns, whose mothers 

declared areca nut consumption during pregnancy. 

A similar percentage of drug-exposed mothers were found between Spanish, American and African women 

(11.5,9.9 and 8.6%, respectively), while only a 6.3% Asiatic women resulted drug consumers. Exposure 

status caused a significant low birth weight in newborns from mothers exposed to cocaine (mean and S.D.= 

2848 ± 315 g in newborns from cocaine mothers vs 3262 ±504 g in newborns from non-exposed mothers). 

Other somatometric differences included crown-heel height. 

Of the 4 newborns exposed to arecoline, one showed a low birth weight, low intrauterine growth, 

hyporefiexia, hypotonia and a second presented a withdrawal neonatal syndrome by Finnegan test. 

In conclusion, these preliminary findings indicate a prevalence of opiates, cocaine and amphetamines 

consumption during pregnancy in this mixed mediterranean and non-european population lower that that 

reported in the North-american pregnant women. The higher sensitivity of meconium analysis, already 

demonstrated, in comparison to maternal interview, was confirmed making this test ideal for screening 

maternal and fetal exposure to conventional and non-conventional drugs of abuse during pregnancy. 

Keywords: Drugs ofAbuse, Exposure, Pregnancy 

Page 329

M47 

METHAMPHETAMINE AND AMPHETAMINE CONCENTRATIONS IN MECONIUM OF 

NEONATES OF WOMEN ENROLLED IN THE IDEAL STUDY OF IN UTERO 

METHAMPHETAMINE EXPOSURE 

2 

Zirong Zhao!, Jing Liu , Linda L. LaGasse 2 , Chris Derauf, Penny Gran!, Rizwan Shah 2 Amelia Arria" 

William Haning 2 , Lynne M. Smith 2 , Barry Lester" and Marilyn A. Huestis l *: I Che~istry and Dru~ 

Metabolism, IRP, NIDA, NIH, Baltimore, MD 21224; .2 IDEAL Community Research Network 

Background: Tobacco, cannabis and methamphetamine (MA) use by pregnant women can negatively impact 

child development. The Infant Development, Environment, and Lifestyle (IDEAL) study is a multi-center, 

longitudinal investigation of the effects of prenatal methamphetamine exposure. Meconium, a useful matrix for 

identifYing in utero drug exposure, was employed to identify gestational drug use. Little published data are 

available on MA detection in meconium. 

Objective: To characterize the detection and quantification ~f MA, tobacco and cannabis in meconium compared 

to self-report. 

Method: Mothers who used tobacco, cannabis or alcohol during pregnancy were included in both MA-exposed 

and control groups. Meconium samples were analyzed by EMIT (USDTL) for cannabinoids, cocaine, opiates, 

amphetamines with cutoff concentrations of 40, 75, 150 and 500 nglg, respectively. Positive samples were 

confirmed by GC/MS with cutoffs of 2 nglg for II-nor-L'l.9-carboxy-THC and 5 nglg for cocaine, 

benzoylecgonine (BE), m-OH-BE, and cocaethylene, morphine, codeine, hydrocodone, hydromorphone, and 

MA, amphetamine (AMP), and MDMA. Tobacco use was identified with an ELISA cotinine screen 

(International Diagnostics Systems Corp., cutoff to nglg). 

Results: Of the 13,808 mothers screened, 1631 were consented and 176 enrolled. MA exposed mothers (n=84) 

were identified by self-report of gestational MA use and/or GCIMS confirmation of MA, AMP, and/or MDMA 

in infant meconium. Comparison participants (n=92) were matched by race, birth weight, maternal education 

and type of insurance, denied amphetamines use and had negative meconium results. Among the 1631 mothers, 

self-reported use rates were 5.2% (amphetamines), 25% (tobacco) and 5.9% (cannabis). Positive meconium 

screening rates were 3.6% for any amphetamine, 20% cotinine and 11.2% cannabis. For specimens that screened 

positive, 40.7% of amphetamines and 20.2% of cannabis specimens were confirmed. EMIT sensitivity and 

specificity were checked by reanalyzing 35 specimens from babies of women who reported MA use but had a 

negative screen and 35 specimens from the control group that also tested negative. All controls' meconium 

specimens were negative by GClMS (5-nglg), while 4 of35 specimens from self-reporters that screened negative 

were found positive by GCIMS, albeit at low concentrations (MA 8 52 and AMP 5 - 26 nglg meconium). 

Users reported administering MA by the fonowing routes: 48% smoked; 9% injected, II % sniffed, 6% ingested 

and 26% used multiple routes of administration. On average, 68% of the meconium from neonates whose 

mothers reported 3 nl trimester use had detectable MA, while detection rates were :::;10% for self-reported use 

during the 1 st and/or 2nd trimesters. Mean±SD, median and range ofMA concentrations were 3674 ± 3406, 2623, 

479 to 13,431 nglg meconium and AMP 569 ± 543, 403, 30 to 2000 nglg meconium in infants whose mothers 

reported 3 nl trimester use. However, the highest MA (19,376 and 16,976 nglg) and AMP (2765 ng/g) 

concentrations were found in offspring born to women who reported MA use only in the 1 st or 1 5t and 2 nd 

trimesters, raising questions about the self-report. The log transformed meconium MA concentrations 

significantly correlated with the frequency ofMA use in the 3 nl trimester (r=0.645, P=O.004), although variability 

prevents prediction of frequency of use for an individual mother. AMP was always detected in MA positive 

meconium. In 55% of the GCMS positive samples, the ratios of amphetamine to MA were 0.1 to 0.2; 14% were 

less than 0.1 and 18% were 0.2 to 0.3. 

Limitations: Data are preliminary as the IDEAL study continues to recruit; larger samples in the future may 

strengthen or refute interim observations. 

Conclusions: Meconium analysis for MA is a useful adjunct to self-report for identification of MA exposure; 

however, the [reatest sensitivity was achieved with specimens collected from offspring of women who reported 

use in the 3 r trimester. Further research is needed to determine if there are additional MA metabolites in 

meconium that could improve the identification of MA-exposed infants. 

Keywords; Methamphetamine, Meconium, In Utero Exposure 

Page 330

M48 

PASSIVE CANNABIS SMOKE EXPOSURE AND ORAL FLUID TESTING 

1* I I I 1 

Keith Kardos ,Sam Niedbala, Sal Salamone, Dean Fritch, Matth Bronsgeest and Edward J. Cone 2 ; I 

OraSure Technologies, Bethlehem, PA, and 2 ConeChem Research, LLC, Severna Park, MD 

Illicit cannabis use continues to be highly prevalent in the United States. Oral fluid testing for /19 

tetrahydrocannabinol (THe) provides a convenient means of detection of recent usage. In this study, the 

risk of positive oral fluid tests from passive cannabis smoke exposure was investigated by housing four 

cannabis-free volunteers in a small, unventilated and sealed room with an approximate volume of 36 m 3 • 

Five active cannabis smokers also were present in the room and each smoked a single cannabis cigarette 

which contained an average of 1.75% THC. Cannabis smoking occurred over the first 20 minutes of the 

study session. All subjects remained in the room for approximately 4 hrs and provided oral fluid and urine 

specimens at designated times. Oral fluid specimens were collected with the Intercept DOA Oral Specimen 

Collection Device before the start of the session and periodically throughout the session. Three urine 

specimens were collected (0, 20 and 245 min), In addition, three air samples were collected (first 5 min 

during smoking, for 20 min immediately following smoking, and at 65-100 min after start of session) for 

measurement of THC concentrations in air. All oral fluid specimens were screened by enzyme 

immunoassay (EIA) for cannabinoids (cutoff concentration == 3 ng/mL) and tested by GC-MS-MS for THC 

(LOQILOD = 0.25 ng/mL). All urine specimens were screened by EIA for cannabinoids (cutoff 

concentration =50 nglmL) and tested by GC-MS-MS for THCCOOH (LOQ/LOD 1 nglmL). Air samples 

were measured for THC by GC-MS (LOD = I ng/L). A total of eight oral fluid specimens (collected 20 to 

50 min following initiation of smoking) from the four passive subjects screened and confirmed positive for 

THC at concentrations ranging from 3.6-26.4 ng/mL. Two additional specimens from one passive subject, 

colIected at 50 and 65 min, screened negative but contained THC in concentrations of 4.2 and 1.1 ng/mL, 

respectively. All subsequent specimens for passive participants tested negative by EIA and GC-MS-MS for 

the remainder of the 4 hr session. All urine specimens collected during the session from the passive 

participants screened and confirmed negative at LOQ/LOD by GC-MS-MS with the exception of one 

subject whose specimen collected at the end ofthe session contained 3.4 ng/mL ofTHCCOOH by GC-MS 

MS. In contrast, oral fluid specimens collected from the five cannabis smokers generally screened and 

confirmed positive for THC throughout the session at concentrations substantially higher than observed for 

passive subjects. Urine specimens from cannabis smokers also screened and confirmed positive at 

conventional cutoff concentrations. Based on the limited air sample measurements, it was estimated that 

passive subjects inhaled a total of approximately 0.05 mg of THC during the exposure session. A biphasic 

pattern of decline for THC was observed in oral fluid specimens collected from cannabis smokers, whereas 

a linear decline was seen for passive subjects suggesting that initial oral fluid contamination is cleared 

rapidly and is followed by THC sequestration in the oral mucosa. It is concluded that the risk of positive 

oral fluid tests from passive cannabis smoke inhalation is limited to a period of approximately one hour 

following exposure. 

Keywords; Cannabis, THC, Passive, Oral Fluid 

Page 331

M49 

DISPOSITION OF NJ-TETRAHYDROCANNABINOL IN ORAL FLUID AND PLASMA AFTER 

CONTROLLED ADMINISTRATION OF SMOKED CANNABIS 

Edward J. Cone 1 * and Marilyn A. Huestis 2 : I ConeChem Research, LLC, Severna Park, MD; 2 National 

Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD' 

Understanding the relationship of A9-tetrahydrocannabinol (THC) concentrations in oral fluid and plasma 

is important in interpretation of oral fluid test results. Current evidence suggests that THC is deposited in 

the oral cavity during cannabis smoking. This "depot" represents the primal)' or sole source of THC found 

when oral fluid is collected and analyzed. In this research, oral fluid and plasma specimens were collected 

from six subjects following smoking of cannabis cigarettes containing 1.75% and 3.55% THC. There was 

at least one week between each cannabis administration. Plasma specimens were analyzed by GC-MS and 

paired oral fluid specimens were analyzed by radioimmunoassay (RIA). In addition, one individual's oral 

fluid specimens were also analyzed by GCIMS. These data are unique in that they represent simultaneous 

or near simultaneous collection of oral fluid and plasma specimens in subjects following controlled 

cannabis dosing. The first oral fluid specimen, collected from one subject at 0.2 hr following initiation of 

smoking, contained a THC concentration of 5800 ng/mL (GC-MS). By 0.33 hr, the THC concentration in 

oral fluid had fallen to 81 nglmL. From approximately 0.3 hr through 4.0 hr, the mean (± SD) THC ratio of 

oral fluid to plasma THC concentrations was LI8 (0.62) with a range of 0.5 to 2.2. Within 12 hr, both oral 

fluid and plasma THC concentrations generally declined below 1 nglmL. RIA analyses of oral fluid 

specimens for six subjects demonstrated the same pattern of initial high levels of contamination 

immediately after smoking, followed by rapid clearing, and a slower decline over 12 hr. Mean THC oral 

fluid concentrations by RIA at 0.2 hr were 864 nglmL and 4167 nglmL compared to plasma concentrations 

of 52 ng/mL and 230 ng/mL at 0.27 hr following the low and high dose cannabis cigarettes, respectively. 

The similarity in oral fluid and plasma THC concentrations following the dissipation of the initial 

"contamination" indicates the likelihood of a physiological link between these specimens. Recent studies 

have shown that sublingual or transmucosal administration of pure THC results in direct absorption of'~' 

intact THC into the bloodstream, thereby bypassing the gastrointestinal tract. The current study 

demonstrates that THC is deposited in the oral cavity and remains for up to 24 hr following cannabis 

smoking. The decline in THC oral fluid concentration over this time suggests that there may be absorption 

of THC into blood as previously shown with pure THC. Passive cannabis exposure studies appear to 

indicate that positive oral fluid tests for THC can occur shortly after cannabis smoke exposure, but results 

were negative within one hour. Consequently, when vel)' recent passive exposure to cannabis smoke can 

be ruled out, it is concluded that a positive oral fluid test provides credible evidence of active cannabis use. 

Keywords: Cannabis, THC, Oral Fluid, Depot 

Page 332

M50 

DETECTION OF DRUGS OF ABUSE USING ORAL FLUID WITHIN A PAROLE SETTING 

Ann Snyder'*, Tiffany Miller\ Eileen Sevem 2 , Chris CiottiZ, Dean Fritch 2 , Sal Salamone 2 , Keith Kardos 2 , 

R. Sam Niedbala 2 :: 'Cuyahoga County Court of Common Pleas Laboratory, 1276 West Third St, 

Cleveland, OH 44113; 2 OraSure Technologies, 150 Webster St, Bethlehem PA 18015. 

A study was conducted using rapid on-site and lab based oral fluid tests as well as urine for drugs of abuse 

detection in a probation facility that requires routine testing of participants. Individuals in this facility are 

required to routinely visit with a probation officer. As part of their visit a urine specimen is collected, any 

presence of drug is punishable and grounds for revocation of probation. This study was organized to 

compare new technologies for on-site and lab-based oral fluid tests comparing them to routine urinalysis. 

On-site oral fluids were determined using the UPlink Test System. UPlink is based on instrumented 

analysis of lateral flow chromatography using Up-Converting Phosphors. Lab-based oral fluids were 

determined using the Intercept System. The Intercept oral fluid collection device gathers an average of 

OAmL of oral fluids, which is express-sent to a laboratory. After isolation of the sample, Intercept 

specimens are screened using microplate assays followed by GCMSMS confirmation of positive samples. 

Urine analysis was performed using Microgenics Inc. reagents on a Hitachi 917. A total of217 specimens 

were tested in oral fluids. A total of 207 specimens were tested with urine due to problems with subjects 

unable to provide a specimen. Positive samples were found in all fluids for all major classes of drugs 

including cocaine, THC, opiates, PCP and amphetamines. True Positives and True Negatives were judged 

based upon manufacturers recommendations for oral fluids and Substance Abuse and Mental Health 

Services Administration Federal workplace drug testing guidelines for urine, with the exception of opiates, 

which use a 300 nglmL cutoff. Overall, the % agreement when comparing on-site UPlink and lab-based 

Intercept results to GCMSMS was 98.7% and 99.6% respectively. Overall % agreement between urine 

screening results and GCMS was 99.8%. These results demonstrated the analytical performance of either 

oral fluid test system was comparable to lab-based urine testing. However, collection of oral fluid negated 

the requirements for special facilities allowing probation officers to directly observe collections. 

Keywords: Oral Fluid, Urine Drug Testing, Drugs ofAbuse 

Page 333

MSl 

DISPOSITION OF COCAINE AND METABOLITES IN HUMAN SWEAT FOLLOWING 

CONTROLLED COCAINE ADMINISTRATION 

Sherri L. Kacinkol*, Allan J. Barnes l , Eugene W. Schwilke 1 , Jonathan M. Oyler 2 , Edward J. Cone\ Eric T. 

Moolchan\ and Marilyn A. Huestis l : IChemistry and Drug Metabolism Section, Intramural Research 

Program, NIDA, NIH, 5500 Nathan Shock Drive, Baltimore, MD 21224; 2Department of the Army, US 

Army Research, Development and Engineering Command, 5183 Blackhawk Road, Aberdeen Proving 

Ground, MD 21010 ; 3ConeChem Research, LLC. 441 Fairtree Drive, Serverrna Park, MD 21146 

The analysis of sweat for drugs of abuse offers a convenient, non-invasive technique for monitoring drug 

exposure. Sweat can be collected using the Pharmchek® Sweat Patch; an absorbent cellulose pad with 

adhesive backing that is worn on the body. The comparison of sweat test results to urinalysis indicates that 

sweat may be an acceptable matrix for drugs of abuse screening for drug treatment, criminal justice, and 

employment screening purposes. 

Healthy volunteers (n=8), with a history of cocaine (COC) use, provided informed consent to participate in 

this IRB approved research study and resided on the closed ward throughout the 12-week study. After an 

initial three-week washout period, volunteers received 3 low dose (15 mg/10 kg) subcutaneous injections of 

cocaine HCI on every other day and, three weeks later, 3 high doses (150 mgl10 kg). 

Pharmchek® Sweat Patches collected for up to 48 hours following the first low (n=82) and high (n=18) 

doses were isolated for cocaine and eleven metabolites by solid phase extraction and analyzed by GCIEI 

MS. LOD and LOQ were 2.5 ng/patch for COC, ecgonine methyl ester (EME), benzoylecgonine (BE) and 

meta- and para-hydroxycocaine (m- poOH-COC)o 

COC was the primary analyte detected in patches worn for 1-15 hours after the first low (26 of 82, 32% 

positive patches) and high (36 of18, 46% positive patches) doses. COC Cmax ranged from 6-64 ng/patch 

and 5-315 ng/patch following low and high doses, respectively. Not only was there large variability in 

Cmax, but there also was wide variability for the time of Cmax. Several patches (2 of 21 for low dose and 

6 of 26 for high dose) worn for four or less hours were positive for COC 24-48 hours after administration. 

Half of the participants had positive COC results for both low and high dose sweat patches applied 32 

hours after dose and worn for 15 hours, demonstrating improved detection in patches worn for extended 

periods. 

EME was detected in sweat patches collected from 3 participants after low dose (1% positive patches) and 

4 participants after the high dose (13% positive patches) and was detected only in patches collected for the 

first 24 hours after dosing. Maximum EME concentrations ranged from 8-41 ng/patch and 5-51 ng/patch 

for the low and high dose, respectively. BE was detected in 6% of the low and 8% of the high dose patches. 

with concentration ranges of 5-13 ng/patch after low dose and 6-12 ng/patch after high dose. One 

participant had a positive m-OH-COC patch (3 ng/patch) after high dose administration. In addition, m 

and p-OH-COC were detected concurrently in the 2-4 hour (3 ng/patch m-OH-COC, 6 ng/patch p-OH 

COC) and 8-23 hour (3 ng/patch m-OH-COC and 5 ng/patch p-OH-COC) patches collected from one 

participant after the high dose. 

COC, BE and EME can be detected within 2 hours of drug administration but there is wide intra- and intersubject 

variability in the amount detected and the time at which Cmax is reached. During the 24 hours after 

exposure COC can be detected in patches worn for short periods (1-2 hours) but after 24 hours the patch 

must be worn for longer periods to detect COCo EME was detected more often and at higher 

concentrations than BE; both were detected only within the first 24 hours after exposure. 

Sweat testing for COC and BE has been proposed by the Substance Abuse and Mental Health Services 

Administration for federally-mandated workplace drug testing with patches worn for 1-14 days. In this 

study patches were only worn for 15 hours. It should be noted, however, that EME was detected more 

frequently and at higher concentrations than BE. 

Keywords: Cocaine, Sweat, Controlled Cocaine Administration 

Page 334



Postmortem 

Toxicology 

Page 335

PI 

NEW GENERATIONS OF ANTIDEPRESANTS IN FATAL INTOXICATIONS 

Marie Stankova*, Petr Kurka, 1. DvoraCek 

Institute of Forensic Medicine, University Hospital, Ostrava, Czech Republic 

New generations of antidepresants are widely used for treatment of various types of depressive and other 

disorders. These drugs have a relatively low toxicity, and fatalitiesexclusively from these newer drugs are 

uncommon. 

The authors describe six cases of fatal intoxications with a combination of some newer antidepresant with 

other drugs. Although these newer antidepresants are relatively safe, in combination with other drugs 

(especially with psychoactive drugs) they can cause death. 

There drugs were isolated from biological specimes using either a diethyl ether extraction for qualitative 

analysis or by solid phase extraction for quantitative analysis. 

Detection of drugs in biological specimes (urine, gastric contents, liver, kidney) was performed by thin 

layer chromatography/colour reactions (TLC/CR) and confirmed by gas chromatography/mass 

spectrometry method (GC/MS). Concentrations of drugs in postmortem bloods (subclavian artery blood) 

were determined using GC/MS method. 

' 

The results are presented in the table: 

case gender/age antidepressant blood lev. other drugs blood lev. 

1. F/41 citaloprarn 0.80 Promethazine 10.20 

2. F/47 moclobemide 18.20 7.44 

3. M/50 mianserin 1.33 0.51 

Dosulepin 2.76 

4. M/38 moclobemide 32.00 Promethazine 19.00 

Alcohol 

5. M/51 citalopram 1.24 Levomepromazine 

ketamine 

6. M/37 citaloprarn 2.39 Morphine 

blood (subclavian artery) level: mg/I - drugs, g/kg - alcohol 

NQ - no quantification 

Keywords: new antidepresants, overdose, GCMS 

Page 336

P2 

IT'S ONLY JUST BEGUN A POSTMORTEM TISSUE DISTRIBUTION OF STRATTERA® OR 

A TOMOXETINE IN TWO FATALITIES 

Dan Anderson"', Kristina Fritz, Michelle Sandberg, Jamie Lintemoot, Sara Kegler, and Joseph J. Muto 

Los Angeles County Dept. a/Coroner, 1104 N. Mission Road, Los Angeles, CA 90033 

Stratterl~ or Atomoxetine is a selective norephinephrine reuptake inhibitor approved by the Food and Drug 

Administration in December 2002. It is considered the first non-stimulant treatment of attention deficit 

hyperactivity (ADHD) in both children and adults. Atomoxetine is prescribed in lO, 18,25,40, and 60-mg 

dose capsules. Since Strattera® has entered the market, the Los Angeles County Dept. of Coroner 

Toxicology Laboratory has only encountered two cases where atomoxetine was detected. Presented are the 

case histories, cause and manner of death, the postmortem tissue distribution of atomoxetine, all designed 

to aid the forensic toxicologist with the interpretation of their own casework. 

The analysis of atomoxetine from postmortem specimens consisted of a basic, Iiquid/liquid (nbutylchloride) 

extraction procedure with screening and quantitation by GC/NPD. Linearity was achieved 

from 0.05 to 5.0 mgIL with the limit of quantitation at 0.05 mgIL. Confirmation of atomoxetine was 

determined using a GCIMS by comparison with an analytical standard. 

The tissue distribution of atomoxetine was as follows: 

... 

Atomoxetine ._._,.-->-' (mg/L r:-:-- or mg/kg) _.,...__.__._-- ---, 

Chest Blood Femoral Blood Vitreous Liver Gastric Bile Urine 

I Case No. 1 0.08 0.04 -- - -- -- 0.16 

I Case No.2 1.3 0.23 0.34 4.2 1.2 or 0.17 mg total 1.5 0.62 

It's the author's belief that these are one of the first cases of atomoxetine being detected and reported from 

postmortem specimens. 

Keywords: Atomoxetine; Tissue Distribution, Postmortem 

Page 337

P3 

POSTMORTEM REDISTRIBUTION OF THE ENANTIOMERS OF CITALOPRAM AND ITS 

METABOLITES IN A RAT MODEL 

Kugelberg FC i ', Kingback M i , Carlsson B i , Druid H2 

iDepartment of Clinical Pharmacology, Linkoping University, SE-581 85 Linkoping, Sweden 

2Department of Forensic Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden 

Enantioselective drug analysis is used to study the variation in the disposition of the enantiomers of 

racemic drugs. This type of analysis may give additional information in interpreting forensic toxicological 

results, provided that the postmortem redistribution is of the same magnitude for the enantiomers. One 

example of a racemic drug is the widely used antidepressant citalopram (CIT) that belongs to the selective 

serotonin reuptake inhibitors (SSRIs). Different therapeutic properties have been shown for the CIT 

enantiomers, and the S-(+)-enantiomer is responsible for the clinically relevant effects. Recent data indicate 

that the R-(-)-enantiomer instead counteracts the SSRI effect that is produced by the S-(+)-enantiomer. The 

major CIT metabolites, demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT), are less potent 

than the parent compound with regard to SSRI properties. In recent years, several reports on CIT 

concentrations in human postmortem cases have become available. However, only limited data are 

available describing postmortem concentrations of the separate enantiomers of CIT and metabolites. There 

is a general awareness that the concentration of a drug in an autopsy blood sample may not necessarily 

reflect the in vivo concentration just before death, and this difference is explained by postmortem drug 

redistribution. Thus, in order to accurlltely interpret the postmortem concentrations of CIT a general 

understanding of the changes in the enantiomer concentrations after death is required. Hence, the aim of the 

present study was to investigate if postmortem redistribution of the enantiomers of CIT, DCIT .and DDCIT 

occurs in an experimental rat model after three different dosing procedures with racemic CIT. Two rat 

groups underwent chronic administration (20 mglkg daily) using subcutaneously (s.c.) implanted osmotic 

pumps. After 10 days, one of these groups received an acute-on-chronic drug challenge by a single s.c. 

injection of 100 mg/kg. The third group received the single 100 mg/kg dose only. Heart blood and brain 

samples were collected antemortem and 1,3 or 24 h postmortem after storage in room temperature. In an 

additional experiment, we examined the role of the lungs as a reservoir of postmortem drug release and if 

the early-phase postmortem redistribution was different in room temperature (21°C) as compared with a 

cold environment (4°C). The samples were analyzed with an enantioselective HPLC method with 

fluorescence detection. Increased postmortem blood drug and metabolite concentrations compared to 

corresponding antemortem concentrations were observed in all groups (p

P4 

INFLUENCE OF PUTREFACTION AND WATERY CIRCUMSTANCES ON THE 

CONCENTRATIONS OF ACONITINE IN TISSUES 

Hideyuki Hayakawa 1 *, Makoto Nihira zl , Makiko Hayashida 2l , Morihisa Sagil), Toshiji Mukai l ) and 

Youkichi Ohn0 2 ; 1) Department ofLegaJ Medicine, St. Marianna University School of Medicine 

2) Department ofLegaJ Medicine, Nippon Medical School 

Aconitium plants (Ranunculaceae) are showy herbaceous flowering perennials that are widely distributed in the mountainous or 

cooler parts of northern temperate zones. They contain highly toxic Aconitum alkaloids such as aconitine, mesaconitine, 

hypaconitine and jesaconitine in the root, leaf and stem. Because of their high toxicity, Aconitum alkaloids have been used in 

suicides and homicides. In such cases, quantitative analysis ofAconitum alkalo ids in the body fluids and tissues is necessary to 

determine the cause of death. For correct diagnosis of poisoning in cases where only putrefied material is available, it is 

necessary to investigate the influence of putrefaction on the concentrations ofAconitum alkaloids in body tissues. Moreover, we 

sometimes encounter cases of putrefied cadavers floating in the water, where toxicological analysis is necessary. Although the 

rate of non-enzymatic hydrolysis of aconitine dissolved in phosphate buffer has been studied, no reports are available on 

postmortem changes of Aconitum alkaloids in putrefied tissue. Therefore, we performed animal experiments to determine the 

influence of putrefaction over a 4-weekperiod on the concentrations of Aconitum alkaloids .in body tissues after oral 

administration of aconitine; one group of animals has been kept in the water for putrefaction, as a model of putrefied humans 

found in the water. ICR mice were divided into a whole-body storage group and an excised-organ storage group. Aconitine was 

administered orally at a dose of 4 mglkg body weight to the mice, which were sacrificed within 35 min; for the whole-body 

storage group, each cadaver ofthe animals was placed in a bottle containing 750 ml water, followed by incubation at 20 or 35 "C 

for 1, 3, 7, 14 and 28 days. For the excised-organ storage group, the liver and brain were separately incubated without any water 

at 20 'C for the same intervals. The concentrations of aconitine and its hydrolysis products were measured by gas 

chromatography-mass spectrometry in the selected ion monitoring mode. In the whole-body storage group, the concentration 

of aconitine markedly increased in the liver and brain, showing postmortem diffusion of aconitine from the stomach. 

Concentrations of benz oyla conine and aconine also increased in the liver; but when expressed as ratios of the amounts of these 

substances to the total amounts of alkaloids, the ratios of aconitine gradually decreased, while those of its hydrolysis products 

increased. In the excised-organ storage group, no increase ofaconitine concentration was observed; while benzoylaconine in the 

liver increased significantly on days 3 and 7. Only small amounts of aconitine were detected in the bottle water. The above 

results show that aconitine changes through hydrolysis and postmortem diffusion. But aconitine can be detected in the liver even 

on day 28. Our results of the whole-body storage group suggest that the postmortem diffusion of aconitine from the stomach to 

the brain via tissues and body fluids may occur in watery circumstances. Caution should be taken against the postmortem 

diffusion of aconitine for victims found in the water. 

Keywords: Aconitine, Putrefilction, Postmortem diffusion 

Page 339

PS 

POSTMORTEM DETERMINATION OF SILDENAFIL IN BLOOD 

Hwakyung Choi, SangHee Woo, Juseon Lee, Yooshin Park, Myungyun Pyo, Heesun Chung* 

National Institute of Scientific Investigation, Seoul, Korea 

School ofPharmacy, Sookmyung Women's. University, Seoul, Korea 

Sildenafil is sold as a remedy for an oral therapy for erectile dysfunction by the name of ViagraTM. It is an 

. inhibitor of cGMP phosphodiesterase and has a potential for cardiac risk during sexual activity in patients 

with preexisting cardiovascular disease. Therefore, patients who have cardiovascular problems should not 

use sildenafil citrate. 

There have been 10 sildenafil related deaths in Korea. All were males in their late thirties to sixties. To 

investigate the cause of death, the gastric contents and blood underwent toxicological screening. To Iml 

blood 50[J of 100/0 tadanafil (lnt.std) were added. Blood samples were purified by solid phase 

extraction using a Bond Elut-Certify .column (Varian®). Blood was loaded into a cartridge that was prewashed/conditioned 

with methanol and O.IMphosphate buffer. The elution was performed with ethyl 

acetate:ammonia (98:2). After the solid phase extraction of blood, sildenafil was analyzed with high 

performance liquid chromatography with photodiode array detector (HPLCIPDA). The mobile phase 

consisted of 0.05M triethylamine-phosphate (pH 3.0), methanol and acetonitrile (580:250: 170,v/v). 

Postmortem bloods were analyzed at a flow rate of 1.0mllmin with a detection wavelength of 290nm. 

Standard curve of spiked sildenafil into blank blood was linear with a correlation coefficient of 0.998. The 

limit of detection and the limit of quantification for sildenafil were 0.02 llg/mL and O.IIlg/mL, respectively. 

The relative recovery was 99.4%. 

The concentration range of sildenafil of postmortem blood was 0.00-0.70 Ilg/mL.(mean 0.166±0.067) in all 

ten cases. In case 6, the blood concentration of sildenafil was below the limit of quantification (0.1 Ilg/mL), 

however, sildenafil was confirmed in the gastric contents. In case I, 2 and 5, the victims had a medical 

history of cardiac disease. 

Key words: Sildenafil; postmortem blood concentration, HPLC/PDA 

Page 340

P6 

TWO CASES INVOLVING CLOMIPRAMINE INTOXICATION 

Joseph Avella*, Michael Lehrer, Michael Katz and Edward Minden. 

Suffolk County Office of the Medical Examiner, Toxicology Laboratory, Hauppauge NY. 

Clomipramine and its active metabolite norclomipramine were identified and quantitated in multiple tissues 

recovered from two postmortem cases using liquid chromatography/mass spectrometry (LCIMS). The 

LCIMS method afforded the consistent chromatography needed to accurately determine both the parent 

drug and the more polar metabolite while simultaneously providing the specificity associated with mass 

spectral data. 

In the first case a 23-year-old female with a history of depression presented to her attending pulmonologist 

with a respiratory tract infection. She was diagnosed with pneumonia upon admission to the hospital. The 

patient was placed on antibiotics and oxygen and appeared stable. Within twelve hours the patient became 

confused and anxious. She began to have difficulty breathing and subsequently coded and expired despite 

resuscitative efforts. An autopsy was conducted and tissues were submitted for toxicological analysis. 

Clomipramine and norclomipramine was detected in the following concentrations: Heart blood; 1.39 mg/L 

and 2.64 mg/L, Brain; 8.02 mg/kg and 26.74 mg/kg, Liver; 14.25 mg/kg and 41.12 mg/kg, Urine; 0.48 

mg/L and 1.15 mg/L and Gastric Contents 3.99 mg/total and 0.61 mg/total. 

~\ 

Case #2 was a 56-year-old female with a history of depression who had previously attempted suicide with 

prescription painkillers. The evening prior to her death, the decedent had no complaints and retired for the 

night. The following morning she was found dead in her bed. Autopsy revealed evidence of medication 

around her mouth and in her gastric contents. No significant natural disease was present and toxicology 

was requested to assess the possibility of an overdose. In this case clomipramine and norclomipramine was 

detected in the following .amounts: Femoral blood; 0.70 mg/L and 0.66 mg/L, Pulmonary artery blood; 1.00 

mg/L and 1.11 mg/L Brain; 4.86 mg/kg and 7.43 mg/kg, Liver; 11.68 mg/kg and 20.76 mg/kg, Gastric 

21.20 mg/total and 1.34 mg/total and Small Intestinal contents; not detected and 0.10 mg/total. The other 

significant finding was the presence ofhydro cod one in femoral blood at a concentration of0.13 mg/L. 

In both cases the determination of clomipramine and metabolite in the brain samples was considered 

especially useful for assessing toxicity. The author's found the brain to be helpful for several reasons. 

First, although the blood results were potentially toxic, the liver results were not conclusively indicative of 

an overdose. Patients that have undergone long-term tricyclics antidepressant (TCA) therapy can sequester 

substantial amounts of parent drug and metabolite in liver, a particular concern because the TCA's may 

exhibit postmortem redistribution. In some cases of TCA overdose the liver results are unmistakable (i.e. 

clomipramine concentrations exceeding 200 mg/kg after acute poisoning). In the cases detailed in this 

report the liver results were considered insufficient to confidently establish fatal toxicity. Blood specimens 

(especially central) were viewed critically while interpreting results due to the concern of possible 

postmortem changes. The use of brain was advantageous because concentration changes are unlikely to 

occur in brain tissue due to postmortem redistribution. In these cases the analysis of brain provided further 

insight with respect to toxicity by establishing the concentrations of clomipramine and norclomipramine at 

the site of action without complications arising from postmortem redistribution. 

This communication supplements the database on clomipramine and norclomipramine by providing 

quantitative determinations of both parent drug and metabolite in multiple tissues. A literature search 

revealed a paucity of data on clomipramine and norclomipramine levels in general and an absence of 

documented brain levels. When conducting these postmortem investigations, the inclusion of brain 

determinations provided valuable information in assessing the magnitude of toxicity in these cases 

involving clomipramine and its active metabolite norclomipramine. 

Keywords: clomipramine, postmortem redistribution, tricyclic antidepressants 

Page 341

P7 

A COMBINED DRUG INTOXICATION INVOLVING METAXALONE (SKELAXIN®) 

Juraj Cervenak*, Sarah M. Colvin, and Fiona J. Couper 

Office of the Chief Medical Examiner, 1910 Massachusetts Ave SE, Bldg 27, Washington, D.C. 20003. 

We present a case of a 24-year old female who was found dead on her bedroom floor, in a secure 

apartment. She had a history of anxiety and had been prescribed antidepressants for several years. A scene 

investigation revealed empty beer cans and numerous prescription and over-the-counter medications. 

Prescribed medications included Lexapro® (citalopram), Skelaxin® (metaxalone), Ritalin® 

(methylphenidate), and Restoril® (temazepam). An autopsy was performed and the findings were 

unremarkable. Specimens were submitted for a full toxicological analysis, including an alcohol analysis by 

headspace gas chromatography with flame ionization detection; a screen for drugs of abuse and several 

prescription drug classes using an enzyme-linked immunosorbent assay technique (ELISA); and a screen 

for basic compounds using gas chromatography-mass spectrometry (GC-MS). Positive findings were 

confirmed and quantitated using GC-MS. The following drug concentrations were measured in postmortem 

blood: metaxalone 38 mg/L, citalopram 0.49 mglL, diphenhydramine 0.10 mgIL and ethanol 0.02 g/100 

mL. Methylphenidate and temazepam were not detected. Further investigation revealed the following 

prescription history for the decedent prior to her death: 

# Prescribed # Remainin 

100 tablets 66 tablets 

180 o 

60 o 

30 5 

Metaxalone is a centrally acting muscle relaxant prescribed for the relief of pain associated with acute, 

musculoskeletal conditions. The recommended dose for adults is 2 x 400 mg tablets, three to four times a 

day. The circumstances and drug results in this case were similar to those reported in the only other 

metaxalone-related fatality cited in the literature: a 29-year old female with a history of depression had 

postmortem blood metaxalone and citalopramconcentrations of 39 mglL and 0.77 mgIL, respectively. In 

the present study, the cause of death was determined to be due to "combined effects of metaxaione, 

citaiopram, diphenhydramine and ethanof', and the manner of death was "undeterminec!'. A discussion of 

the case circumstances, the toxicology findings and the pharmacology of metaxalone will be presented. 

Keywords: Metaxalone, Skelaxin, Intoxication 

Page 342

P8 

A CASE OF FATAL OVERDOSE WITH LABETALOL 

Karen Woodall"', Centre of Forensic Sciences, Toxicology Section, 25 Grosvenor Street, Toronto, Ontario, 

Canada 

Labetalol (Normodyne®, Trandate®) is an alpha and beta-adrenergic and beta-adrenergic receptor 

antagonist used in the treatment of hypertension. A typical dose oflabetalol ranges from 200 400 mg/day 

however some individuals may require up to 2100 mg/day. Symptoms of labetalol overdose are excessive 

hypotension and bradycardia. There is very limited information available regarding labetalol concentrations 

and toxicity and no reports where the cause of death has been attributed solely to a labetalol overdose. This 

study describes a case where the cause ofdeath was attributed to an acute overdose of labetalol. 

A 79-year-old female was admitted to hospital for high blood pressure and chest pain. She was stabilized 

and after remaining in hospital for 7 days was being prepared for discharge. At this time she was observed 

to have swallowed numerous tablets by another patient. She subsequently became hypotensive and 

bradycardic, and resuscitation attempts were unsuccessful. A blood sample taken during the attempted 

resuscitation was tested at the hospital and the drug screen showed that labetalol was significantly higher 

than the therapeutic range. Empty prescription bottles of labetalol, lorazepam and furosemide (medications 

she brought with her to the hospital) were found at the scene, however the actual number of tablets taken is 

not known. There were no other significant findings in the hospital drug screen except for the elevated 

labetalol concentration. 

A postmortem was conducted and toxicology testing was performed on a heart blood sample. Labetalol was 

measured by high-pressure liquid chromatography (HPLC) and the detected concentration was 15 mg/L. 

Lorazepam was measured by mass spectrometry (MS) and was detected at a concentration of 73 ng/mL. In 

addition to specific testing for labetalol and lorazepam, a general basic drug screen using gas 

chromatography and mass spectrometry (GC and GCIMS) was performed and traces of sertraline and 

metoprolol were detected. This laboratory does not have a method for the analysis of furosemide. 

Therapeutic concentrations of labetalol in serum have been shown to range from 0.02 - 0.25 mg/L, and 

levels over 0.5 mg/L are considered to be toxic. In one case report, a woman who suffered renal failure 

following an acute ingestion of labetalol (16g) had a serum concentration of 29 mg/mL. The concentration 

of labetalol in this case is approximately 60 times higher than the therapeutic levels. The autopsy showed 

signs of chronic hypertension but no gross or microscopic cause of death. In the absence of other 

significant drug findings the cause ofdeath was reported as acute labetalol intoxication. 

Key words: labetalol, fatality, overdose 

Page 343

P9 

DETERMINATION OF OXCARBAZEPINE AND ITS METABOLITES IN POSTMORTEM 

SPECIMENS INCLUDING HAIR BY MEANS OF LIQUID CHROMATOGRAPHY WITH MASS 

DETECTION (HPLC/APCIIMS) IN AN EPILEPTIC PATIENT AFTER A FATAL DRUG 

POISONING 

Malgorzata Klys* and Sebastian Rojek 

Grzegorzecka str 16, Krakow, Malopolskie, 31-531, Poland 

One use ofhair analysis in forensic toxicology is the documentation of prior drug administration. This is 

illustrated in a suicidal death of a 58-year-old epileptic patient who was treated with oxcarbazepine and 

probably with levomepromazine. 

The toxicological analysis carried out by HPLC/APCIIMS included also the hair (6cm length) besides of 

postmortem blood and liver samples. The method was validated for oxcarbazepine (OXCBZ) and its two 

metabolites, 10-hydroxycarbazepine (CBZ-100H) and trans-diol-carbazepine (CBZ-diOH) in various 

biological matrices. 

Thetoxicological findings are as follows: 

Hair 

Autopsy 

Segment (2 Segment (2 

Segment (cm) 

cm) 

cm) 

I 

II 

III 

Blood Liver 

Concentration (I!Wg) 

Oxcarbazepine 3.9 10.4 13.0 0.79 n.d. 

18.4 

53.9 

105.9 13.96 15,28 

i CBZ-100H 

4.7* 

5.1 * 

8.1 * 17.6* - 

.0.5 

1.2 

3.0 0.23 n.d 

CBZ-diOH 

0.12* 

0.12* 0.23* 0.29* 

- 

Levomepromazine - - - 1.96 30,25 

*Relatlve concentration metabolite / prekursor 

n.d. - not detected 

The analysis of the postmortem blood indicated oxcarbazepine and its two main metabolites were present at 

therapeutic concentrations; levomepromazine was detected at a fatal concentration. In three 2-cm segments 

ofhair,.oxcarbazepine and its two metabolites were detected; however, levomepromazine was not detected 

in this . specimen. A complex chemical-toxicological investigation confirmed the information that the 

decedent. was an epileptic patient and was treated with oxcarbazepine for at least 6 months before death. In 

addition, he took a toxic dose of levomepromazine in order to commit suicide. The analysis revealed 

differences between the' concentration levels of oxcarbazepine and its active metabolite CBZ-IOORin 

postmortem specimens and hair, suggesting different mechanisms of penetration of certain metabolites and 

their precursors into these materials. 

Keywords: oxcarbazepine, hair, LClAPCIIMS 

Page 344

PIO 

STABILITY OF SULFONYLUREAS IN STORED POSTMORTEM BLOOD SPECIMENS AND 

WATER STANDARDS 

A.H. Battah, M.D, Ph.D*, Forensic Medicine and Toxicology Division, Faculty of Medicine, University of 

Jordan, Amman 11942, Jordan. 

K.A. Hadidi, Ph.D, Toxicology Laboratory, Forensic Medicine and Toxicology Division, Faculty of 

Medicine, University of Jordan, Amman 11942, Jordan. 

Stability of three commonly prescribed sulfonylureas (gJipizide, chlopropamide and glibenclamide) in 

postmortem blood and water stored at -20,4, and 25°C were evaluated over a I-year period. The drugs 

were analyzed simultaneous by utilizing solid phase extraction techniques and were identified and 

quantified by high performance liquid chromatography. The data showed that, significant loss of 

g/ibenclamide (GB) was noticed at second and third week of storage at 25 and 4°C respectively. The 

decrease was gradual but no clear pattern could be established. At the end of the I-year storage period at 

either 4 or 25°C, the level of GB was about 37% and 46% of its initial value in aqueous solution and 

postmortem blood samples respectively. There was no significant loss of both glipizide and chlopropamide 

in all stored samples over the first 6-month period. After that time a significant decrease in their levels 

were noticed, and at the end of the I-year of storage concentrations ranged from 67.5 to 84.1 % of their 

initial values. Storage at -20°C showed the least loss of drug concentration. Authentic samples of these 

drugs showed similar results. This study supports the importance of paying careful attention to using 

prepared aqueous drug standards even when stored in cold conditions. In order to obtain reliable results of 

drug analysis and drug stability in biological samples, such studies should be considered after prolonged 

sample storage time. The best storage temperature to keep aqueous standards and blood samples for the 

investigated drugs with sufficient stability was found to be at -20°C. 

Keywords: Stability, Sulfonylurea, Blood. 

Page 345

PH 

DISTRIBUTION OF QUETIAPINE IN NINE POSTMORTEM CASES 

Tara J. Valouch, B.S.'", Dwight Flammia, Ph.D., James K. Kuhlman, Ph.D., Virginia Division of Forensic 

Science 

Quetiapine (Seroquel ®) is an antipsychotic drug approved by US FDA in September 1997 for the 

treatment of schizophrenia. It is classified as a dibenzothiazepine and structurally related to clozapine. The 

molecular formula is C21H2sN30zS with a molecular weight of 383.6. It is believed to exert its 

pharmacological effect through antagonism of sertonergic (5HT2) and dopaminergic (D2) receptors in the 

brain. Quetiapine is extensively metabolized, but the pharmacological effect is primarily attributed to 

parent drug. It is rapidly absorbed after oral administration, reaching peak plasma concentrations in 1.5 h. 

The elimination half life is 6 h. Peak serum quetiapine concentrations range from 0.17 - 0.37 mgIL 

following a single oral 75 mg dose to 0.19 0.63 mg/L following a 450 mg dose. 

Nine postmortem quetiapine cases identified by the Virginia Division of Forensic Science toxicology 

laboratories during 2003 are presented. Quetiapine was identified by full scan electron impact gas 

chromatography-mass spectrometry (GCMS) following a basic drug extraction from blood. Quantitation 

was performed using a basic solid-phase extraction followed by the addition of BSTF A and selected ion 

monitoring GCMS. The following mean tissue concentrations and ranges were determined: heart blood 

18.9 mgIL (0.53 - 76 mgIL, n = 4), peripheral blood 11.5 mg/L (0.46 - 37 mg/L, n = 6), urine 16.4 mg/L 

(1.9 - 37 mg/L, n 6), liver 123 mg/Kg (7.2 510 mg/Kg, n = 9), and gastric 48.2 gIL (15-130 gIL, n = 4). 

Medical examiners certified the cause of death in eight of the nine cases as combined drug toxicity. In one 

case the cause of death was quetiapine toxicity and the manner of death was suicide. The quetiapine 

concentrations in this case were 15 mglL in antemortem blood, 18 mgIL in peripheral postmortem blood, 

and 89 mglKg in liver. Only one case contained both heart and peripheral blood (7.0mgIL and 3.4 mg/L 

respectively). The heart/peripheral blood ratio was 2.0 suggesting postmortem redistribution. 

Keywords: quetiapine, postmortem, distribution 

Page 346

P12 

DETERMINATION OF ATRACTYLOSIDE IN CALLILEPIS LAUREOLA USING SOLID-PHASE 

EXTRACTION AND LIQUID CHROMATOGRAPHY - ATMOSPHERIC PRESSURE 

IONISATION MASS SPECTROMETRY 

Paul A. Steenkamp"', Nial M. Harding', Fanie R. van Heerden b .•, Ben-Erik van Wyk C 

"Forensic Chemistry Laboratory, Department ofHealth, P.O. Box 1080, Johannesburg 2000, South Africa; 

bDepartment of Chemistry and Biochemistry, Rand Afrikaans University, P.O. Box 524, Auckland Park 

2006, South Africa; CDepartment of Botany, Rand Afrikaans University, P.O. Box 524, Auckland Park 

2006, South Africa 

A selective analytical method based on high-performance liquid chromatography, combined with 

atmospheric pressure ionisation mass spectrometry, was developed for the detection of atractyloside. The 

tuberous root of the plant Callilepis laureola is used in traditional medicine by the Zulu and Xhosa people 

of South Africa and it is well known by the Zulu name impila, which means 'health'. However, C. laureola 

has also been implicated in the death of numerous patients who used medication prepared from impila in 

which atractyloside, the dipotassium salt of a sulfonated kaurene glycoside, was identified as the toxic 

principle. This compound, together with carboxyatractyloside, was also identified as the toxic principle of 

Atractylis gummifera L. (Asteraceae), a plant linked to human fatalities in Mediterranean countries since 

ancient times. 

The analysis was performed on an Xterra Phenyl column utilising a gradient elution profile and a mobile 

phase consisting of 10 mM aqueous ammonium acetate buffer-MeOH-acetonitriIe. The developed method 

showed a good linearity for the calibration curve spanning the 10 ng/mL to 1 j.ig/mL (r2 = 0.997) range. 

The limit of detection and limit of quantitation were determined and found to be 100 pglmL and 10 ng/mL, 

respectively. The method was successfully applied to the analysis of C. laureola tuber, herbal medicine 

mixtures and viscera samples (stomach, stomach contents, liver, kidney, blood, urine, bile) for the presence 

of atractyloside. 

Keywords: Atractyloside, CallUepis laureola, HPLC-MS 

* Corresponding author. Tel: +27-11-725-2279; fax: +27-11-725-4731. E-mail address: fcIjhb@icon.co.za 

Page 347

P13 

MIRTAZAPINE-RELATED DEATHS 

R A Braithwaite* and S P Elliott 

Regional Laboratory for Toxicology, City Hospital, Birmingham. UK 

Mirtazapine is a novel antidepressant drug which has a pharmacological action on both noradrenergic and 

serotonergic systems. The drug has been on the US and European markets for about 10 years, but 

relatively few deaths associated with mirtazapine have been reported in the literature. Post-mortem blood 

mirtazapine concentrations of up to 0.3 mglL have been reported in cases where the drug is not implicated 

in the cause of death. There is also evidence to show that the drug undergoes post-mortem redistribution, 

which can make interpretation offindings more difficult. 

Relatively few deaths reported in UK have noted the presence of mirtazapine. However, in the last few 

years (2001-2003) the Laboratory has investigated a total of 8 cases (5 male, 3 female) aged between 17 

and 77 years where mirtazapine has been found to be present in significant concentrations, but generally in 

association with other drugs. Post-mortem blood mirtazapine and its desmethyl metabolite 

(normirtazapine) have been measured using liquid chromatography with U.V-diode array detection. In 

addition, extensive screening of blood and other fluids was undertaken to determine the presence of other 

drugs and alcohol. 

Details of the 8 cases investigated are shown in the Table below. In 7 of the 8 cases blood was taken from 

a peripheral site at post-mortem. Post-mortem blood mirtazapine concentrations ranged between 1.0 - 404 

mglL (mean 2.1 mglL) The ratio of mirtazapine to normirtazapine concentrations ranged between 1.9 and 

20. 

Age Sex Site oj Mirta:: Normirta= MirtiN ormirta:: Other drugs present and 

Collect (mgIL) (mgIL) Ratio (concentrations mglL) 

/~... 

75 M Femoral 2.2 OJ 7J Temazepam, (2.2), venlafaxine 

(3.6). Found dead at home 

46 M Femoral 2.0 20 Temazepam (Ll) ethanol 

(214 mg/dL). Found dead at home 

60 M Unknown 1.6 0.8 2.0 No other drugs detected. Found 

dead in river 

56 M Femoral 1.0 OJ 3.3 Diazepam (1.5), Sildenafil (trace), 

alcohol (72 mg/dL). Found dead at 

home 

77 M Femoral 2.1 1.1 1.9 Zopic1one (1.6). Found dead in bed 

51 F Femoral 2.5 004 6.7 Zopiclone (1.7), Lorazepam (004), 

chlorpromazine (trace), alcohol 

(54 mgldL). Found dead in bath 

21 F Common 1.3 0.3 5.0 Dextropropoxyphene (2.4), 

Iliac 

Paracetamol (124), Venlafaxine 

(1.4) Zopidem (trace). Found dead 

at home 

17 F Femoral 4.4 0.7 6.1 Diltiazem (4.2), paracetamol (70), 

Zopic1one (trace). Found dead at 

home with suicide note 

In conclusion, mirtazapine appears to be more commonly seen in recent post-mortem cases, probably due 

to prescribing trends. In all but one of the cases, other drugs or alcohol were present. However, 

mirtazapine overdosage appeared to be a significant factor in at least half of the deaths reported and were 

associated with a parent blood mirtazapine concentration >2.0 mgIL and a ratio of mirtazapine to 

normirtazapine >2. 

Keywords: blood, mirtazapine, postmortem 

Page 348

.~. 

P14 

TWO FATAL INTOXICATION CASES WITH IMIDACLOPRID: LC~MS ANALYSIS 

P.Proen~a*, H.M. Teixeira, F. Castanheira, P. Monsanto, J.Pinheiro, S. Santos, E.P. Marques, D.N. Vieira 

National Institute of Legal Medicine, Coimbra, PORTUGAL . 

Aim: Imidacloprid [1~(6-chloro-3pyridylmethyl)-N-nitroimidazolidin~2-ylideneamine] is a new and potent 

nitro methylene insecticide with high insecticidal activity at very low application rates. It is the first highly 

effective insecticide for which the mode of action has been found to deviate from the almost complete and 

irreversible blocking of the postsynaptic nicotinergic acetylcholine receptors. The authors present two fatal 

cases due to this pesticide, in two males, 33 and 66 years old. 

Methods: An LC~MS with electrospray method is described for measuring imidacloprid and its metabolite 

in postmortem samples. In the chromatographic separation, a reverse-phase column CIS (2.1xI50mm, 

3.5Jlm) was used and the mobile phase composed with acetonitrile and 0.1 % formic acid (15:85), at a 0.25 

mllmin flow rate. The samples were prepared by a liquid-liquid extraction procedure with dichloromethane. 

Results: Calibration curves for imidacloprid were performed in acetonitrile in blood and urine, achieving 

linearity between 0.2Jlg/ml and 15Jlg/ml. The mean recovery was 86%, coefficient of variation was ±5.9% 

and the detection limit 2.0ng/ml. Quantitative results were obtained for all postmortem matrices available 

of the two fatal cases: blood, urine, stomach contents, liver and kidney. The concentrations found ranged 

between 0.29Jlglml (urine) and 350Jlg/mi (stomach contents). Postmortem blood concentrations found were 

12.5Jlglml and 2.05Jlg/mL 

Conclusions: The authors developed a sensible and validated method to detect and quantify imidacloprid in 

postmortem samples and emphasize the lack or even absence of literature with fatal cases related to this 

insecticide. 

Keywords: Imidacloprid, fatal intoxication, LC-MS 

Page 349

PIS 

LEVELS OF LEVETIRACETAM IN POSTMORTEM BLOOD 

Ashraf Mozayani* and Terry J. Danielson. Harris County Medical Examiner, 1885 Old Spanish Trail, 

Houston, Texas, 77054 

Levetiracetam (LEV) is a recently introduced anticonvulsant agent and little information is available to 

assist in the interpretation of levels seen in post-mortem cases. Our objective is to report our observations 

from three Medical Examiner cases in which LEV was detected. 

LEV is said to have "ideal" pharmacokinetic characteristics for clinical application. It is well absorbed after 

oral administration and is distributed approximately into body water. It is not appreciably bound to plasma 

protein and its apparent plasma elimination half-life is near to 6 hours. 

LEV was extracted from acidified blood into dichloromethane and was assayed by gas chromatography I 

mass spectrometry using hexobarbital as internal standard. The retention factor, relative to hexobarbital, 

was 0.79 on a HP-5 column (initial temp, 50 ·C; time 1, 1 min; ramp 1,50 ·C Imin to 100 ·C; ramp 2, 20 

·C/min to 285 "C). The electron impact mass spectrum contained an intense ion at mlz 126, minor ions at 

mlz 170 (m*), 98 and 69. 

Case summaries and levels of LEV in postmortem blood are reported in Table 1. The data suggest that 

postmortem levels of LEV near 60 mgIL may be within the normal therapeutic range, and might be 

encountered, without toxicological consequence. A lower level, near 10 mgIL as seen in case number 3, 

may be sub-therapeutic. 

Table 1: Levels of Levetiracetam (LEV) in postmortem blood, Case 1, femoral. Cases 2 and 3, source not 

indicated 

Case # Age Sex LEV Other drugs 

Cause ofDeath 

Manner 

(mg/L) (mg/L blood) 

1 22 M 60 None detected Idiopathic epilepsy, Natural 

cerebral sclerosis 

2 58 M 61 Hydrocodone, 0.25 Hypertensive 

Natural 

Metoprolol, 1.4 

Amitriptyline, 0.38 

Nortriptyline, 0.57 

Paroxetine, 0.33 

cardiovascular disease 

Post -cerebral vascular 

infarct seizure disorder 

I 

Trazadone, 0.93 

Phenobarbital, < 10 

3 35 M 11 None detected Drowning, 

Accident 

Seizure due to remote 

leptomeningitis 

Keywords: Levetiracetam, blood, postmortem 

Page 350

P16 

OPTIMIZING AN AUTOMATED SOLID-PHASE EXTRACTION PROCEDURE FOR 

POSTMORTEM TISSUE SAMPLES 

T. Stimpf1*, Department of Forensic Medicine, Medical University of Vienna, Sensengasse 2, A-1090 

Vienna, Austria 

Quality assurance regulations in forensic laboratories, efforts to reduce systematic errors, costs, time spent per case as 

well as efforts to improve technician-safety all lead to the consideration ofautomated procedures for sample extraction. 

In contrast to liquid-liquid extraction, solid-phase extraction (SPE) can be more easily automated by incorporating 

robotics improving the sample quality in respect to the analyte yield and increasing consistency and laboratory 

productivity . 

Today, automated SPE procedures are widely used for the extraction of body fluids such as urine, serum, plasma and 

whole blood. In postmortem forensic toxicology, however, these specimens are not always available and, with regard to 

the investigation of the cause of death, human tissue samples - especially brain - can be highly important. 

Drummer and Gerostamoulos pointed out that"... there is little evidence that extraction efficiencies ofdrugs from solid 

tissues are likely to be much worse than with fluid specimens ifsuitable precautions are taken. These include a suitably 

fluid homogenate prepared from the solid tissue with sufficient water or buffer" [I]. 

In an automated SPE procedure published by our working group, a crude extract from postmortem tissue samples could 

be achieved, avoiding protein precipitation and the resulting loss ofanalytes by adsorption or occlusion. 

In respect to the problem that tissue homogenates often do not easily pass through tightly-packed cartridges, it could be 

shown that - after homogenization and dilution with large volumes of buffer solution - the resulting colloidal solution 

could be directly applied to the polymeric sorbent [2]. 

For routine solid-phase extraction of tissue samples, several adaptations of the automated system ASPECTMXL, 

operated under 735-sampler software V 5.1 for Win NT 4.0 (Gilson Inc., Middleton, WI, USA), were necessary. 

• A large volume ofsample had to be applied in order to dilute the sample and dissolve protein bonds. 

• The upper frit ofthe extraction cartridge had to be removed to minimize the risk ofclogging. 

• A 5mL stainless steel sample loop for HPLC (Supeico, Bellefonte, PA, USA) was installed on the ASPEC 

XL to avoid adsorption of analytes to synthetic plastic materials (transfer tubing). 

• Special cleaning routines with 2N NaOH and 2N RN0 3 were used to remove any precipitated proteins. from 

the system. 

• The sample was applied very slowly to make use of "micellar chromatography", thus avoiding the adsorption 

ofmacromolecular interferences with larger than pore-sized diameters to the polymeric sorbent. 

• A pressure sensor was installed to compensate for inconsistent flow rates during loading, washing and 

eluting. 

When postmortem samples of tissue have to be extracted by automated SPE, monitoring of the overall system pressure 

is essential. Pressure control allows for high-pressure release if clogging of the cartridge occurs, avoiding 

contamination of the working area with the infectious sample; the aborted sample is then replaced to the sample tube. 

1. O.H. Drummer, J. Gerostamoulos, Postmortem drug analysis: analytical and toxicological aspects. Therap. 

Drug Monit. 24 (2002) 199-209. 

2. T. Stimpfl, J. Jurenitsch, W. Vycudilik, General unknown screening in postmortem tissue and blood samples: 

a semi-automatic solid-phase extraction using polystyrene resins followed by liquid-liquid extraction. J. 

Anal. Toxieo!. 25 (2001) 125-129. 

Keywords: automation, solid-phase extraction (SPE), tissue samples 

Page 351

P17 

POSTMORTEM DISTRIBUTION OF TRAMADOL, AMITRIPTYLINE AND THEIR 

METABOLITES IN A SUICIDAL OVERDOSE 

Nichole D. Bynum*, Justin L. Poklis, Maryanne Gaffney-Kraft and Jeri D. Ropero-Miller. Office of the 

Chief Medical Examiner, Chapel Hill, NC 27599 U.S.A. 

A case report involving a 34 year old white male who was found dead at home by a roommate is presented. The 

decedent's history included a suicide attempt and pain as result of a motor vehicle accident occurring 5 months 

earlier. At the time of death, he was being treated with tramadol/acetaminophen, metaxalone, oxycodone and 

amitriptyline. The descendant's mother stated that he had been taking increasing amounts of pain medication in 

order to sleep at night. 

Further investigation of this case was based on the fact that seven tissues were available for a fatal multi-drug 

ingestion concerning several analytes of interest, one of them being tramadol, which has limited information 

concerning tissue and metabolite concentrations. Toxicology tests yielded acetaminophen, oxycodone and 

cyclobenzaprine aortic blood concentrations of 140, 0.42, and 0.97 mg/L, respectively. Metaxalone and 

ibuprofen were detected at below therapeutic concentrations. The following matrices were analyzed for tramadol, 

n-desmethyltramadol, o-desmethyltramadol, amitriptyline and nortriptyline: aortic and iliac blood, urine, liver, 

vitreous humor, bile, brain, heart, kidney, lung, muscle and spleen. Analytes were quantitated by electron impact 

gas chromatography/mass spectrometry (GeIMS). 

The following table contains the postmortem analyte concentrations obtained for each tissue analyzed in this 

study. 

Matrix Tramadol N-Desmethyl O-DesmethyI Amitriptyline Nortriptyline 

Tramadol Tramadol 

Blood (aorta) mgIL 31.0 BQL* 0.901 5.79 

1.49 

Blood (iliac) mglL 6.21 0.20 

0.68 2.33 0.88 

Liver (mg/kg) 14.0 BQL 2.47 82.3 

29.1 

Urine (mg/L) 55.3 1.45 8.27 27.1 

6.63 

Vitreous Humor (mglL) 3.32 BQL 

0.25 0.25 BQL 

Bile (mgIL) 2.95 BQL 0.45 

41.4 

4.59 

Brain (mglkg) 4.62 BQL 

0.73 24.3 

9.29 

Heart (mg/kg) 4.95 BQL 1.08 17.4 

8.98 

Kidney (mglkg) 9.61 BQL 2.54 

25.8 8.40 

Lung (mg/kg) 87.0 BQL 1.15 

134 

22.2 

Muscle (mglkg) 22.0 BQL BQL 

22.6 2.47 

Spleen (mglkg) 29.1 BQL 

2.61 28.8 16.9 

, ~l,!L' Below quanbtat",n IIm.t 01 11.1; mglL (mSlkgJ. 

For all analytes, the parent-to-metabolite ratios support an acute toxicity with the parent concentrations being 5 

10 times greater than its major metabolite. For amitriptyline and nortriptyline, the order of highest to lowest 

concentrations detected are lung, liver, bile, urine and other solid tissues (spleen, kidney, brain, muscle and 

heart), central and peripheral blood, and finally vitreous humor. On the other hand, tramadol and its metabolites 

demonstrate a similar concentration trend with the exception of central blood and liver. It has been suggested that 

tramadol does not sequester into liver tissues or demonstrate postmortem redistribution from .drug-rich tissues. 

This case does not support or refute these observations since gastric contents were not collected and we cannot 

confirm the possibility of diffusion from the gastrointestinal tract to the central blood or timing of the ingestion 

of these drugs with respect to one another. There were no significant findings at autopsy; however, toxicology 

results supported a cause and manner of death resulting from suicidal mixed tramadol and amitriptyline toxicity. 

This case reports the tissue and fluid distribution of tramadol, amitriptyline, and their metabolites in an acutely 

fatal ingestion in an effort to document concentrations of these analytes in 12 matrices with respect to one 

another to assist toxicologists in difficult interpretations. 

Keywords: postmortem distribution, tramadol, amitriptyline 

Page 352

P18 

SCREENING BY ELISA FOR CARISOPRODOL IN POST-MORTEM BLOOD 

F. Nunes-Daniel and D. Boehme 

Ventura County Sheriff Forensic Sciences Laboratory; 800 S. Victoria Ave; Ventura, CA 93009 

Carisoprodol is an unscheduled substance widely prescribed in the USA to promote skeletal muscle 

relaxation. The use of carisoprodol is associated with CNS depression, which can lead to impairment in 

driving and other psychomotor activities. Therefore, it is very important to have a reliable method to test 

post-mortem blood sample for carisoprodol. Using Immunalysis Direct Elisa Kit and the PersonnalLABTM 

as the analyzer we developed an application to screen carisoprodol in post-mortem blood. We tested a 

variety of samples using different sample volumes and cutoffs. Using 25uL as the sample volume and 

500nglmL as the cutoff, thirty-two samples were analyzed by ELISA v. GCfMS and GCfNPD. Ten were 

positive by both the immunoassay and the chromatographic methods. One sample screened between the 

negative control and the cutoff calibrator, and was found to contain 4.78 mglmL of carisoprodol. The 

concentrations of carisoprodol in samples that screened positive ranged from 0.12 mgIL to 45.2 mgIL. 

These samples also contained meprobamate, ranging in concentration from 2.26 mg/L to 20.2 mgIL. None 

of the ELISA-negative samples contained carisoprodol at greater than 500 ngimL. The twenty-one samples 

that were negative by both the ELISA and by the chromatographic methods contained a broad range of 

other drugs, including cocaine, benzoylecgonine, codeine, morphine, hydrocodone, methadone, 

amitriptyline, nortriptyline, diazepam, nordiazepam, temazepam, alprazolam, acetaminophen, bupropion 

and its metabolites, norchlorcyclizine, nicotine, cotinine, caffeine, olanzapine, fluoxetine, norfluoxetine, 

norpropoxyphene, doxylamine, tripolidine, and metoclopramide. A precision study was conducted by 

analyzing 10 replicates each of the negative calibrator, a 250 ng/mL control, the 500 ng/mL calibrator, and 

a positive control containing 1000 nglmL carisoprodol. The OD's were tracked and the results were as 

follows: negative calibrator: 1.386 ±..0.051 (CV= 3.7%); 250 ng/mL control: 0.510 ± 0.015 (CV= 2.9%); 

500 nglmL calibrator: 0.440 ± 0.024 (CV= 5.4%); and 1000 ng/mL control: 0.410 ± 0.014 (CV= 3.4%). 

Keywords: Carisoprodol; immunoassay; postmortem blood 

Page 353

P19 

EVALUA TION OF DEBATED QUESTIONS: DO POSTMORTEM METHADONE 

CONCENTRATIONS OF AL TERNA TIVE SPECIMENS HAVE INTERPRETIVE VALUE 

Jeri D. Ropero-MiIler* and Ruth E. Winecker. Office of the Chief Medical Examiner, Chapel Hill, NC 

27599 U.S.A. 

Deaths due to methadone ingestion are escalating in many geographical locations nationwide. Often these deaths 

are the result of naIve users ingesting methadone either recreationally or as prescribed, however, their systems do 

not tolerate the dose taken. With the increased use of methadone, either as a recreational drug or to treat chronic 

pain, the population dying from methadone toxicity has radically changed over the last decade. Many factors 

contribute to the difficulty of interpreting methadone's role in a death. Some postmortem laboratories do not 

practice analyzing alternative matrices to interpret methadone's contribution to a death. In fact, limited 

publications discuss whether postmortem redistribution occurs and if so to what degree. Furthermore, some 

postmortem laboratories have traditionally ignored methadone liver concentrations based on the belief that their 

interpretative values are minimal. Moreover, overlapping therapeutic and toxic ranges make it difficult to 

interpret methadone's contribution to a death without having additional history, pathological findings (e.g. 

bronchopneumonia or peri mortem aspiration), and/or dosing regime. Given these observations, the Office of the 

Chief Medical Examiner (NC-DCME) retrospectively reviewed all deaths in the State of North Carolina between 

200 I and 2002 in which methadone was detected during toxicological analysis. This presentation evaluates 223 

death investigations in which methadone was detected in several postmortem tissues and fluids to ascertain 

whether or not data from alternative specimens are more useful than originally thought. 

After careful review of the history, pathology and toxicology findings, it was determined that 180 (80%) cases 

could be evaluated given the data obtained. Exclusion criterion included cases in which the prescription status 

was unknown or multiple drugs were detected in toxic concentrations. To assist in interpretation, these cases 

were divided into several groups based on their drug use pattern and the assigned cause of death. These groups 

are as follows: 1) methadone-related death in a methadone-prescribed!tolerant individual 2) methadone-related 

death in a methadone-not prescribed or -recently prescribed! non-tolerant individual 3) methadone unrelated to 

death or indirectly contributed to death (i.e. controls). The following table presents data from representative 

observations of this study: 

Group 

Central Peripheral Central! Liver Liverl Presence of Presence of 

Blood Blood Peripheral (mglkg) Central Broncho 

(mglL) (mglL) 

aspiration 

Ratio Ratio pneumonia 

mean 

(n= 46) median 0.68 0.36 1.5 2.6 4.7 10 (21%) 3 (6.5%) 

range 0.1-9.9 0.07-1.9 OAI-2.3 0.44-17 0.72-19 

2 mean OA9 0.34 1.8 2.5 7.5 

(n= 105) median 0.38 0.35 1.2 2.2 6.3 42 (40%) 14 (13%) 

range 0.05-2.5 0.047-0.82 OA7-7.3 0.36-9.3 1.4-25 

mean 

(n= 29) median 0.25 2.2 1.7 4 (13%) 2 (6.8%) 

range 0.03-0.72 1.2-3.8 0.19-27 

This investigation supports that postmortem redistribution of methadone can occur and may be significant in 

some cases. Liver concentrations when compared to central blood do not readily distinguish a methadone 

overdose from a methadone-unrelated death, especially if the dosing regime is unknown. Bronchopneumonia and 

perimortem aspiration, although they can support methadone ingestion, do not always occur (13-40% and 6-13%, 

respectively). These data suggest that while collection and analysis of blood from a peripheral source, in addition 

to review and consideration of pathological findings and drug use history, can improve the interpretation of 

toxicological data in methadone-related deaths, liver concentrations may have less interpretive value. 

Keywords: Methadone, Interpretation, Postmortem Fluids and Tissues 

Page 354

P20 

A COMPARISON OF METHADONE, HYDROCODONE, AND OXYCODONE ASSOCIATED 

MORTALITY IN CUYAHOGA COUNTY, OHIO: 1998 - 2003 

Daniel D. Baker IV*, and Amanda J. Jenkins, The Office ofthe Cuyahoga County Coroner 

11001 Cedar Ave., Cleveland, OH 44106, U.S.A. 

Significant increases in methadone related deaths have been recently documented across the United States. 

Historically, the substantial rise in oxycodone associated mortality is well documented as opposed to 

hydrocodone, where a paucity of literature remains. In response to these reports, the authors investigated 

cases over a six-year period in which postmortem toxicological analyses revealed the presence of 

methadone, hydrocodone, and oxycodone in any matrix type. The study was designed to detennine 

whether methadone associated mortality in Cuyahoga County reflected national trends and more 

specifically, to distinguish methadone trends from other commonly used opioid analgesics. 

All records of decedents that were found to be positive for methadone, hydrocodone, and oxycodone from 

1998-2003 were reviewed. Demographic information including age, sex, race, and location of residence 

was collected. The cause and manner of death was compiled and the cases were divided into drug 

overdoses and those cases where a positive result was detennined to be an incidental finding. Overdoses as 

a result of only methadone, hydrocodone, and oxycodone were separated from poly drug overdoses. The 

data was calculated for each type of drug based on quantitative heart blood drug concentrations for 

comparison. 

Review of21,460 deaths in Cuyahoga County, from 1998-2003, revealed that a total of 55 decedents were 

positive for methadone and of these, 29% were ruled overdoses. 202 cases were found to be positive for 

hydrocodone in which, 28% were due to a lethal dose. Oxycodone was discovered in 190 decedents of 

which, 29% were caused by an overdose. Mortality caused by methadone, hydrocodone, and oxycodone 

increased 400%,183%, and 366% respectively from 1998 to 2003. 

Demographic data revealed 286 males and 161 females were positive for the studied opioids. 351 opioid 

positive decedents were white, as opposed to 96 black. Decedent ages ranged from 2 to 101 years. 

Caucasian males between the ages of 34 and 51 (mean 42.5 ±2SD) were the demographic group 

predominantly positive for each of the three opioids. Decedents in which methadone was detected were 

shown to live within Cleveland city limits vs. the suburbs 3:1, as opposed to hydrocodone and oxycodone 

positive decedents who principally resided in the suburbs. 

Heart blood methadone overdose concentrations ranged from 0.11-1.31 mg/L (mean=0.67 mgIL, n=19) 

Similarly, heart blood methadone incidental finding concentrations ranged from 0.08-4.26 mgIL 

(mean=0.76 mgIL, n=35). Lethal. hydrocodone heart blood concentrations ranged from 0.01-1.66 mgIL 

(mean=0.29 mg/L, n=54). Incidental hydrocodone findings ranged from 0.01-2.56 mgIL (mean=0.11 mgIL, 

n=112). Oxycodone overdose concentrations ranged from 0.01-36.54 mg/L (mean=1.80 mg/L, n=48). 

Oxycodone positive concentrations that were detennined to be incidental findings ranged from 

0.01-1.78 mg/L (mean=0.31 mg/L, n=81). 

During the study period, an increase was observed in the number of positive cases for all three opioid 

analgesics. In conflict with recent national data however, although the number of methadone positive cases 

increased from 4 cases in 1998 to 18 cases in 2003, this did not result in a substantial increase in methadone 

overdoses, 1 death in 1998 to 4 deaths in 2003. No methadone level of lethal toxicity was discemable by 

comparing decedents whose death was caused by methadone intoxication as opposed to, an incidental 

finding. In contrast, the mean lethal hydrocodone and oxycodone blood concentrations were definable, at 

two and five times greater than non-overdose mean blood concentrations, respectively. Methadone, 

hydrocodone, and oxycodone overdoses equally comprised 28-29% of cases in which these drugs were 

detected. 

Keywords: Methadone, Oxycodone, Hydrocodone 

Page 355

P21 

"STUDENT ON ALPHA-METHYLTRYPTAMINE DISCOVERS mE SECRET OF THE 

UNIVERSE.•• AND DIES" 

Diane M. Boland*, Wilmo Andollo, George W. Hime, and William L. Hearn, Dade County Medical 

Examiner, Number One on Bob Hope Rd, Miami, FL, 33136 

Alpha-methyltryptamine (AMT) is a synthetic indole analog of amphetamine initially investigated as a 

monoamine oxidase inhibitor. In the 1960s, the Soviet Union marketed AMT as an antidepressant under 

the name oflndopan. During the same period, the Upjohn Company studied the alpha-ethylated analog for 

use as a commercial antidepressant known as Monase, but found it to be of little medicinal value. 

Although clinical use of AMT is obsolete today, recreational use has gained popularity due to its intense 

hallucinogenic properties lasting up to 16 hours. To illustrate recreational use of AMT in the 1960s, 

Alexander Shulgin in his book Tihkal, references the author Ken Kesey and his experiences with AMT and 

other hallucinogenic drugs. 

Today, AMT is recognized as a powerful psychedelic drug among high school and college-aged men and 

women who may have experienced the effects of other hallucinogenic amphetamines. Its popularity is 

partly due to the legality and availability of AMT for purchase via the Internet prior to April 2003. 

Emergency designation of AMT as a Schedule 1 controlled substance by the Drug Enforcement 

Administration occurred shortly after the Miami-Dade Medical Examiner reported the death of a 22 yearold 

college student who ingested a large amount of AMT. Prior to death, the deceased advised his 

roommate that he was ''taking hallucinating drugs" and as a result had "discovered the secret of the 

universe". The roommate reported that the deceased was shaking and sweating profusely. Approximately 

12 hours later, the roommate discovered the deceased lying in bed unresponsive. An empty Igram vial of 

AMT was recovered from the scene and sent to the Toxicology Laboratory. An autopsy by the Medical 

Examiner did not reveal any significant gross findings of any organs, and no evidence oftraumatic injury. 

Initial screening of urine by enzyme-multiplied immunoassay technique was positive for amphetamines, 

and the basic drug blood screen detected a small peak later identified by mass spectrometry as AMT. For 

quantitation, AMT was isolated using solid phase extraction, derivatized with pentafluoropropionic 

anhydride, and analyzed using gas chromatography/mass spectrometry. Ions monitored for AMT included 

mlz 276, 303, and 466. Quantitative analysis was based upon mJz 276 for AMT and mJz 306 for the 

internal standard 5-methoxy-alpha-methyltryptamine. A linear calibration curve from 50 to 500ng/mL was 

used to calculate· the concentration of AMT in the samples and controls. Blood, tissue, and gastric 

specimens were diluted to bring the observed concentration within the limits of the standard curve. Matrix 

matched controls were extracted and analyzed with each run. Postmortem blood revealed 2.0mg/L, gastric 

contents 9.6mg total, liver 24.7mgikg, and brain 7.8mgikg. 

Keywords: Alpha-methyltryptamine, 5-Methoxy-alpha-methyltryptamine, hallucinogens 

Page 356

!~. 

P22 

TILL DEATH DO US PART: FENTANYL POISONING IN A HUSBAND AND WIFE 

George S. Behonick, Ph.D:, Tara Valouch, B.S., Virginia Division of Forensic Science Western 

Laboratory and Susan E. Venuti, M.D., Assistant Chief Medical Examiner, Western District Office of The 

Chief Medical Examiner and Assistant Clinical Professor, Department of Legal Medicine at Virginia 

Commonwealth University, 6600 Northside High School Road, Roanoke, V A 24019 

One treatment modality for chronic pain is the application of fentanyl transdermal patches (Duragesic®). 

However, the transdermal therapeutic system, designed for the rate-controlled delivery of drug, is 

increasingly favored by members of the opioid-misusing/abusing population. Deliberate misuse or abuse 

may include cutaneous application of mUltiple patches, transmucosal absorption (buccal cavity), inhalation 

of volatilized fentanyl and intravenous injection of contents extracted from trans dermal patches. The selfdelivery 

of drug also occurs by simultaneously combining different routes of administration. Herein we 

describe a case report of two fatal fentanyl poisonings which occurred in a married couple within a five 

hour time interval. We discuss the available medical histories of the two decedents (including prescribed 

medications) together with first responder witness reports and significant findings from both autopsies and 

the postmortem toxicological analyses in each case. Physical evidence included a 75 jlg/h fentanyl 

transdermal patch retrieved from the oral cavity or airway of the female decedent during resuscitative 

efforts at the scene and a "disrupted" (e.g., cut) 75 jlg/h fentanyl transdermal patch recovered in the 

personal effects of the male decedent at autopsy. The postmortem blood fentanyl concentrations measured 

in the 45-year-old white female decedent and the 52-year-old white male decedent were 0.050 mg/L and 

0.030 mg/L, respectively. Fentanyl was extracted from blood using solid phase extraction and quantitated 

by 81M GCMS using a previously published procedure. 

Key Words: fentanyl, poisoning, blood 

Page 357

P23 

CASE REPORT: THE USE OF AMITRIPTYLINE IN DRUG FACILITATED SEXUAL ASSAULT 

Kristina Fritz* 

Los Angeles County Department of Coroner, 1104 N. Mission Road, Los Angeles, CA 90033 

Amitriptyline is a tricyclic antidepressant with sedative effects that has been available in the United States 

since the early 1960's. It is administered in 10-150 mg tablets or as a 10 mgIL injectable solution. While 

amitriptyline is commonly encountered by the Los Angeles County Department of Coroner Toxicology 

Laboratory, the circumstances of its use in four Southern California homicides is unique. 

Four murders occurred between April 2000 and April 2001. The victims, two females and two males, were 

initially invited for consensual sex. They were then drugged, sexually abused, bound with ligatures, and 

ultimately killed. This report reviews four cases of drug-facilitated sexual assault using amitriptyline. The 

presence of amitriptyline in high concentrations coupled with unique criminalistics evidence aided in the 

linking of this series of homicides across county jurisdictions. 

The analysis of amitriptyline from postmortem. specimens consisted of a basic, liquid/liquid (nbutylchloride) 

extraction procedure with screening and quantitation by GC/NPD. Linearity was achieved 

from 0.10 to 5.0 mg/L with the limit of quantitation at 0.10 mgIL. Confirmation of amitriptyline was 

determined using a GCIMS by comparison with an analytical standard. 

The following amitriptyline concentrations are reported. 

~..,__,__,.._. ,~.._..,_..__~!!tr_~~,~~~..._..._,__ -,_.._......._._... 

Central Hood Peri(Deral Hood liver Gastric Brain 

Case # 1 - - 86 19rrg 62 

Case #2 - - 76 - 83 

Case #3 O.IX> 0.11 1

P24 

"THE DOSE MAKES THE POISON", DISCUSSION OF THREE CASES OF LETHAL HEROIN 

INTOXICA TIONS 

W Bemhard*, B Aebi, M Gasser, St. Bolliger, Institute of Legal Medicine, Bern, Switzerland, 

W. Vycudilik, Institute ofLegal Medicine, Vienna, Austria 

Introduction: The lethal dose of heroin differs widely. Tolerance plays a key role. To the persons in the 

official Swiss heroin program, the drug is often prescribed in high dose. In spite of the medical 

surveillance, lethal poisoning is not an uncommon finding. 

Results: Case A) A 35 y.o. male administered himself (intravenously) under medical observation in 30 min. 

400 mg chemically pure heroin and ingested 500 mg Morphine-retard. He then went back home. Two days 

later he was found death in his flat. Toxicological findings: heart blood: free morphine 1605 nglmL, M-3-G 

686 ng/mL, M-6-G 390 ng/mL. Interpretation: the deceased did not swallow the Morphine-retard capsules. 

He smuggled them out of the injection place, the so-called "Fixerstilbli" and injected them instead. Cause 

of death: heroin and morphine intoxication. Manner of death: suicide or accident. Case B) A 39 y.o. male 

came by himself to a hospital with stomach pain. During the medical treatment he received O.2mg of 

fentanyl and 5mg diazepam by the medical staff. During his stay he snorted two times a powder which was 

believed by the nurses to be cocaine. Four hours after the start of the medical treatment he died. 

Toxicological findings: serum (peripheral) sample taken at the hospitalization: free morphine 267 nglmL, 

Codeine 32 nglmL. Serum from heart blood collected at the autopsy: free morphine 726 nglmL, codeine 

120 ng/mL. Findings: The patient snorted, instead of cocaine, "street heroin". Cause of death: heroin 

poisoning. Manner of death: suicide or accident. Case C) A 28 y.o. female was found dead in her flat. An 

injection mark was found in her right antecubital fossa. Toxicological findings: heart blood: free morphine 

140 nglmL, total morphine 693 ng/mL. Results of police investigation: a stranger injected approx. 400 mg 

of street heroin (purity approx 15%) intravenously on this female on her request. Cause of death: heroin 

poisoning. Manner of death: killing on request. 

Conclusions: High doses of heroin and morphine are needed to satisfy the patients in the official heroin 

program, and are typically tolerated by them. However, in spite of medical surveillance lethal overdoses are 

observed. Quite low heroin doses may be lethal for opiate-naive subjects. 

Keywords: heroin, morphine, postmortem 

Page 359

P25 

ECSTACY MANUFACTURE: A CASE FOR QUALITY CONTROL 

Jim Gerostamoulos*, Matthew Lynch, and Olaf H. Drummer. 

Victorian Institute of Forensic Medicine, Department of Forensic Medicine, Monash University, 57-83 

Kavanagh St., South bank, Victoria 3006, AUSTRALIA 

This case report involves the death ofa 25 year old male who was found deceased in his boarding room some 

2!t2 days after last being seen alive. The decedent's body was located face down on his bed. There were no 

signs of drug use and no medication/illicit drugs were discovered at the scene by attending police. 

A full autopsy was performed on the deceased to determine the cause of death. Macroscopic findings 

indicated putrefactive changes with extensive autolysis of internal organs in keeping with early stages of 

decomposition. There was marked pUlmonary oedema and bilateral hydronephrosis. There were no other 

significant pathological findings. Specimens submitted for toxicological analysis revealed a free morphine 

concentration (cavity blood)-60 mgIL; 4.2 mg/kg (liver); total morphine 76 mgIL (urine); and 190 mg of 

morphine in stomach contents. No 6-acetyl morphine was detected in blood or urine, and no other drugs or 

alcohol were detected. The cause of death was given as morphine toxicity. 

Additional information provided by the family indicated a family history of sleep apnoea. Tablets, located in 

the decedent's bedroom and which according to the girlfriend belonged to the deceased, were submitted to 

the laboratory for analysis some weeks after the death. The 3 tablets (green, scored with the markings CK on 

either side) and initially purchased presumably as ecstacy, were identified as containing 100 mg of morphine 

with trace amounts of methamphetamine. The decedent had consumed at least two of these tablets or more 

resulting in his death. This case illustrates the danger of experimenting with illicit drugs and serves as a 

reminder that information germane to medicolegal death investigation can come from a variety of sources. 

The Coroner found that the death was an accidental overdose. 

Keywords: ecstacy, morphine, death. 

Page 360

P26 

A RETROSPECTIVE STUDY OFOXYCODONE DEATHS IN ONTARIO 1999-2003 

lCentre of Forensic Sciences, 25 Grosvenor St, Toronto, Ontario, M7A 208. 2Northem Regional Forensic 

Laboratory, Centre of Forensic Sciences, 70 Foster Dr, Sault Ste. Marie, Ontario, P6A 6V3. 30ffice of the 

Chief Coroner, 26 Grenville St, Toronto, Ontario, M7A 2G9. 

This report presents the results of a retrospective study aimed at determining the role of oxycodone in 

deaths investigated by the Office of the Chief Coroner in Ontario, Canada, between 1999 and 2003. The 

objectives of the study were 1) to assess changes over this period in the prevalence of oxycodone in deaths 

investigated by the coroners, and 2) to assess the extent to which these deaths were oxycodone-induced or 

oxycodone-related. The cases included in this study were death investigations in which specimens obtained 

from a medico-legal autopsy, were submitted to the Centre of Forensic Sciences for a toxicological 

examination. In all but three cases, examinations consisted of analysis for alcohols, a general drug 

screening procedure that detects a range of therapeutic agents and illicit substances that exhibit central 

nervous system toxicity and are encountered in postmortem toxicology, and targeted analyses for the 

quantitation and confirmation of screen findings. Cases were included in this study only when a blood 

oxycodone finding was available. In the majority of cases, analysis was performed on heart blood, and in 

others, on samples identified as femoral or peripheral venous, or from an unspecified source. Quantitation 

of total oxycodone in whole blood was carried out using a liquidlliquid basic extraction, followed by a gas 

chromatographic analysis using a nitrogen/phosphorous detector and capillary fused silica columns. 

A total of 291 cases met the inclusion criteria for this study, representing 194 males and 97 females. 

Oxycodone-positive cases submitted to this laboratory in 1999 numbered 22 and increased steadily each 

year to reach 101 in 2003. The increase in annual submissions was markedly disproportionate to the 

overall increase in submissions. Although no attempt was made in this phase of the study to determine 

whether the detected oxycodone was due to the ingestion of Oxycontin® or any other oxycodonecontaining 

formulation, it is noteworthy that the increase in the prevalence of oxycodone findings in death 

investigations coincides with the introduction of Oxycontin® to the Canadian market in about 1996. In 

order to assess the involvement of oxycodone in these deaths, the coroner's conclusion regarding the cause 

of death was recorded. In 39 cases over this five-year period, death was attributed to natural causes or 

trauma, but not related to the presence of oxycodone. Blood oxycodone concentrations ranged from less 

than 0.01 to 0.9 mg/L. 

In the remaining cases (n == 252), death was determined by the coroner to be drug-induced or drug-related. 

Based on the reported postmortem blood toxicology findings, cases were divided into four categories: 1) 

oxycodone alone (2%); 2) oxycodone and other drugs in therapeutic concentrations, with ethanol present at 

or below 50 mg/IOO mL (20%); 3) oxycodone and other drugs in potentially toxic concentrations, with 

ethanol in excess of 50 mg/l00 mL (59%); and 4) oxycodone and other drugs in potentially fatal 

concentrations (19%). The blood oxycodone concentration ranges for each group were 0.1 to 0.92 mg/L, 

0.1 to 5.5 mg/L, less than 0.01 to 9.8 mglL, and 0.03 to 4.4 mg/L, respectively. 

The observed overlap in blood oxycodone concentrations across these groups underscores the importance 

of other factors such as polydrug use, drug formulation, and the experience of the user in assessing the 

contribution ofoxycodone to the death. 

Keywords: oxycodone, postmortem, toxicology 

Page 361

P27 

ANALYSIS OF POSTMORTEM BONEIBONE MARROW SPECIMENS FOR DRUGS OF 

IMPORTANCE IN FORENSIC TOXICOLOGY 

Kelly K. McGrath* and Amanda 1. Jenkins, Ph.D, The Office of the Cuyahoga County Coroner, 11001 

Cedar Avenue, Cleveland, OH 44106, U.S.A. 

To date, there is a paucity of literature published on the determination of drugs in bone and bone marrow. 

This type of research is important forensically because the extraction of bone specimens may be useful 

when traditional postmortem samples are not available for toxicological analysis. Postmortem bone 

samples, taken from the iliac crest in adults and vertebrae in infants, were extracted and analyzed for the 

presence of benzodiazepines and cocaine and metabolites. The purpose of this study was to correlate the 

concentration of drugs found in postmortem blood with the concentrations in boneibone marrow samples. 

The specimens were thoroughly cleaned by removing all traces of skeletal muscle and tissue and then 

rinsing with deionized water until the wash ran clear. Two grams of each bonelbone marrow sample was 

cut into slivers and soaked in 4 milliliters of methanol for sixteen hours (± 0.5 hours). Cocaine and 

metabolites were extracted from the methanol by liquid/liquid extraction followed by solid phase extraction 

(SPE), derivatization with MSTFA,· and analysis by GCIMS in single ion monitoring (SIM) mode. 

Benzodiazepines (BDP) were isolated from the methanol extract by liquid/liquid extraction and analyzed 

by GC/ECD. 

The concentration of drugs found in the bonelbone marrow samples was compared to postmortem blood 

toxicology results. Three of seven cases analyzed for cocaine and metabolites were positive. In one case, 

cocaine was quantitated at 120 nglg bone, cocaethylene 80 nglg bone, and ecgonine methyl ester (EME) 

was qualitatively positive. The heart blood results in this case were benzoylecgonine (BE) 332 ng/ml, 

cocaine 144nglml, cocaethylene 32 nglml, and EME qualitatively positive. In another case, BE was 

quantitated at 666 nglg bone and in the heart blood BE was 522 nglml. The third case had 48 nglg bone 

cocaethylene and EME was qualitatively positive. The heart blood in this case contained 211 ng/ml BE, 29 

nglml cocaine, and EME was qualitatively positive. The remaining four cases were negative for cocaine 

and metabolites in the boneibone marrow and the blood concentrations for BE and cocaine ranged from 

123 nglrnl-394 nglml and 24 ng/ml-29 nglml respectively. These positive cases demonstrate that cocaine 

and metabolites can be detected in bone, but the presence of specific analytes may differ from those present 

in the blood and the concentration of the analytes in boneibone marrow may be higher or lower than that in 

the blood. Therefore, these results should be interpreted cautiously. 

Six cases were examined for the presence ofBDP. Ofthese six cases, four were positive for diazepam with 

values ranging from 0.60 ug/g bone-2.4 ug/g bone, four were positive for desmethyldiazepam (DMD) with 

. values ranging from 0.32 uglg bone-l.6 uglg bone, and one case was unsuitable for analysis. In the cases 

where diazepam was positive in the bonelbone marrow, the heart blood concentrations ranged from 0.13 

mg/L-0.39 mg/L. DMD in the heart blood ranged from 0.10 mg/L-0.67 mg/L. In every case where 

diazepam and/orDMD were present in the heart blood, both were also recovered in the boneibone marrow. 

These results demonstrate that· bonelbone marrow specimens may be utilized as an alternative matrix for 

postmortem toxicological analysis where traditional matrices are not available. The preliminary data on 

cocaine and metabolites and BDP failed to show a linear correlation between boneibone marrow and blood 

drug concentrations. It appears that the likelihood of detecting a drug in boneibone marrow specimens 

depends on the type of drug being analyzed. Future investigation of other drug classes such as opiates, 

basic, and acidic/neutral drugs may provide additional information for the elucidation of correlation data 

between bonelbone marrow and blood specimens. 

KEYWORDS: Bone, Cocaine, Benzodiazepine 

Page 362

P28 

DRUG RELATED DEATHS IN THE CITY AND COUNTY SAN FRANCISCO 

NP Lemos*, PhD, SB Karch, MD, E Lin, MS, G Nazareno, MD, V Azar, MD, A Hart, MD and BG 

Stephens, MD 

Aims: The timely detection of drug-related health problems and emerging drug trends depend, at least in 

part, on information provided by coroners and medical examiners. To assist in this process we reviewed all 

drug-related deaths occurring in our jurisdiction during 2002. 

Design: Retrospective analysis of all death investigations carried out by the San Francisco Office of the 

Chief Medical Examiner (SFOCME), from January 1, 2002 until December 31, 2002, where postmortem 

toxicology revealed the presence of psychoactive or abused drugs, and complete autopsy performed. 

Methods: If available, urine is first screened with EMIT testing and results confirmed by GCIMS or 

LCIMS. In the absence of urine, GCIMS is used to screen blood, liver, CSF or other biological specimens 

directly. 

Findings: The SFOCME serves a population of approximately 750,000, and this number has been stable for 

several decades. In 2002, 1463 cases came under the jurisdiction of our office, and drugs were detected in 

257 of these cases. Cocaine or benzoylecgonine was found in the blood and/or urine of 153 (59%) cases. 

Morphine and/or codeine were detected in the blood or urine of 119 (46%), and methamphetamine was 

present in 19% (48/257). Poly-drug abuse was common among the cases which came under our 

jurisdiction. Morphine was present in nearly a third (72/257, 28%), of the cocaine users. The occurrence 

rate for other drugs was much lower; methadone was present in 33 cases, oxycodone in 23, fentanyl in 7, 

hydrocodone in 6, MDMA in 5, and PCP in 2 cases. 

Conclusions: The number of methamphetamine-related deaths continues to be remarkably stable within our 

jurisdiction and is, in fact, lower than in 1994, when 52 deaths were recorded (J Forensic Sci. 1999 Mar; 

44(2):359-68). The rate for methadone related deaths also appears to be stable, while the number of 

oxycodone-related deaths appears to be increasing, suggesting that allocating additional resources to treat 

methadone diversion may be unnecessary in our jurisdiction. On the other hand, the increasing number in 

fentanyl-related deaths suggests that diversion of Duragesic® patches in our jurisdiction is an increasing 

problem worthy of further investigation. 

Keywords: Drug, Epidemiology, San Francisco 

Page 363

P29 

THE ROLE OF COCAINE IN HEROIN RELATED DEATHS. HYPOTHESIS ON THE HEROIN 

COCAINE INTERACTION 

A. Polettini*, V. Poloni, A. Groppi, C. Stramesi, C. Vignali, L. Politi, M. Montagna 

Department of Legal Medicine & Public Health, University of PaVia, Via Forlanini 12, 27100 PAVIA, 

Italy 

In recent years, drugs-of-abuse related deaths involving cocaine observed at the Department of Legal 

Medicine & Public Health of Pavia have shown an increase, probably reflecting the rising trend in cocaine 

use in Western Europe: "pure" cocaine deaths have increased from 6 cases in 1979-1991 (1.5% of drug-ofabuse 

deaths) to 13 in 1992-2002 (3,2%), and in the same periods heroin related deaths (HRDs) involving 

cocaine amounted to 8 (1.9%) and 22 (5.4%), respectively. In the attempt to investigate the role of cocaine 

in HRDs, acute narcotism cases testing positive for cocaine and/or metabolites in blood (>10 ngtml, 

COC+) were examined. Only cases occurred from 1997 to 2001 were considered as in this period all data 

were obtained using the same analytical procedures (free morphine and total morphine by DPC Coat-A 

Count radioimmunoassay before and after enzymatic hydrolysis, cocaine and metabolites in blood by SPE, 

TMS derivatization and GC-MS). The median, minimum and maximum concentration of free morphine 

(FM) and total morphine in blood (TM), urine (UM) and bile (BM) in the COC+ group (n=9) were 

compared with those calculated in the group of "pure" HRDs (no other drugs detected in blood, COCo, 

n=30). Differences among the two groups were statistically evaluated using the two-tailed Mann-Whitney 

U Test. Statistical analysis was also carried out including in both groups cases testing positive (>2 mgtdL) 

for blood alcohol (COC+, n=19; COCo, n=76). The median TM was found to be lower in the COC+ group 

(0.32 mg/l vs. 0.90 mg/l, P=0.0214) and also the median FM (0.08 mgtl vs. 0.28 mg/l, P=O.l064). The 

FMITM ratio was similar in the two groups (0.33 and 0.35) and also UM (21.0 mg/J and 18.0 mg/J), 

whereas BM was higher (90.0 mgtl vs. 49.0 mgtl, P=0.0268). Similar results were obtained by repeating 

statistical analyses after including in the two groups cases with positive blood alcohol concentration 

(BAC). This picture is very different from what was previously observed for the heroin-ethanol interaction 

in HRD cases CA. Polettini et al., J. Anal. Toxico!., 23, 570, 1999), and updated with more recent data: in 

the high-ethanol (HE, BAC >100 mg/dl) group TM was lower than in the low-ethanol (LE, BAC:5100 

mgtdl) group (0.59 mg/l vs. 0.90 mgtl, P=0.0180), the FMlTM ratio was higher (0.66 vs. 0.43, P=0.0038), 

FM was equal, UM was lower (0.21 mgtl vs. 26.5 mg/J, P=O.OOOl), and so BM (10.0 mgtl vs. 26.5 mg/l, 

P"-'0.0156). Indeed, the observed lower TM in the COC+ and in the HE groups support the hypothesis ofan 

interaction of both cocaine and ethanol with heroin in the occurrence of death. Ethanol results suggest that 

a pharmacokinetic interaction is prevalent (inhibition of heroin metabolism, as suggested by the increased 

FM/TM ratio, resulting in reduced urinary and biliary excretion). In the case of cocaine, a 

pharmacodynamic interaction seems to prevail, as the FMITM ratio remains unchanged and UM and BM 

are not lower in the COC+ group. This hypothesis could not be confirmed owing to the paucity of data and 

the many uncontrolled variables involved. 

Keywords: heroin related death, cocaine, interaction 

Page 364

P30 

DRUG EXPOSURE PATTERN IN HAIR AND FEMORAL BLOOD IN DECEASED DRUG 

ADDICTS 

Henrik Druid!', Joakim Strandberg!, Ingrid Nystrom 2 , Kanar Alkassl, Fredrik Kugelberg 3 and Robert 

Kronstrand 2 

IDepartment of Forensic Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden; 2Department of 

Forensic Chemistry, National Board of Forensic Medicine, SE-581 85 Linkoping, Sweden; 3Departrnent of 

Clinical Pharmacology, University ofLinkoping, SE-581 85 Linkoping, Sweden 

Blood drug levels of illicit drugs are often not particularly high in postmortem overdose cases. This is true 

for amphetamine and cocaine as well as for opiates. Thus, the postmortem investigations do not support the 

use of the word "overdose", since the dose taken in these cases is probably not much larger than is usually 

taken by chronic addicts. One explanation for this outcome may be reduced drug tolerance following a 

period of abstinence, but - although some epidemiological reports suggest a recent interruption in drug use 

in some cases -there are no firm data to confirm this hypothesis. It is difficult to estimate the degree of drug 

tolerance in deceased subjects, but hair analysis of illicit drugs may at least provide a map of the past and 

recent drug use pattern. In the present study, segmental hair analysis was applied on hair samples collected 

from 40 deceased, supposedly drug addicts, including both cases of probable drug overdoses, and cases 

with other causes of death. Portions of the hair samples were analyzed with an LC-MS-MS screening 

method, which covers amphetamine and -derivatives, cocaine and metabolites, morphine, 6 

monoacetylmorphine (6-MAM), codeine, diazepam, flunitrazepam, nicotine and cotinine. Positive findings, 

except nicotine and cotinine, were confirmed with GC-MS verification methods. For these analyses, the 

hair was carefully aligned and cut in short segments (the first three segments 5 mm each, and additional 

segments 10 mm long). The verification methods also included washing steps, and analysis of washings 

was carried out to control for external contamination. Femoral blood, urine and vitreous humor were also 

collected at the postmortem examinations for the routine toxicological drug screen. Twelve of the cases 

were classified as heroin overdoses by the responsible forensic pathologist. In Table 1 the number of 

detections of seven different drugs/metabolites in hair and blood (results for benzodiazepines not shown) in 

these 12 cases are displayed. In most cases, a marked change in abuse pattern was evident. Hence, a 

gradual decrease in morphine and/or 6-MAM levels was observed in 6 of 12 cases. It was also found that 

the drug use at time of death, as reflected by the drugs detected in femoral blood, more often than not 

differed from the drug use pattern seen in hair. Amphetamine, cocaine, benzoylecgonineand 6-MAM were 

more often detected in hair than in blood. The opposite was true for codeine and morphine. In four cases, 

fentanyl was found in femoral blood in concentrations that suggest that this drug most likely was the main 

or a contributory factor for the demise. The results from the segmental hair analyses showed that the final 

heroin dose in four cases obviously had been preceded by a period of lowered drug use, or abstinence, 

whereas in other cases, a continuous drug use before death was disclosed. Thus, reduced tolerance may be 

of importance in certain cases, but in the majority of the cases, other factors seem to be more important. 

Interestingly, in at least four of these twelve "overdose" cases, there was evidence of a delayed death, 

leading to a drop in blood morphine levels. This observation underscores the importance of considering 

autopsy findings and circumstantial information when interpreting postmortem toxicological data - the 

finding of low morphine levels, and/or lack of 6-MAM does not exclude an acute opiate intoxication. It is 

concluded from the present study that hair analysis can assist in the toxicological evaluation of postmortem 

cases, and that segmental analysis can improve the interpretation further. 

Table 1. Number of detections of different drugs of abuse in hair and blood from 12 deceased heroin 

addicts classified as overdoses. 

L Codeine Morphine 6-MAM Amphetamine Cocaine Benzoylecgonine Fentanyl 

I Hair 5 5 9 5 2 2 n.a. 

I Blood 7 12 3 3 0 1 4 

Key words: Hair analysis, opiates, substance abuse 

Page 365

P31 

TRENDS IN THE DETECTION OF DRUGS OF ABUSE IN POSTMORTEM SAMPLES 

Bernard Croal"', Stephen Duncan, Daniel Docherty and Patricia Rooke, Department of Clinical 

Biochemistry, Aberdeen Royal Infirmary, Aberdeen, Aberdeenshire, AB25 2ZD, UK 

Introduction: Drug misuse is a major social and economic problem facing the Grampian and Highland 

regions of Northern Scotland. The Oil and Fishing industries have brought wealth and reduced 

unemployment in these regions. Consequently, drug dealers have targeted these areas as a potential market 

for the selling of drugs. As a result, there has been a dramatic increase over the last decade in the number 

of samples screened for drugs-of-abuse in Grampian. Unfortunately, the rise in drug misuse has also seen 

an increase in the number of drug related deaths in Grampian and Highland over this time. Fatalities from 

suspicious causes are frequently associated with illegal drug misuse. We have assessed the trends of the 

presence of commonly abused illicit drugs in post-mortem specimens from individuals who died under 

suspicious and accidental circumstances in Grampian and Highland from 1994 to 2000. 

Methods: Post-mortem data was obtained from an archival laboratory database of forensic cases for 

accidental or suspicious death over the period 1994 to 2000. Drug trends were assessed in cases in which 

at least one drug was detected. The drugs chosen for this investigation included Amphetamine, 3,4 

Methylenedioxyamphetamine (MDA), 3,4-Methylenedioxymethamphetamine (MDMA; Ecstasy), 

Cannabinoids, Benzoylecgonine, Cocaine, Codeine, Diazepam, Dihydrocodeine, Methadone, Morphine and 

6-Monoacetylmorphine, since they represented the most commonly abused drugs found in Grampian and 

Highland. Drug detection was assessed from post-mortem blood and urine specimens collected into plain 

tubes and extracted by liquid-liquid or solid-phase extraction using either GC-MS or HPLC for detection. 

Results: A total of 2993 forensic cases were referred for analysis over the six year period. There was no 

evidence that the number ofcases was increasing (mean =458 cases per year, regression coefficient:: 9.57, 

p 0.51). From these cases, 641 positive drug screens were obtained from a total of 330 individuals. A 

rising trend (regression coefficient; significance level) from 1994 to 1999 was observed for the presence of 

Cannabinoids (6.29; 0.002), Codeine (5.77; 0.006), Diazepam (6.89; 0.002), Dihydrocodeine (3.26; 0.002) 

and Morphine (7.43; 0.004). Overall, a rising trend was observed for the presence of at least one drug 

(regression coefficient 13.89; p :: 0.001), with the finding of more than one drug being present also 

increasing. 

Conclusions: From 1994 to 2000 the number of positive drug tests in post-mortem specimens from this 

source are increasing on average by 37 cases per year. Ofthese, the number of patients with more than one 

positive drug is also increasing. This would fit in with the view that drug abuse is increasing within society, 

as is multiple drug use. Overall this study provides evidence that drugs of abuse are increasingly implicated 

or associated with death from suspicious causes. This evidence ofthe increasing involvement of drug abuse 

in fatalities arising from suspicious and accidental causes will strengthen the demand for increasing 

specialised toxicological analysis by the Procurator Fiscal, which will have profound economic and 

resource implications upon laboratories both in Grampian and nationally. 

Page 366

.. ~.. 

P32 

METHOD FOR CARBON MONOXIDE DETERMINATION IN BLOOD USING GAS 

CHROMATOGRAPHY WITH METHANE AS AN INTERNAL STANDARD 

H.Samkova*, J.Pivnicka, M.Spackova 

Institute of Forensic Medicine of St. Anne's University Hospital Brno, Czech Republic. 

A gas chromatographic procedure for determination of carboxy haemoglobin in blood with a thermal 

conductivity detector is commonly used. Processing of blood samples for the measurement of liberated 

carbon monoxide and obtaining reproducible results, especially in cases of postmortem blood samples, is 

performed using a method with methane as an internal standard. An apparatus for postmortem blood 

sample preparation for CO liberation and subsequent GC determination is presented together with a 

description ofthe method that includes schematic drawings. A core part ofthe device is a simple reaction 

tube partly filled with saturated NaCI solution. Blood, diluted 1: 1 with distilled water, is added into the 

tube. Then the tube is closed and deaerated through a rubber septum. Through the same septum, a 

ferricyanide reagent and a gas mixture of H2 and CH 4 (10:1) are added. The addition of reagents and gas 

phase is performed in a system of communicating vessels. Methane serves as an internal standard. The 

reaction mixture is shaken and an aliquot part of the gas phase is injected into the gas chromatograph 

(chromatographic column packed with molecular sieve 5A, isothermal conditions 80'C , TCD, carrier gas: 

hydrogen). A blood sample saturated with carbon monoxide up to 100% is processed in the same way. 

Total concentration of bonded CO in the sample is calculated from peak areas of analysed sample and 100 

% saturated one. 

Aco 

ACH4 

%CO = ( **--*--.- )samPle X ( ---.---.- )100% X 100 % 

ACH4 

Aco 

The calibration curve was obtained on the basis of measurements of intermixture fully saturated blood 

sample and negative one in required ratios. 

The method was validated with following parameters: 

COHb calibration in the range of 0 - 100% 

Y= 0,0031x + 0,0045 

R2=0,9973 

LOD 0,44 % COHb 

LOQ = 1,47 % COHb 

The method is applicable even in cases of decomposed blood. Replicate injection of the homogenous gas 

phase into the gas chromatograph may be performed. The precision of the determination expressed as 

coefficient ofvariation deteriorates in cases oflower concentration ofCOHb. 

Keywords: blood sample preparation, carbon monoxide, internal standard 

Page 367

P33 

POSTMORTEM CASES RELATED TO COCAINE IN THE NETHERLANDS 

Ll. Bosman*, M. Verschraagen, KJ. Lusthof 

Netherlands Forensic Institute 

Cocaine is a popular drug and is often encountered in postmortem toxicology cases. In this study, we 

present cocaine-'related deaths in the Netherlands from October 2002 to October 2003. In this period, the 

presence of cocaine, benzoylecgonine and methylecgonine was demonstrated in 45 cases in which cocaine· . 

concentrations were 0.02 mg/I or higher. Of these 45 cases, 34 were male. The 45 deaths were divided into 

the following categories: 

I. anatomical cause of death; cocaine present with or without ethanol and other drugs (ACD, n=17) 

2. toxicological cause of death, cocaine as most likely cause (TCD, n=12) 

3. toxicological cause of death, cocaine and other drugs present, other drug(s) as most likely cause or 

combination (TCD+, n=6) 

4. no clear cause of death, cocaine present with or without ethanol and other drugs (NCD, n=8) 

In 12 ofthese 45 cases, cocaine-overdose (TCD) was the most probable cause of death. In these 12 cases, 

mean concentrations (± standard deviation) of cocaine, benzoylecgonine and methylecgonine in femoral 

blood were 8.0 (±4.9) mg/I, 9.4 (±4.0) mg/I and 7.0 (±3.9) mg/I, respectively. In eleven of these cases, 

packages were found in the stomach. The other case was most probably a fatal case of cocaine delirium 

with a cocaine concentration in femoral blood of 0.23 mg/I, a benzoyJecgonine concentration of 7.0 mg/! 

and a methylecgonine concentration of 1.64 mg/1. 

The mean concentrations of cocaine and metabolites in femoral blood in the TCD+ category were much 

lower compared to the TCDcategory: the concentrations of cocaine, benzoylecgonine and methylecgonine 

. in these cases were 0.54 mg/I, 1.99 mg/l and 1.21 mg/I, respectively. In these cases of TCD+, combinations 

with ethanol, amphetamines, benzodiazepines, methadone, morphine and GHB were found. 

Compared to the TCD and TCD+ category, the mean concentrations of cocaine and metabolites in femoral 

blood in the categories ACD and NCD were much lower: the mean concentrations were 0.10 mg/I and 0.18 

mg/I for cocaine, 1.11 mg/I and 1.91 mg/I for benzoylecgonine and 0.25mg/l and 0.45 mg/I for 

methylecgonine , respectively. In the cases of anatomical cause of death (A CD), there were two cases in 

which cocaine was the only drug present. In the other 15 cases of ACD, combinations of cocaine with 

ethanol and other drugs (amphetamines, benzodiazepines, methadone and morphine) were found. Out of 8 

cases where no clear cause of death (NCD) was found, 7 cases had combinations of cocaine and ethanol or 

other drugs (benzodiazepines, methadone and morphine). There were no striking differences between the· 

categories TCD+; ACD and NCD in combinations of drugs found together with cocaine. 

In two of the total 45 cases, the cause of death could be anatomical as well as toxicological. One of these 

cases was a woman with packages in the stomach and one package in the trachea (mechanical obstruction): 

the concentrations of cocaine, benzoylecgonine and methylecgonine in femoral blood were 3 mg/I, 4.3 mg/I 

and4.7 mg/I respectively. The other case was a shooting; a cocaine concentration in femoral blood of 1.9 

mg/I was found. 

Keywords: postmortem blood concentrations, cause of death, cocaine 

Page 368

P34 

DETERMINA TION OF PERMETHRIN, ETHYLBENZENE, XYLENE AND THEIR METABOLITES IN 

AN ACUTE INGESTION OF INSECTICIDE 

Justin L. Poklis* I, Jeri D. Ropero-Miller l , Diana Garside l , William KeIly2 and Ruth E. Winecker l . IOffice 

of the Chief Medical Examiner, 100l Brinkhous-Bullitt Building, Chapel Hill, NC 27599 U.S.A. 2Coastal 

Pathology Associates, PO Box 317 Western Boulevard, Jacksonville, NC 28546 U.S.A. 

The case history and toxicological findings of an acute ingestion of the insecticide permethrin in a 7 year 

old female found in bed with vomit and blood on her face are presented. Permethrin and its carriers, 

ethylbenzene and xylene, are all components found in the commercial product, Atroban. The concentration 

of permethrin was measured in the liver, aortic blood and gastric content using gas chromatography-mass 

spectrometry (GCIMS). Concentrations of ethylbenzene and xylene were measured in the same biological 

samples by headspace-gas chromatography. Blood and liver concentrations of permethrin, ethyl benzene 

and xylene were detected at concentrations below the lowest curve calibrator. Therefore, permethrin and 

ethylbenzene were reported as < 1.0 mgIL(kg), and xylene as

P35 

CARBON MONOXIDE AND ETHANOL IN FIVE VICTIMS OF A FIRE FATALITY 

Ferrari,L.A.* ,Arado M.G. , Nardo,C.A., .Mirson,D Krbavcic,I & Garrote, LV. 

Laboratory of Toxicology & Legal Chemistry, Supreme Court of Justice Province, Buemos Aires, 

Argentina. Depart. Toxicology, University of Mor6n, Argentina 

Several reports have been published about carbon monoxide and ethanol in fire fatalities, but possible 

physiological interaction between these toxic agents is still under discussion. We report a tragic mattress 

fire that occurred in a small room at a prison where five young men under the influence of alcohol died. 

Blood specimens were obtained from heart of victims and tested for ethanol by headspace gas 

chromatography ( HS-GClFlO). Shimadzu GC-14, CR 4A with FlO detector, stainless steel column 

packed with 0.3% carbowax 1500 graphapac 60/80 and tert butanol internal standard were used. Percent 

of carboxihemoglobin (%COHb) was determined by microdifussion methods. Additionaly, micro 

quantitative determination of cyanide was determined according to the method proposed by Gelttler and 

Goldbaum. Comprehensive testing for drugs of abuse, psychotopic drugs and other therapeutic drugs was 

performed by HPTLC. The %COHb and ethanol (giL) in the five victims were: 60% and 0.54; 35 % and 

3.99; 55% and 0.39; 45% and 0.70, 50% and 0.81 respectively. No other components with toxic relevance 

were found. According to this study we concluded that there was no evidence for an ethanol protective 

effect against carbon monoxide poisoning. 

Keywords: carbon monoxide, ethanol, fire 

Page 370

P36 

METHAMPHETAMINE IN HAIR AND INTERPRETATION OF FORENSIC FINDINGS IN A 

FATAL CASE 

Katei'ina Benmkova, Vilma Habrdova, Marie Balikova" Pl'emysl Strejc 

Institute of Forensic Medicine and Toxicology, 1st Medical Faculty, Charles University in Prague, 121 08 

Prague 2, Czech Republic, (mba\i@lfl.cunLcz) 

Hair analyses for drugs are considered to be a significant tool for distinguishing between recent and long 

term drug abuse in forensic and clinical toxicology. Chronic consumption of drugs can gradually induce 

certain harmful effects on the human organism and it can exacerbate some pre-existing diseases. Neither 

analyses for drugs in blood or urine in isolation can yield sufficient information about the history of a 

person and their results can not be correlated directly with the toxic effects displayed. The chronic abuse of 

methamphetamine is known to be connected with cardiovascular diseases. During or after autopsy certain 

types of morphologic alterations are found in the hearts of stimulant addicts. The rapid increase of 

blood pressure after a methamphetamine intravenous dose can be risky for addicts with arteriosclerosis. 

However, the anamnestic data about a deceased person may not always be available to explain the 

pathological findings and to clasify the cause of death correctly. 

The aim of this presentation is to demonstrate the usefulness of hair analyses for drugs in the context of 

explaining the development of pathological cardiovascular alterations found after the autopsy of a case 

where methamphetamine consumption was involved. In this case (31 year old man) only methamhetamine 

and metabolites were detected with traces of ephedrine which is the precursor in illicit synthesis ofPervitin. 

The femoral blood level of methamphetamine was 1500 nglml. It was documented by a witness that the 

man died within one hour after an intravenous injection of the drug. The cause of death was 

established as cerebral edema due to cerebellar bleeding shortly after an intravenous 

dose of methamphetamine. Findings of methamphetamine in the first three 2 cm hair segments 

numbered from the roots were nearly equal (132 nglmg), in the fourth 2 cm segment it was approximately 

a half of previous values. In the remaining, distal 7 cm hair segment sample the value of methamphetamine 

was comparable to the first one. These results provide clear evidence that the man had been a chronic 

methamphetamine abuser for about more than a . year and this information can help to explain the 

pathology, the consequence of which could be the bleeding into the cerebellum after the last single 

methamphetamine dose. 

Keywords: forensic toxicology, amphetamines in hair, methamphetamine chronic toxicity 

Page 371

P37 

QUANTITA TIVE APPROACH TO DRUG ALCOHOL INTERACTION BASED ON 

POSTMORTEM DATA 

A Koski*, I Ojanpera, E Vuor, Department of Forensic Medicine, University of Helsinki, FINLAND 

Our objective was to investigate the drug-alcohol interaction in terms of postmortem concentrations of both 

drugs and alcohol in fatal poisonings. As our target analytes, we chose the three drugs most commonly 

causing fatal single-drug poisonings in Finland: the tricyclic antidepressant amitriptyline, the opioid 

analgesic propoxyphene and the phenothiazine antipsychotic promazine. The effect of benzodiazepines 

(BDZs) on drug and alcohol concentrations was also evaluated in the study. 

The laboratory database was searched for fatal poisonings caused by A) amitriptyline, propoxyphene or 

promazine alone, B) one of these drugs with alcohol present, or C) alcohol (ethanol) with one of these 

drugs detected. In order to increase sample size, cases with therapeutic or subtherapeutic concentrations of 

some of the most frequently occurring BDZs were also included, but separate statistical treatments were 

performed to evaluate their effect. The drug and alcohol concentrations included in the study had been 

measured in femoral venous blood. The final data set consisted of 116 amitriptyline cases, of which 60 

(S2%) BDZ positive, 120 propoxyphene cases, of which 43 (36%) BDZ positive, and 96 promazine cases, 

of which 42 (44%) were BDZ positive. 

The blood drug concentrations found in the unmixed amitriptyline poisonings were compared with those 

detected in the amitriptyline-alcohol cases, and likewise for propoxyphene and promazine. The blood 

alcohol concentrations found in the drug-alcohol poisonings were in turn compared with those in a 

previously compiled reference group consisting of fatal alcohol poisonings caused by alcohol alone 

(n=61S). 

The median drug concentrations in unmixed drug poisonings (BDZ+) and drug-alcohol poisonings (BDZ+) 

were 3.S (2.1) mg!l and 1.9 (0.7S) mg!1 for amitriptyline, 8.2 (4.9) mg/l and 6.1 (2.7) mg/I for 

propoxyphene, and 6.3 (7.6) mg/l and 6.7 (S.I) mg/l for promazine. Regarding the effect of BDZs, the 

difference between the median drug concentrations was statistically significant in amitriptyline poisonings 

with and without BDZs (p

P38 

TREND OF ACUTE NARCOTISM DURING THE LAST TEN YEARS 

Nadia Fucci"', Nadia De Giovanni, Fabio De Giorgio, Marcello Chiarotti 

Istituto di Medicina Legale e delle Assicurazioni, Universita Cattolica del Sacro Cuore, L.go F. Vito,1 

00168 Roma (Italy) 

Since 1993 the Laboratory of Forensic Toxicology at the Catholic University of Rome has evaluated the 

trends of deaths from narcotism ..We have already studied previous periods and our data were compared 

with D.C.S.A. (Dipartimento Centrale Servizi Antidroga - Central Antidrug Department) and GTF (Gruppo 

Tossicologi Forensi - Italian Forensic Toxicologist Association) informations. In this paper we present a 

statistical analysis of the lethal intoxications in the period 2000-2003 and compare to two prior periods 

(period A 1993-1996; period B 1997-1999). 

Annually cruel deaths observed by the Legal Medicine Institute of Catholic University range between 300 

to 400. About 18-22% of these cases are submitted for toxicologic analyses; less than 10% of total deaths 

could be referred to lethal intoxications. 

Analysis of data showed interesting observations relative to the last ten years: 

• A decreasing in overdose cases 

• Morphine alone is stiil the main drug accounting for death 

• An increase in methadone-related-deaths 

• A slight reduction in cases of acute cocaine intoxications 

A separate study on morphine and ethanol related deaths was performed; this study showed that the 

presence of ethanol higher than 0.5 gil was more significant when morphine concentrations ranged 

between 20 and 250 ng/m!. 

Our study suggests the usefulness of periodic review of mortality phenomena related to drug intoxications. 

Keywords: acute narcotism, statistical evaluation, postmortem 

Page 373

P39 

FORMALIN-INDUCED METHAMPHETAMINE DECOMPOSITION IN HUMAN LIVER 

l 

Diaa M. Shakleya , James C. Kraner 2 , James A. Kaplan 2 , and Patrick S. Callery*l,! Dept. of Basic 

Pharmaceutical Sciences, West Virginia University, Morgantown, WV 26506 and 20ffice ofChief Medical 

Examiner, Charleston, WV, 25302. 

Methamphetamine is methylated in the presence of aqueous formalin mixtures within hours at room 

temperature. The product, N,N-dimethylamphetamine, is also found in human liver exposed to 

methamphetamine followed by incubation with formalin. In the present study, we determined if liver 

samples from confirmed methamphetamine abuse subjects would produce N,N-dimethylamphetamine upon 

formalin fixation. Human liver samples were obtained from four deaths that were investigated by the West 

Virginia Office of the Chief Medical Examiner. Full toxicological analysis was conducted on samples 

from the decedents and methamphetamine was among the positive findings in each case. The method used 

to expose the tissue to formaldehyde involved treating a small piece of liver (100-200 mg) from each case 

with formalin solution (20% v/v) for 24 hrs at room temperature. The formalin treated tissues were 

homogenized and the resulting suspension was sonicated for 5 min and then centrifuged. Supernatant 

aJiquots (l0 f.l.1) were added to 500 f.l.1 of 0.1 % formic acid in acetonitrile for mass spectrometric analysis on 

a Finnigan LCQ DECA ion trap mass spectrometer with an electrospray ionization source. Positive ion 

spectra recorded in MS, MS2 and MS 3 modes were used to confirm the presence of N,Ndimethylamphetamine 

in the mixture, as well as the absence of methamphetamine. Liver tissue not treated 

with formalin did not contain a detectable level of N,N-dimethylamphetamine. The results suggest that 

embalmed tissues may give false negative findings for methamphetamine. Detection of N,Ndimethyl 

amphetamine in embalmed liver is a potential surrogate indicator of methamphetamine ingestion. 

This project was supported by Award 2001-RC-CX-K003 through the National Institute of Justice, Office 

of Justice Programs. The opinions, findings, and conclusions or recommendations expressed in this 

publication are those ofthe authors and do not necessarily reflect the views of the Department ofJustice. 

Key words: Formalin-induced, Methamphetamine, Methylation 

,,-", 

Page 374

P40 

FATAL METHADONE INTOXICATION IN AN INFANT 

Fiona J. Couper*, Kiran Chopra and Marie Lydie Y. Pierre~Louis. 

Office of the Chief Medical Examiner, 1910 Massachusetts Ave SE, Bldg 27, Washington, D.C. 20003. 

We present the case history and toxicological findings of an infant fatality involving methadone. A lO~ 

month old infant was found unresponsive in a crib by her mother. The infant was taken to hospital; 

however, she was cold and stiff on arrival and was pronounced dead. Few details regarding the case history 

were known at the time, and the autopsy findings were unremarkable. Specimens were submitted for a full 

toxicological analysis, including an alcohol analysis by headspace gas chromatography with flame 

ionization detection; a screen for drugs of abuse and several prescription drug classes using an enzyme~ 

linked immunosorbent assay technique (ELISA); and a screen for basic compounds using gas 

chromatography~mass spectrometry (GC-MS). Positive findings were confirmed and quantitated using GC 

MS. Methadone was detected in subclavian blood at a concentration of 0.67 mglL. This unexpected result 

initiated a police investigation, and it was revealed that the mother had fed methadone to her infant in the 

baby's formula for several months to stop the infant from crying. The mother was on a methadone 

maintenance program and was given take-home doses of liquid methadone. On the day of the infant's 

death, the mother admitted to administering twice the amount of methadone she normally gave her child. 

The cause of death was determined to be "methadone intoxication", and the manner of death was 

"homicide". A discussion of the case circumstances, the toxicology findings and methadone 

pharmacokinetics will be presented. 

Keywords: Methadone, Infant, Intoxication 

Page 375

P41 

SURVEY OF ABUSED DRUGS IN PERIOD 1986-2003 IN WEST BOHEMIA (MIDDLE EUROPE) 

V. Petrov;i*, D. Petr, M. Balvin 

Institute of Forensic Medicine, University Hospital in Pilsen, Czech Republic 

West Bohemia's area is about 11 000 km 2 and has a population of850 000 people. The center of the region 

is Pilsen. After the fall of Communistic regime in 1989, the number and spectrum of drug abuse cases has 

been gradually increasing. Before 1989, abuse of volatile substances (like toluene) and opiates (especially 

codeine) was prevalent. After 1989, the production and abuse of methamphetamine (pervitin) has 

significantly increased. The amphetamine derivatives are the most frequent abused drugs in this region as 

well as in the whole country. The number of cases has increased seven fold in the last decade to a total of 

620 cases .. Most of deaths connected with drug abuse and overdosage are caused by methamphetamine. 

Occurrence of opiates (mostly heroin) and cocaine is less common - often in Asiatic communities (202 

cases of opiates and 12 of cocaine). In the last two years, occurrence of cannabinoids has also increased; 

reports have doubled from 2001 to 2003 to a 388 cases. The overall increase in drug abuse has also caused 

an increase in drugged drivers. From statistics it follows that the most dangerous drug in traffic are 

cannabinoids. Four fatal road accidents caused by drivers under the influence of THC were reported in the 

year 2003 (three drivers and three other road users died). There was one fatal road accident caused by 

methamphetamine (the driver and his front passenger died) in 2003).The first and so far unique case of 

lethal paramethoxyamphetamine (PMA) intoxication in the Czech Republic was recorded in our laboratory 

in 200 I. It was a 21 year old man who had a blood PMA concentration of 1.3 mg/I. Included in these cases 

are two veterinary surgeons (39 and 41 years) who died in connection with ketamine abuse. The first one 

had been abusing this substance for a long time and shot himself. The other one committed suicide with 

combined ketamine, succinylcholine and alcohol intoxication. 

Keywords: paramethoxyamphetamine, methamphetamine, cannabinoids 

Page 376

P42 

ENANTIOMERIC ANALYSIS OF EPHEDRINES AND NOREPHEDRINES 

2 

Sheng-Meng Wang l • 2 , Russell J. Lewis' 2 , Dennis Canfield 2 , and Ray H. LiU .J. 4 , 'Central Police University, 

Taoyuan, Taiwan. 2Toxicology and Accident Research Laboratory, FAA Civil Aerospace Medical Institute, 

Oklahoma City, OK, U.S.A. JDepartment of medical technology, Fooyin University, Kaohsiung Hsieh, 

Taiwan. 4Department ofJustice Sciences, University ofAlabama at Birmingham, Birmingham, AL, U.S.A. 

Concerned with variations in abuse potential and control status among various isomers of ephedrines and 

norephedrines, this study was conducted to develop an effective method for the simultaneous analysis of 

nine structurally related ephedrine-type compounds. Select cold medications were then analyzed to 

characterize the enantiomeric compositions of ephedrine, norephedrine (phenylpropanolamine or PPA), 

pseudoephedrine, norpseudoephedrine (cathine), and cathinone. 

Among various approaches investigated, a 60-m HP-5MS (0.25 mm!D, 0.25 !!m film thickness) was found 

to successfully resolve the following compounds of interest that were derivatized with (-)-a-methoxy-a. 

trifloromethy lpheny lacetic acid (MTPA): (+ )-cathine, (+ )-cathinone, (-)-cathinone, (+)-ephedrine, (-) 

ephedrine, (+)-PPA, (-)-PPA, (+)-pseudoephedrine, (-}-pseudoephedrine. A (-}-cathine standard was not 

available, but should also be resolvable using this analytical procedure. The injector temperature was set at 

250°C and the oven profile was 160-220°C at 5°C/min, hold 1 min, 220-250°C at 25°C/min. This method 

was applied to the analysis of various over-the-counter cold medications and the results derived from three 

samples are shown in Table 1. These compounds were isolated from the cold medications utilizing a 

simple liquid/liquid extraction with ethyl acetate. This method has proven to be an efficient procedure for 

the separation and identification ofvarious enantiomeric ephedrine and norephedrine-type compounds. 

Table 1. Enantiomeric composition (JlglmL) of various ephedrines and norephedrines found in various cold 

remedies 

(+)-Cathinone 

PPA thinone PPA Cathine (+)-Ephedrine 

(+) H-Ca 

(-) (+)- 

SamJ;!le 

H-Ephedrine 

(-)-PseudoeJ;!hedrine 

(+)-PseudoeJ;!hedrine 

2 1.84 0.565 

16 0.549 0.414 48.1 

19 1.35 1.19 26.8 2130 340 

References 

1. Liu J-T, Liu RH: Enantiomeric Composition of Abused Amine Drugs--Chromatographic Methods of 

Analysis and Data Interpretation Matrices; J Biochem Biophys Methods 54:115-146; 2002. 

2. Iwanicki RM, Maier K, Zlotnick JA, Liu RH, Kuo T-L, Tagliaro F: Separation of Enantiomeric 

Ephedrine and Pseudoephedrine-High Pressure Liquid Chromatography and Capillary Electrophoresis; 

J Forensic Sci 44:470--474; 1999. 

Key Words: Ephedrine, Norephedrine, Enantiomer 

Page 377

P43 

"ECSTASY" IN THE A.M. AND P.M. 

MDMA CONCENTRATIONS IN FATALITIES FOLLOWING HOSPITAL ADMISSION 

Simon Elliott*, Regional Laboratory for Toxicology, City Hospital, Birmingham, U.K. 

Over the last 15 years numerous deaths involving "Ecstasy" (MDMA) have been reported and described in 

the literature. With most cases, either ante-mortem (AM) or post-mortem (PM) concentration data are 

available. Due to the wide range of results and potential idiosyncratic nature of MDMA toxicity, 

interpretation of either data set is difficult. The possible influence ofpost-mortem redistribution may also 

be a factor, however it has been suggested that most amphetamine analogues are less influenced by this 

phenomenon than some other drugs. The aim of this study was to evaluate the concentrations of MDMA 

found in 5 fatalities admitted to hospital where both ante-mortem and post-mortem blood samples were 

available. Concentration ratios between PM and AM samples were also calculated to compare case data 

and evaluate redistribution. 

Case 1: 31 year old male admitted to hospital following suspected injection of crushed amphetamine and 

"Ecstasy" tablets. He developed malignant hyperpyrexia and later died. An AM serum MDMA 

concentration of 1.22 mgIL was measured (45 minutes prior to death) with a corresponding PM (brachial) 

blood concentration of2.37 mg/L. This produced a PM:AM concentration ratio of 1.9. Amphetamine and 

ethanol were also detected. 

Case 2: 30 year old male recovered from a river after a night out drinking. He was admitted to hospital but 

later died. 6 AM samples were analysed (between 9-20 hours prior to death). An AM plasma MDMA 

concentration of 0.31 mg/L was measured (9 hours before death) with a PM (trunk) blood concentration of 

0.47 mg/L (PM:AM ratio 1.5) and PM, (arm) concentration of 0.52 mgIL (PM:AM ratio 1.7). 

Chlordiazepoxide and ethanol were also detected. 

Case 3: 26 year old male found collapsed in the street after having taken several "Ecstasy" tablets. He was 

admitted to hospital but later died after suffering hyperpyrexia. An AM blood MDMA concentration of 

2.04 mg/L was measured (1 hour prior to death) with a corresponding PM (femoral) blood concentration of 

2.25 mgIL) and PM Ougular) blood concentration of 2.99 mg/L. These produced PM:AM concentration 

ratios of l.l and 1.5, respectively. A trace of par aceta mol was also detected but no ethanol. 

Case 4: 22 year old female admitted to hospital with hyperthermia following ingestion of approximately 12 

"Ecstasy" tablets. An AM serum MDMA concentration of 4.33 mg/L was measured (1 day prior to death) 

with corresponding PM blood concentrations of7.25 mgIL (left femoral), 6.19 mgIL (right femoral), 28.39 

mgIL (heart) and 11.93 mgIL (vitreous humor). These produced PM:AM concentration ratios of 1.7, 1.4, 

6.6 and 2.8, respectively. Cocaine metabolite and ethanol were also detected. 

Case 5: 63 year old male found collapsed at home having allegedly ingested 4 "Ecstasy" tablets. He was 

taken to hospital but later died following a cardiac arrest. 3 AM samples were analysed (between 0-2 days 

prior to death). An AM serum MDMA concentration of 0.44 mg/L was measured (day of death) with a 

corresponding PM (femoral) blood concentration of 1.14 mg/L (PM:AM ratio 2.6). Cannabinoids, cocaine 

metabolite and ethanol were also detected. 

Overall, the PM blood cOncentrations were higher compared to the AM concentrations in all 5 cases 

(PM:AM ratio l.l to 2.0 in 4 cases). In one case, there also appeared to be a significant difference in the 

PM blood concentrations between anatomical sites (heart and femoral). Consequently, the data suggest that 

post-mortem blood MDMA concentrations may not accurately relate to the concentration either at the time 

of, or prior to death and calculations based on this assumption (e.g; dosage) should not be made. 

Keywords: Ecstasy, MDMA, redistribution. 

Page 378

P44 

FATAL CASES DUE TO CYANIDE INTOXICATION THROUGH A LOCAL SUICIDE 

FOSTERING WEBSITE 

SK Lee l , JY Yang l , KW Kiml, SY Lee!, YS Park l , HS Chung*l, YC Y002; INational Institute of Scientific 

Investigation, Seoul, KOREA; 2College ofPhannacy, Chungbuk National University, KOREA 

With the rapid development of the infonnation technology industry, new opportunities for committing 

suicide have appeared and become a social problem. With internet availability at most schools, companies 

and homes, committing suicide using the information from internet websites has been increasing. An 

individual opens a local suicide-fostering website that provides the infonnation for suicide, selling poisons, 

recruiting members who want to commit suicide and promoting them to kill themselves together. The 

poisons mainly introduced on the suicide websites are cyanide salt and pesticides. Two cases related to 

cyanide salt were reported here. One man and two women in case 1 and three men and two women in case 

2 were found dead in a motel on two separate occasions. Experimental grade potassium cyanide was also 

discovered in both cases. Identification and quantification of cyanide ion were performed by microdiffusion 

method after blood and gastric contents were collected at autopsy. Postmortem blood concentrations of 

cyanide ion ranged from 53.8 to 208 flglml in case 1 and from 5.4 to 194 flg/ml in case 2. The cyanide ion 

concentrations in case 2 were all over 100 flg/ml except one of the victims, where the blood concentration 

was 5.4 flglml. Based upon the history, autopsy and toxicology findings, (1) the victims were in their teens 

and 20s who use the internet, (2) experimental grade KCN was used in these cases while industrial grade 

NaCN was used in most of the previous cases, and (3) the postmortem blood concentrations in these cases 

were generally much higher than previous cases in Korea (0.4-74 flg/ml) and those reported in other 

countries (1.1-53 flg/ml). The government should strengthen education for human dignity as well as poison 

control in order to protect youth prone to suicide seduction. 

Keywords: Cyanide, Intoxication, Internet 

Page 379

P45 

ARSENIC IN NAPOLEON'S HAIR: IS EXTERNAL CONTAMINATION A POSSIBLE SOURCE 

I. Ricordel l , S. Pirnay*\ A. Marechal l , P. Chevallier, G. Meyer 3 , N. Milani, 1. Plesse l 

I Laboratoire de toxicologie de la Prefecture de Police, 75012 Paris, France " 2 LURE 918980rsay France' 

3 ' " 

INSTN-CEA, 91190 GifNvette, France 

Since the end of the 20 th century, forensic toxicology experts have extensively studied the precise cause of 

Napoleon's death. Several toxicological studies have shown that there were significant concentrations of 

arsenic in Napoleon's hair, leading to the suspicion that he was poisoned with arsenic. In our work, we 

confirmed the presence of arsenic in the hair; however, the heterogeneity of the results between laboratories 

and high level of arsenic found in Napoleon's hair encouraged us to question the interpretation of these 

results. In a previous study by non-destructive X-ray fluorescence analysis, we could determine the 

presence of arsenic at high concentration in Napoleon's system. Another complicating factor was that there 

were no clinical symptoms of arsenic intoxication as reported by witnesses at that time. In this context, our 

objective was to determine if an arsenic external contamination of Napoleon Bonaparte's hair may be a 

plausible cause (only or associated with other factors) to explain arsenic rates of Emperor hair. 

From aqueous solutions of arsenic, our protocol was to establish the quantity of arsenic capable of 

penetrating the hair (cut extremity protected). For this purpose, we chose varied parameters: 1) the 

composition of the aqueous solutions (5 different solutions), the time of contact with the hair (from 24 h to 

I month), the color of the hair (blonde, brown, and black) and the ethnic origin of the hair. Arsenic 

concentrations were determined on the hair, before and after four washes (five minutes successively with 

water, acetone, water and water sonicate), and on the liquids of washing by atomic absorption spectrometry 

in a pyrolytic oven with Zeemann correction (GTA 110 Zeemann, Varian®). 

Major results show that: (a) Trivalent arsenic can penetrate and remain in the hair, (b) Washing the hair 

induces the decrease of the arsenic rate, but a significant quantity of arsenic is still firmly fixed after 

different treatments. (c) The time of immersion of the hair in a trivalent arsenic solution, does not change 

the quantity of arsenic fixed on the capillary between 24 hours and 1 month, whatever its ethnic origin. (d) 

The additives, likely used to prepare the disulfides bonds of the keratin for facilitating arsenic penetration, 

do not significantly modifY the qualitative and quantitative arsenic binding, whatever the additive and the 

ethnic origin ofthe hair. 

In conclusion, our study confirms that arsenic can penetrate the hair through an external source as other 

reports. Arsenic may remain at least partly fixed at high and toxic levels into the hair, even after full 

washes. Although arsenic intoxication of Napoleon cannot be excluded, our data suggests that external 

contamination from arsenic may provide an alternate explanation for the pres.ence of arsenic in the hair. 

Keywords: Arsenic, Napoleon, external contamination. 

Page 380

P46 

FATAL GHB INTOXICATION FOLLOWING RECREATIONAL USE 

Susan Mazarr-Proo, B.U.S.* and Sarah Kerrigan, Ph.D. 



Gamma-hydroxybutyrate (GHB) is encountered in biological specimens both as an endogenous 

neuromodulator, and as a recreational drug. Illicit GHB is currently classified as a schedule I drug in the 

United States due its abuse among recreational drug users. Therapeutically the drug is used for the 

treatment of cataplexy. Illicit doses are typically 2-4 g and the onset of action is rapid, occurring 15-30 

minutes following oral ingestion. Dose dependent effects include drowsiness, euphoria, dizziness, 

vomiting, respiratory depression, coma and death. 

GHB was isolated from biological samples using a simple liquid-liquid extraction. Trimethylsilyl 

derivatives were analyzed using gas chromatography mass spectrometry using positive chemical ionization 

(GCIMSIPCI). Deuterated internal standard and selective ion monitoring is used throughout. Samples were 

analyzed using an HP 5973 MSD equipped with a 30m DB-5 capillary column. 

We report a GHB fatality involving a 35 year-old male who was "partying" with friends. Subjects at the 

party ingested unknown quantities of wine and GHB. A female companion at the party reported seeing the 

male alive earlier in day before she passed out. She awoke to find the individual decedent cold and stiff. 

Postmortem specimens were submitted to our laboratory for comprehensive toxicology testing. No alcohol 

or common drugs of abuse were detected in the femoral blood. A targeted analysis revealed GHB in urine, 

brain, vitreous fluid, femoral blood, heart blood and liver at concentrations of 1665 mg/L, 102 mg/kg, 48 

mg/L, 461 mg/L, 276 mg/L and 52.2 mg/kg respectively. Concentrations of the drug in urine and vitreous 

fluid are extremely important in death investigations due to significant postmortem production of GHB in 

blood specimens. The cause of death was attributed to GHB intoxication and the manner of death was 

accidental. 

Keywords: GHB, postmortem toxicology, fatality 

Page 381

P47 

INTERPRETATION OF POSTMORTEM ALCOHOL CONCENTRATIONS: A CASE STUDY 

Curtis Caylor, B.S.,* Ruth Luthi, B.S. and Sarah Kerrigan, Ph.D. 

New Mexico Department of Health, Scientific Laboratory Division, Toxicology Bureau, PO Box 4700, 


Our laboratory performs drug and alcohol testing on approximately 2800 medical examiner cases each 

year. The preferred specimen for alcohol analysis was blood, and until recently, additional specimens were 

not routinely tested unless the submitting agency requested additional analyses. A new policy required that 

all positive blood alcohol measurements in postmortem cases were confirmed using an alternative 

specimen. Although this resulted in a 43% increase in alcohol casework, these analyses provided important 

interpretive information and also identified a number of unusual results that would not have been otherwise 

identified. 

Postmortem blood and vitreous alcohol concentrations were determined in a total of 295 alcohol-positive 

cases. The vitreous alcohol concentration (V AC) exceeded the blood alcohol concentration (BAC) in 209 

cases (71%). Blood alcohol concentrations exceeded vitreous concentrations in 81 cases (27%) and the 

concentrations were equivalent in 5 cases (5%). Samples that were negative in both specimens were 

excluded, as were cases where the confirmatory test was performed using a specimen other than vitreous 

fluid (e.g. urine, liver, blood from an alternative source). In casework where the VAC>BAC, linear 

regression analysis indicated an R2 value of 0.958 (n=295) and a V AC approximately 18% higher than the 

BAC. The VACIBAC ratio was more variable at lower BACs «0.1 glIOOmL). Although VACIBAC ratios 

were more consistent at concentrations of0.1 gllOOmL and above, the overall ratio ranged from 1.01-2.20. 

Ofthe 81 cases where the BAC>VAC, a total of24 cases indicated no vitreous alcohol. The range ofblood 

alcohol concentrations among these cases was widely variable (0.01 to 0.30 glIOOmL). All analyses were 

conducted using dual column gas chromatography with flame ionization detection (GC-FID) with a 

reporting limit of0.0 1 g/l00mL for postmortem samples. 

A series of case studies are used to demonstrate postmortem interpretive issues and the benefits associated 

with multiple specimen analysis. Cases include in-situ contamination of specimens due to trauma, 

postmortem production of ethanol, death during the absorptive phase among others. Actual case studies 

involving other volatile organic compounds are also discussed including isopropanol and acetone from 

endogenous and exogenous sources. These cases studies highlight the importance of analyzing multiple 

specimens in postmortem casework. 

Keywords: ethanol, postmortem, blood, vitreous fluid 

Page 382

P48 

DETERMINATlON OF FENTANYL IN POSTMORTEM BLOOD SAMPLES BY LIQUID 

CHROMA TOGRAPHY TANDEM MASS SPECTROMETRY 

Vera Coopman l ', Dirk. Van Varenbergh 2 , Karen Pien 2 and Jan Cordonnier l 

lDepartment of Analytical Toxicology, Chemiphar N.V., Lieven Bauwensstraat 4, Bruges, Belgium. 

2Department of Forensic Medicine, Free Brussels University, Brussels, Belgium. 

Fentanyl is a potent synthetic narcotic analgesic administrated in the form of a transdermal patch for the 

management of chronic pain. The authors present a liquid-liquid extraction procedure and a liquid 

chromatography-tandem mass spectrometry method (LC-MS-MS) for the quantitation of fentanyl in 

postmortem blood samples .. The method was applied in a case of acute intoxication due to excessive 

administrated Durogesic® transdermal patch. 

To a 1.0 mL aliquot of blood sample, 50 flL of internal standard solution fentanyl-ds (1 flgJmL) and 1 mL 

1M K 2 C0 3 was added. Extraction was performed with 7 mL of a mixture of n-hexane:ethylacetate (7:3, 

VN). After vortex mixing and centrifugation, the upper organic layer was evaporated and reconstituted in 

1.0 mL of initial mobile phase. The LC-MS-MS analysis were performed using an Alliance® 2695 system 

and a Quatro Micro mass spectrometer (Waters Milford, USA). Chromatography was achieved using a 

Xterra® MS C18 column 2.1 mm x 100 mm with a 3.5 flm particle size (Waters Milford, USA). A gradient 

elution was used with 0.15% formic acid and 0.15% formic acid in acetonitrile. The chromatographic 

duration was 30 minutes. The flow rate was 0.20 mLimin and the column oven temperature was set at 

35°C. A 20 flL aliquot of the sample was injected. Multiple reaction monitoring (MRM) with positive ion 

dectection (ES+) was used for selective detection of fentanyl and fentanyl-ds. The precursor ion and two 

fragment ions were selected as quantifier and qualifier, for fentanyl mlz 336.8 and fragments at m/z 187.95 

and 104.4, for fentanyl-ds mlz 341.9 and fragments at m/z 188.0 and 104.5. Calibration was performed by 

addition of standard solutions to fentanyl-free blood prior to extraction. Precision, accuracy and recovery 

experiments were carried out. The liquid-liquid extraction procedure in combination with LC-MS-MS 

proved to be a good alternative compared with the more labor-intensive solid-phase extractions previously 

described. 

The analytical procedure was applied in a forensic case: a 78-year-old woman was found death in bed, 

lying on het back. The external examination revealed 10 Durogesic® transdermal patches (fentanyl 100 

flglh) on the abdomen. An autopsy was carried out 2 days later. Multiple samples were taken for 

toxicological examination. The following fentanyl concentrations were found; 28.6 flglL (right) and 28.2 

fJg/L (left) in subclavian blood, 21.3 fJglL (right) and 20.9 fJg/L (left) in femoral blood, 37.6 fJglL (right) 

and 33.9 flg/L (left) in ventricular blood. The comprehensive systematic toxicological analysis did not 

reveal the presence of an other substance which contributed to the death of the woman. We concluded that 

death was caused by intoxication with fentanyl. The manner was presumed to be suicide. 

Keywords: fentanyl, LC-MS-MS, fatality 

.~. 

Page 383

P49 

ALFENTANIL FATAL INJECTION: A CASE REPORT 

V.Dumestre-Toulet* I, M. Villain 2, P. Kintz 2 

ILaboratoire TOXGEN - Bordeaux; 2Institut de Medecine Legale - ChemTox - Strasbourg 

Introduction: We report a case of fatal intoxication with alfentanil (RAPIFEN,ALFENT A ampoules of 

1 and 5 mg), an intravenous narcotic analgesic, chemically related to fentanyl and characterized by a very 

rapid onset and short duration of action. Alfentanil is used as an adjunct for surgical anaesthesia under 

assisted ventilation in medical units. 

Case-report: A 46-year-old anaesthetist male nurse was found dead in the toilets of an hospital. A syringe 

was found near the body. He was suspected of narcotic theft and abuse because a lot of ampoules were 

missing since several months in the unit. Autopsy findings revealed injection marks on the left ann, an 

oedematous and inflammatory aspect of various organs and a bilateral pulmonary oedema. Biological 

samples (blood, urine and hair) were collected and sent to the laboratory for forensic toxicological analysis. 

Methods: Screening analyses were performed by immunoassay, gas chromatography/mass spectrometry 

(GC/MS) and liquid chromatography/diode array detection (LCIDAD) using standardized methods of our 

laboratory. GC/flame ionisation detector with headspace injection (HS) was used to detennine blood 

alcohol concentration. Alfentanil and fentanyl were screened with GCIMS and quantification was 

performed with GC/MS/MS after liquid-liquid extraction with chlorofonn/2-propanol/heptane (33:17:50, 

v/v/v) and phosphate buffer (Iml, pH 9.5). Fentanyl d S was used as internal standard. 

Results: Alfentanil was identified in blood and urine at 45.1 and 2.7 nglml, respectively. Blood alcohol 

concentration was measured at 1.32 gil. Hair analysis revealed chronic use of alfentanil (2 pglmg) and 

fentanyl (8 pg/mg). No other drugs were detected. 

Adult surgical patients who received a bolus dose of 8 to 50 jlg/kg alfentanil by intravenous infusion 

developed plasma concentrations of 200 to 1000 nglml. Alfentanil is capable of producing severe 

respiratory depression with hypotension and coma during surgical operations even with assisted ventilation 

and plasmatic concentrations above 100 - 200 ng/ml (1). Whereas fentanyl is reported as a commonly 

abused substance by healthcare professionals (2), alfentanil misuse and abuse seems exceptional (3). 

Conclusion: Although the measured concentrations are therapeutic, alfentanil injection without assisted 

ventilation may be the cause of the death. Hair analysis demonstrated chronic abuse of alfentanil and 

fentanyl by the anesthetist nurse. 

References: 

(1) Mahla ME, White SE, Moneta MD. Delayed respiratory depression after alfentanil. Anesthesiology 

1988 ; 69: 527-534 

(2) Villain M, Cirimele V, Ludes B, Kintz P. Toxicomanie d'un anesthesiste au fentanyl: la preuve 

fonnelle par analyse des cheveux en utilisant la CPG/SM/SM. Ann. Toxicol. Anal. 2001; 13 (1): 49 

53, 

(3) Kintz P, Villain M, Cirimele V, Ludes B. Conduites addictives en milieu hospitalier : particularites 

d'un service d'anesthesie. Ann. Toxicol. Anal. 2002; 14(1): 83- 89 

Keywords: alfentanil, chronic abuse, hair, GC/MSIMS. 

Page 384

P50 

THE DETERMINATION OF ETHANOL ORIGIN IN FATAL AVIATION ACCIDENT VICTIMS: 

A MULTI-CASE STUDY 

Johnson, R.D.*!, Lewis, RJ. 1 , and Blank, C.L 

'Civil Aerospace Medical Institute, AAM-61O, CAM I Building, RM 205, 6500 S. Macarthur Blvd, 

Oklahoma City, OK 73169-6901 USA; 2The University of Oklahoma, Department of Chemistry and 

Biochemistry; 620 Parrington Oval, Rm 208, 1';/orman, OK 73019 

Specimens from aviation accident victims are submitted to the FAA's Civil Aerospace Medical Institute 

(CAMI) for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on each 

such case. Care must be taken when interpreting a positive ethanol result due to the potential for 

postmortem ethanol formation. Historically, ethanol distribution in various tissues and fluids from the same 

case and/or the presence of other volatile organic compounds (VOCs) at abnormal concentrations in these 

fluids and tissues has been employed as an indicator of postmortem microbial ethanol formation. However, 

these methods are not always reliable. The consumption of ethanol has been shown to alter the 

concentration of two major serotonin metabolites, 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3 

acetic acid (5-HIAA). While the 5-HTOLl5-HIAA ratio is normally low, previous studies have 

demonstrated that the urinary 5-HTOLl5-HIAA ratio is significantly elevated following ethanol ingestion. 

The S-HTOLl5-HIAA ratio is not affected by the microbial formation of ethanol, by consumption of 

serotonin~rich foods or by the use of SSRl's. 

Recently, our laboratory has developed a single analytical approach to determine concentrations of both 5 

HTOL and 5-HIAA that has provided a convenient, rapid and reliable solution to this problem. This novel 

methodology eliminates the need for two separate and unrelated analytical techniques, GCIMS and LCIEC, 

for the determination of these metabolites. The simultaneous determination of 5-HTOL and 5-HIAA in 

forensic urine specimens was achieved using a liquid/liquid extraction technique in conjunction with 

LCIMS. The ion trap MS used allowed us to perform MSIMS/MS on both 5-HTOL and 5-HIAA, and 

afforded limits of quantitation below 1 ng/mL for each compound. After development of this method, the 

previously established, antemortem, 15 pmol/nmol 5-HTOLl5-HIAA ratio cutoff was investigated and 

subsequently validated for use with forensic specimens. 

Utilizing this newly validated method, we examined the 5-HTOLIS-HIAA ratio in urine specimens 

obtained from six separate fatal aviation accidents where ethanol was present, but its source was unclear. In 

one of these cases antemortem consumption of ethanol was indicated, even though microbial production 

was initially considered. After examining the 5-HTOLl5-HIAA ratio from the other five cases investigated 

we determined that recent ethanol consumption had not occurred. The ethanol present in these cases was 

due to microbial formation, and not consumption. This presentation will discuss, in depth, the difficulties in 

determining the source of ethanol in these six cases and the application of this novel methodology in 

elucidating its origin. 

Keywords: Postmortem Ethanol, LC/MS, Serotonin metabolites 

Page 385

PSt 

UNUSUAL TRAMADOL CONCENTRATIONS IN AN ACCIDENTAL DEATH INVOLVING 

POLY DRUG USE 

Ginger Baker, M.S., * Janice Yazzie, B.S., and Sarah Kerrigan, Ph.D. 

New Mexico Department of Health, Scientific Laboratory Division, Toxicology Bureau, PO Box 4700, 

Albuquerque, NM 87196·4700 

Tramadol is a synthetic opioid-receptor agonist that is used clinically as an analgesic in daily doses of 100 

to 400 mg. Therapeutic concentrations in blood are reported in the range 0.1 to 0.8 mg/L. Overdoses of 500 

mg and higher may cause agitation, hypertension, tachycardia and seizures whereas doses greater than 800 

mg may result in coma and respiratory depression. We report a case involving unusually high postmortem 

tramadol (Ultram) concentrations in a 35 year-old male with a past medical history of bipolar illness, 

anxiety, depression and substance abuse. 

Tramadol concentrations were determined using solid phase extraction (Polychrom Clin II) and gas 

chromatography/mass spectrometry (GCIMS) using full scan analysis with mepivicaine as the internal 

standard. An R2 value in the linear range (0.2 4.0 mglL) was 1.000 for tramadol. The Intraassay CV 

was 6.4 % using laboratory fortified independent spikes. 

Initial toxicology findings of illicit drugs in femoral blood identified cocaine and benzoylecogonine at 

concentrations of 0.04 mg/L and 0.91 mg/L, respectively. Therapeutic prescription medications found in 

femoral blood included paroxetine, trazadone, tramadoland the active metabolite, O·desmethyltramadol. 

Quantitative analysis oftramadol revealed extraordinarily high concentrations of the parent drug. Tramadol 

concentrations in femoral blood, heart blood and vitreous fluid were 74 mg/L, 83 mg/L and 18 mg/L, 

respectively. O-desmethyltramadol concentrations in femoral blood, heart blood and vitreous fluid were 4 

mg/L,5mg/L and Img/L, respectively. Issues that need to be considered when interpreting toxicological 

findings associated with multiple drug use will be discussed. 

Keywords: tramadol, overdose, postmortem 

Page 386

P52 

PEDIATRIC FATALITY DUE TO HYDROXYCHLOROQUINE INGESTION 

H. Nipper*, P. Studts, R. Baltaro, Creighton University Medical Center, Omaha, NE, 68131 

A 7 year-old African-American female was found at home unresponsive and cold approximately 4 hr after 

being given acetaminophen and sent to bed to rest after complaining of stomach ache and headache. 

At autopsy, findings were unremarkable for clues as to cause of death. Heart blood and vitreous humor 

were sent to Creighton University Medical Center for forensic testing. Initially, serum and vitreous humor 

electrolytes, hemoglobin electrophoresis were ordered, but all results were within expected limits. The 

acetaminophen level was 57.6 ~g/mL. 

Further testing was performed using gas chromatography with FIDINPD on an alkaline extract of blood. 

The presence of an unfamiliar peak was noted and the Douglas County (Nebraska) Coroner was alerted to 

return to the home with investigators to perform a full inventory of pharmaceuticals. The inventory 

included numerous arthritis medications including one identified as "hydroxychlor." The parents 

determined with investigators present that eleven 200 mg tablets (2.2g total drug) were unaccounted for 

from the bottle that they thought was stored safely. 

Simultaneously the laboratory was identifYing the unknown GC peak as hydroxychoroquine using GCIMS 

with a dosage form from the hospital pharmacy as a standard. 

Quantitation of the blood level of hydroxy chloroquine performed at National Medical Services revealed 70 

IJ.g1mL. (NMS therapeutic range: 0.1-1.0 IJ.g1mL) The cause of death was subsequently ruled to be acute 

overdose of hydroxy chloroquine. The manner of death was ruled to be accidental. 

According to the PDR (1) "Children are especially sensitive to the 4-aminoquinoline compounds. A 

number of fatalities have been reported following the accidental ingestion of chloroquine, sometimes from 

relatively small doses (0.75-1.0 g in one three-year-old child). Parents should be strongly warned to keep 

these drugs out of reach of children." Early symptoms of overdose are headache and drowsiness, and may 

occur within 30 minutes of ingestion. These symptoms may be followed by cardiovascular collapse, and 

"sudden and early respiratory and cardiac arrest". (1) 

1. Physicians Desk Reference (PDR), 58 th ed., (2004) p. 3031, Thomson PDR, Montvale, NJ 07645. 

Keywords: Pediatric, Postmortem toxicology, Hydroxychloroquine, Fatality 

Page 387

P53 

LORAZEPAM AND DEATH INVESTIGATION 

Jayne E. Clarkson'·, Ann Marie Gordon', Barry K. Logan' 

IWashington State Toxicology Laboratory, Forensic Laboratory Services Bureau, Washington State Patrol, 

2203 Airport Way South, Seattle, WA 98134. 

The objective of this study was to evaluate the role of lorazepam (Ativan®), a benzodiazepine frequently 

used to manage anxiety and to sedate, in death investigations. Lorazepam has been detected with 

increasing frequency in death investigation cases in Washington State. A review of the literature revealed 

there is currently limited data on the significance of lorazepam in death investigation cases. 

We reviewed death investigation cases which were positive for lorazepam at the Washington State 

Toxicology Laboratory between January 1998 and December 2003. The mean drug concentration found in 

the blood of these decedents (n=112) was 0.15 mg/L (S.D. 0.39, median of 0.03 mg/L). Concentrations 

ranged from

P54 

SERTRALINE WITHDRAWAL IN A NEWBORN - COULD THIS BE A FACTOR IN THE CAUSE OF 

DEATH 

Laureen J. Marinetti* and Russell L.Uptegrove. 

Montgomery County Coroner's Office (MCCO), 361 West Third Street, Dayton, OH 45402 

A 4·day-old infant was found not breathing by her mother at about 6am. The infant was wrapped in a blanket 

and lying on her back on a large sectional couch near her sleeping father. The mother confirmed that the infant's 

face and head were several inches from her husband and were not obstructed in any way. The infant was 

transported to the emergency room where a heart rhythm was established and she was placed on a ventilator. 

The infant was later found to be brain dead and was removed from the ventilator. She was pronounced dead 

approximately 12 hours after the mother had first found her. 

The mother received regular pre-natal care and was diagnosed with gestational diabetes. As a result she had bi 

weekly testing for the last several weeks of her pregnancy. In addition she also experienced sleeplessness and 

had been prescribed sertraline, which she took as prescribed. The birth was induced and was an uneventful 

delivery reSUlting in a 71b, 60z infant. The infant was fed formula (similac) and was not breast-fed at any time. 

The infant was released from the hospital 2 days after she was born. Approximately 24 hours after birth the 

infant became irritable, screaming and crying after every feeding. The last two nights prior to her death she was 

awake all night in this highly irritable state. 

Significant findings at the time of autopsy included epicardial hemorrhage on the anterior surface of the heart, 

(consistent with cardiopulmonary resuscitative efforts), and pulmonary and liver congestion. 

The toxicology laboratory at MCCO received the following specimens; hospital blood (3 mL), heart blood (16 

mL), cerebral spinal fluid (CSF), gastric, liver, vitreous fluid, bile and head hair. Analysis of the heart blood 

revealed the presence of 16 nglmL sertraline and 113 nglmL norsertraline, the hospital blood revealed 71 ng/mL 

norsertraline and analysis of the liver revealed 230 nglg sertraline and 4000 ng/g norsertraline. Sertraline was 

detected in the hospital blood but the small amount of specimen received precluded its accurate measurement. 

Sertraline and norsertraline quantitation was accomplished using high performance liquid chromatography 

(HPLC) with a CI8 column and ultra violet detection with confirmation by gas chromatography/mass 

spectrometry (GC/MS). 

A review of the literature reveals neonatal complications when selective serotonin re-uptake inhibitors (SSRl's) 

are used by the mother near term. Paroxetine, sertraline, fluvoxamine and fluoxetine have been associated with 

withdrawal symptoms in the neonate. The withdrawal symptoms are diverse but most commonly include; 

respiratory distress, hypoglycemia, jaundice (Costei et al. 2003) and irritability, constant crying, shivering, 

increased tonus, eating and sleeping difficulties and convulsions, (Nordeng et at. 2001). The symptoms usually 

occur after 2 days and last for an average of 10 days to one month (Salvia-Roiges et aL 2003). While the SSRI's 

have not been associated with teratogenic risks, it is clear that the potential for withdrawal exists - especially if 

the mother does not breast-feed the infant. Hendrick et at. 2003 determined cord blood concentrations of 

sertraline and norsertraline in II newborn babies whose mothers were prescribed the drug. The ranges were as 

follows, sertraline; less than I up to 14 nglmL and norsertraline; less than I up to 72 nglmL. None of these babies 

experienced withdrawal symptoms but it is unknown ifthey were breast-fed. 

Based on the clinical history of the infant's feeding and sleeping difficulties, irritability and crying, and the fact 

that the infant was being fed formula instead of being breast fed, it is possible that the infant was undergoing 

withdrawal from sertraline which it was exposed to in utero. A search of the medical literature revealed no 

reported cases of an infant's death due to exposure to or withdrawal from sertraline but some infants exposed to 

SSRI's in utero required treatment for the withdrawal symptoms. The cause and manner of death was listed as 

undetermined. In conclusion not enough information is known to conclude that SSRl's are safe to administer to 

a pregnant woman, especially in the third trimester. 

Key Words: Sertraline, Neonate, Withdrawal 

Page 389

P55 

QUETIAPINE CONCENTRATIONS IN INTENTIONAL AND ACCIDENTAL DRUG 

INGESTIONS 

Martha J. Burt* and Diane M. Boland 

Miami-Dade County Medical Examiner Department 

Quetiapine (Seroquel) was introduced in 1993 as a neuroleptic agent in the treatment of psychosis. It has 

high affinity for serotoninergic receptor sites and less affinity for dopaminergic, adrenergic and histamine 

receptors. Since introduction, very few cases have been reported on the potential mortality of quetiapine 

overdose, alone or in combination with other drugs. We present our case series of single drug and mixed 

drug quetiapine overdoses from the Miami-Dade County Medical Examiner Department. The case records 

from the years 1998 to 2003 inclusive were searched for those cases where quetiapine was detected and 

quantitated in postmortem blood, gastric contents, or liver. A total of 17 cases were identified; three were 

excluded because no quetiapine was detected during the quantitation process, and one was excluded 

because a complete autopsy was not performed. Thus, 13 cases are reported in this series. The ages ranged 

from 28 to 55 years, with a mean age of 44 years. These cases were grouped into four categories: those 

where the quetiapine was the sole cause of death (Q only), those where quetiapine was related to the cause 

of death (QR), those where the quetiapine was unrelated to the cause of death (QUR) and those where it 

was unclear if quetiapine played any role in the cause of death (UNK). Two deaths were a result of solely 

quetiapine ingestions (Q only cases). One was a 35 year old white male with central blood, gastric and 

liver concentrations of 25 mgIL, 8370 mg total, and 36 mg/kg, respectively. The other was a 48 year old 

white female with blood, gastric and liver concentrations of 9.5 mgIL, 954 mg total, and 34 mg/kg, 

respectively. Two deaths in our series resulted from polydrug ingestions, where more than one agent, 

including quetiapine could have resulted in death (QR group). The blood quetiapine concentrations were 

15.6 mgIL and 13.6 mgIL, and both had significant concentrations of at least one benzodiazepine, in 

addition to other agents. Four cases fell into the unknown group (UNK), where it was unclear ifquetiapine 

contributed to the cause of death. In these cases, the mean quetiapine concentration was 1.86 mgIL, with a 

range of 0.34 - 2.8 mgIL. All four had other drugs detected, such as benzodiazepines, alcohol and/or 

cocaine. The last group, those where quetiapine clearly played no role in the cause of death, consisted of 5 

cases, with a mean quetiapine concentration of 0.88 mg/L and a range of 0.27 - 1.6 mg/L. This report 

provides evidence that quetiapine alone can result in death, and may significantly contribute to mortality in 

poly drug ingestions. Also, this data may assist medical examiners and toxicologists in separating lethal 

from non-lethal quetiapine concentrations in a variety of circumstances. 

Keywords: Quetiapine, postmortem toxicology, overdose 

Page 390

P56 

POSTMORTEM QUETIAPINE: THERAPEUTIC OR TOXIC CONCENTRATIONS 

Dawn R. Parker* and lain M. McIntyre. San Diego County Medical Examiner's Office, 5555 Overland 

Avenue, Bldg. # 14, San Diego, CA 92123. 

Currently, very little literature data are available on postmortem blood concentrations of quetiapine. A 

study was performed to establish relative guidelines for the delineation of therapeutic and potentially toxic 

concentrations. This study was conducted in medical examiner cases in which quetiapine was detected 

with routine screening methods. 

Quetiapine Fumarate (SEROQUEL®) is a dibenzothiazepine derivative (2-(2-(4-dibenzo [bj) 

[1 ,4]thiazepin-ll-yl-l-piperazinyl)ethoxy ]-ethanol fumarate) used in the treatment of psychosis. Fourteen 

fatalities in which quetiapine was detected were selected for investigation. 

Following liquid-liquid basic extraction, high performance liquid chromatography was used to analyze 

blood (peripheral and central), vitreous humor, and liver. The cases were organized into two groups: "nondrug 

related" fatalities and "drug related" fatalities. 

A total of eight "non-drug related" fatality cases were examined. Peripheral blood concentrations ranged 

from: 0.26 to 0.76 mg/L (mean +1- standard deviation 0.38 +1- 0.17 mg/L); central blood concentrations: 

0.23 to 0.73 mg/L (0.46 +1- 0.21 mg/L); vitreous humor concentrations: 0.08 to 0.32 mg/L (0.16 +/- 0.08 

mg/L); and liver concentrations:

PS7 

CAFFEINE INTOXICA nON: MORE THAN JUST CREAM AND SUGAR 

Sarah Kerrigan* and Tania Lindsey. 


Albuquerque, NM 87196-4700, 

Caffeine is a mild central nervous stimulant that occurs naturally in coffee beans, cocoa beans and tea 

leaves. In large doses, it can be profoundly toxic, resulting in arrhythmia, tachycardia, vomiting, 

convulsions, coma and death, The average cup of coffee or tea in the United States is reported to contain 

between 40 and 100 mg caffeine, Over-the-counter supplements that are used to combat fatigue typically 

contain 100 mg caffeine per tablet and doses of 32-200 mg are included in a variety of prescription drug 

mixtures. 

Caffeine was quantitatively determined in five cups of coffee purchased from local retail outlets. The total 

amount of caffeine per 160z (473mL) cup ranged from 211-342 mg, Fatal caffeine overdoses in adults are 

relatively rare and require the ingestion of a large quantity of the drug, typically in excess of 5g, Over a 

period of approximately 12 months our office reported two cases of fatal caffeine intoxication. The first 

fatality may have occurred due to misidentification of the drug, and the second occurred after misuse of a 

dietary supplement. 

Case #1 involved a 39 year-old female with a past history if intravenous drug use. She was found 

unresponsive outside a restaurant. A syringe was found near the body. Blood samples were negative for 

ethanol and common drugs of abuse. Comprehensive toxicology analysis using gas chromatography mass 

spectrometry (GC/MS) revealed a caffeine concentration of 192 mgIL in femoral blood. The cause of death 

was ruled as caffeine intoxication and the manner was accidental. 

Case #2 involved a 29 year-old male with a history of obesity and diabetes who was admitted to the 

hospital with vomiting and seizures. Resuscitation efforts were unsuccessful. The family reported that he 

had taken a dietary supplement the day before. Toxicology results indicated a caffeine concentration of 567 

mglL in the femoral blood. The cause of death was ruled as caffeine intoxication and the manner was 

accidental. 

Keywords: Caffeine, Intoxication, GCMS 

Page 392

P58 

LOW DOSE ACETAMINOPHEN DEATHS 

Olaf H. Drummer*, Victorian Institute of Forensic Medicine, Department of Forensic Medicine, Monash 

University, 57-83 Kavanagh St, Southbank 3006 AUSTRALIA, e-mail olaflal,yifin.org 

Acetaminophen (paracetamol) is a commonly used over the counter analgesic. It is well known that use of 

excessive doses can lead to the development of liver toxicity. This is mediated through the formation ofthe 

toxic metabolite N-acetyl benzoquinone imine. In adults single doses required to initiate liver necrosis is 

believed to start from 6 to 10 grams, which is about 12 to 20 500-mg tablets. Typically blood 

concentrations are well over 200 mgIL. This presentation describes acetaminophen toxicity in three women 

following doses within therapeutic guidelines. 

Case 1. The deceased was a 28 year old female of age with a history of an eating disorder, depression and 

alcoholism. While in hospital for an unrelated condition she became drowsy, weak, ataxic, and had slurred 

speech. She died some hours later. She had received about ten tablets over the previous 3-4 days. Her 

maximum acetaminophen concentration was 38 mgIL. 

Case 2. The deceased was a 21-year-old healthy woman. She developed a laceration after falling on glass. 

Three days later she presented to hospital with severe headache associated with vomiting and mild 

photophobia. A few days later she was weak, was vomiting and had a high heart rate. She was diagnosed 

with hepatic encephalopathy and her acetaminophen concentration was 46 mglL. She had taken about 10 

acetaminophen tablets over a few days. Other causes of liver necrosis were excluded by exhaustive 

toxicology, serology and virology testing. 

Case 3. This 45-year old woman had undergone surgery for an obstructed bowel. A few days later she was 

confused and agitated. This worsened until her death on day six. The day previously she had been 

diagnosed with liver failure. Her maximum acetaminophen concentration was 46 mgIL. She had been 

given one gram acetaminophen two to three times daily for several days prior to her death. 

All cases showed typical liver necrosis at post-mortem and had clinical changes consistent with 

acetaminophen toxicity. None of the cases had circumstances suggesting recent abuse of acetaminophen. 

The low acetaminophen concentration and absence of other causes of liver necrosis suggests that some 

persons may be sensitive to this drug, perhaps because of low glutathione concentrations due to diet or 

genetic factors and/or were genetically predisposed to the development of the toxic metabolite. A full 

account ofthe toxicology and post-mortem findings will be presented. 

Keywords: acetaminophen, low-dose, toxicity, post-mortem 

Page 393

P59 

INTERPRETING ANTIHISTAMINE LEVELS IN POST~MORTEM BLOOD; ESTABLISHING 

INCIDENTAL AND CONTRIBUTORY RANGES FOR EVALUATION PURPOSES 

Michelle Sandberg *, Dan Anderson, and Kristina Fritz 

Los Angeles County Department ofCoroner, Los Angeles, CA 

Over the last ten years, the Food and Drug Administration has approved numerous prescription 

antihistamines safe for over~the~counter (OTC) distribution. Antihistamines are a broad class of drugs, 

frequently taken to offer symptomatic relief from colds, sinus congestion, allergies, and are also a class of 

drugs frequently abused. 

In forensic toxicology, post~mortem (PM) redistribution of drugs complicates the interpretation of results. 

Many of the reference materials available are for clinical interpretation, and extrapolating PM levels for 

comparison to clinical values is not ideal. The substantial volume of casework at the Los Angeles County 

Department of Coroner (LACDOC) Laboratory provided us the ability to establish applicable PM blood 

level ranges for interpretation ofthe first generation antihistamines by a query of the LACDOC toxicology 

database over a three-year period. First generation antihistamines are easily detected using a basic liquidliquid 

extraction with an acidic back extraction. Diphenhydramine, chlorpheniramine, doxylamine, 

promethazine, and hydroxyzine are commonly detected at the LACDOC by GCINPD. 

A brief summary ofthe query results and interpretation are as follows: 

Categories 

Published 

Clinical 

Therapeutic 

Drug Levels 

Post Mortem Central Blood Levels, mgIL 

Diphenhydramine Doxylamine Promethazine Hydroxyzine Chlorpheniramine 

(471 cases) (87 cases) (136 cases) (44 cases) (126 cases) 

-0.11 -0.10 -0.08 -0.02 

- 0.14 

Determined 

om 0.70 0.01- 0.70 0.01- 0.30 0.01 0.50 om 0.30 

"Incidental" 

81% (383) 84% (73) 74% (101) 73% (32) 96% (I21) 

Drug Levels 

0.31 - 0.0.90 0.51-1.1 

Determined 

16% (21) 18% (8) 

"contributory" >0.70 > 0.70 

>0.30 

and 

and 

. to the cause of 17% (80) 15% (1) 4%(5) 

>0.90 >1.1 

death 

10%(14) 9%(4) 

Determined as 

the > 19 > l32 

"sole cause 2%(8) 1%(1) -- -- -- 

of death" 

The query results were categorized as follows: 

• Incidental - Levels that represent over 70% of the quantitated cases for a particular drug, and 

correspond to therapeutic dosing 

• Contributory - Levels that were greater than therapeutic and could not be ruled out as a 

contributing cause ofdeath 

• Sole Cause ofDeath - Due to the drug alone, not influenced by any other drug 

This study determined PM therapeutic central blood levels for five, first-generation antihistamines and 

central blood values that indicated the drug was a contributing factor in the cause of death. The Los 

Angeles County Coroner Laboratory continues to evaluate the cases where levels are greater than the 

"contributing" category and may have been the sole cause ofdeath, ifother weren't drugs present. 

Keywords: Antihistamines, Interpretation, Post-mortem blood levels 

Page 394

P60 

PHARMACOGENOMICS AS MOLECULAR AUTOPSY: GENOTYPING P450 2D6, 2C9 AND 

2Cl9 USING PYROSEQUENCING FOR METHADONE CASES 

B. Charles Schur 2, Elvan Laleli-Sahin 1.2, Steven H. Wong 1.2, Susan B. Gock 1.2, Paul J. Jannetto I, Jeffrey 

M. Jentzen 1.2 

IMilwauke County Medical Examiners Office; 2Department Of Pathology, Medical College Of Wi, 

Milwaukee, Wi, U.S.A. 

Pharmacogenomics, the study of the impact of heritable traits on pharmacology and toxicology, has the 

potential to explain the relationship between drug administration, drug metabolism and adverse drug 

reactions. Genetically, single nucleotide polymorph isms (SNP's) are single base-pair changes in the DNA 

sequence of a gene. Base deletions or substitutions may result in a change in the amino acid sequence of 

the polypeptide. These heritable mutations have the ability to change the structure and specificity of the 

enzyme to the extent that it may not metabolize the drug in question. Depending on the variant alleles 

present, an individuals' metabolic rate may be phenotypically described as poor (PM), intermediate (1M), 

extensive (EM, normal), or ultra-extensive (rapid). PM's have a high risk of adverse effects which can 

potentially be fatal, while 1M's have a metabolic rate between PM's and EM's. 

In Forensic Pathologyrroxicology, molecular techniques can determine the genotype of a decedent and 

may aide in the determination of the cause and manner of drug related deaths. Methadone, for example, is 

metabolized in the liver by cytochrome P450 (CYP) lA2, 3A4. 2D6 and, to a certain extent, 2C9 and 2CJ9 

enzymes. All of the genes encoding these enzymes are polymorphic. In this study we establish the 

technical feasibility of genotyping for known mutations in CYP2D6 (*3, *4, *5, *6, *7 and *8) as well as 

CYP2C9 (*2, *3) and CYP2C19 (*2, *3, *4) using Pyrosequencing. 

Twelve frozen, archived, blood samples from the Milwaukee County Medical Examiners Office 

(MCMEO), were selected as part of a multi-center retrospective analysis of cases from June 2002 to 

December 2002, which was approved by Medical College of Wisconsin IRE. These cases listed 

methadone as a contributing factor in the cause of death. After DNA extraction, PCR was performed and 

amplified product was then interrogated for the presence of specific SNP's using a Pyrosequencing 

PSQ96MA. Briefly, a sequencing primer is hybridized to a single stranded PCR product, and incubated 

with enzymes, DNA polymerase, ATP sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate (APS) 

and luciferin. A deoxynucleotide triphosphate (dNTP) is added to the reaction. If incorporated, a release 

of pyrophosphate occurs that is equimolar to the number of incorporated nucleotides. ATP sulfurylase 

quantitatively converts PPi to ATP in the presence of adenosine 5' phosphosulfate. This ATP converts 

luciferin to oxiuciferin and generates visible light detectable by a CCD camera and evident by a peak in 

the Pyrogram M. Each light signal is proportional to the number of nucleotides incorporated. Apyrase 

degrades the unincorporated dNTP's and excess A TP prior to the addition of another dNTP. Software 

correlates each Pyrogram to a reference sequence and genotype determinations are made. This platform 

has been previously validated for clinical samples, with results showing ] 00% concordance with other 

platforms including conventional PCR with RFLP analysis, direct sequencing using an ABI 3100, and 

rapid-cycle PCR with fluorescent melting curve analysis using a Roche LightCycier. 

Genotyping results for CYP2D6 indicated 8 cases (66.6%) of EM's, 3 cases (25%) of 1M's and ] case 

(8.3%) of a PM. For CYP2C19 SNP's, 8 samples (66.7%) were found to be EM's and 4 samples (33.3%) 

were found to be 1M's. No mutations (0%) were detected for CYP2C9 in any sample. These percentages 

are comparable to the expected prevalence ranges found in the general population. In this preliminary 

study, we demonstrate the feasibility of genotyping forensic samples for multiple SNP's associated with 

methadone metabolism. These protocols will be used in a large (n > 2000) multi-centers methadone study. 

Keywords: Pharmacogenomics, Methadone, Pyrosequencing 

Page 395

P61 

A MULTI-CENTER STUDY OF PHARMACOGENOMICS AS AN ADJUNCT OF MOLECULAR 

AUTOPSY FOR METHADONE DEATH CERTIFICATION - PRELIMINARY FINDINGS OF 

DATA ACQUISITION AND MULTIPLEX GENOTYPING CYP 450 2D6, 2C9 AND 2C19 BY 

PYROSEQUENCING 

Steve Wong*, Susan Gock, and Jeffrey Jentzen, Milwaukee Medical Examiner Office, 933 Highland Ave., 

Milwaukee, WI 53226 and the Forensic Pathologyrroxicology Pharmacogenomics Methadone Study 

Group (FPTPMSG 

Methadone is used in the treatment of heroin addiction and as an analgesic for pain management. Recently, 

there has been an increased incidence of methadone intoxication due to abuse and diversion. One of the 

contributing factors to drug therapy and toxicity is genetic variation. With the Human Genome Project near 

completion, genomic medicine based on genetic profiles has been proposed for personalized medicine for 

drug treatment and other therapeutic approaches. As an extension of genomics medicine in forensic 

science, a previous study examined the potential contribution of genetic variations in methadone death (J 

For Sci. 2003;48:1406-15). The allelic frequency of wild-type, defined for that study as non- CYP 2D6 *3, 

*4 and *5, was 71.4%, lower than that of living controls 82.7%. Due to the limited number of cases (n 

=21), the study was not adequately powered with sufficient cases to reach statistical significance. Studies 

for oxycodone and antidepressants also yielded a similar lower prevalence. Further, there is increasing 

understanding of methadone metabolism, readily characterized as mUlti-pathways with mUlti-enzymes 

(CYP 3A4, 2C9, 2C19, and 2D6). Some of the genes encoding these enzymes are polymorphic. Hence, a 

multi-center study was designed to enroll an adequate number of methadone cases to evaluate the potential 

contribution of genetic variation to methadone/related deaths Pharmacogenomics as an aspect of 

Molecular Autopsy. The study was initiated with several planning sessions to consider all the key covariables 

and risk factors. In the fall of 2003, the following medical examiner/coroner offices and personnel 

formed the Forensic Pathologyrroxicology Pharmacogenomics Methadone Study Group (FPTPMSG): 

Milwaukee - Wong, Jentzen, Gock, Jannetto, Schur, Sahin, Frolov, Rogalska. Cleveland/Cuyahoga 

Jenkins, Balraj, DetroitlWayne Hepler, Isenschmid, Schmidt, MiamilDade - Hearn, Uhlin-Hansen, New 

Hampshire - Wagner, Andrew, New Mexico - Kerrigan, North Carolina - Winecker, Ropera-MiIler, San 

Diego - McIntyre, San Francisco - Karch, Lemos, British Columbia/Canada- Langman, Washington, DC 

Couper, and Washington Logan, Gordon. In addition, drug-drug interaction interpretations will be 

performed by Moody (Univ. Utah) and secondary case reviews by Jortani (Univ. Louisville). The study 

period is 2002 and 2003, with some offices providing samples before and after for trending studies. The 

Medical College of Wisconsin (MCW) approved an " Umbrella IRB", with subsequent endorsement by the 

participating offices. Inclusion criteria are: cases certified with methadone toxicity, methadone related and 

methadone present. For the latter cases, each office would seek informed consent by decedent's family. 

Whole blood samples were mailed to the MeW Pharmacogenomics Lab for multiplex genotyping for CYP 

2D6 *3, *4, *5, *6, *7, *8., 2C9 *2 and *3., and 2C19*2,*3 and *4 by pyrosequencing (Clin Chern. 

2003;49:A] 2). By using a combination of Microsoft ACCESS and Excel database programs, case history 

and toxicology results were entered, followed by statistical analysis of transformed Excel data. At this 

preliminary stage, the study demonstrated the feasibility of coordinated planning and initial data entry and 

transfer to the MCW via Internet by 5 centers. The projected total caseload for FPTPMSG is more than 

1900. For a limited number of samples (n=22), the following variants were identified in 14 samples: 1 

homozygous for CYP 2D6*4, and the number for heterozygous variants 2 for 2D6*4 and 2C19*2., 1 for 

2D6*31*4., 2 for 2C9*2, 1 for 2C9*3, 4 for 2C19*2, Ifor 2D6*3, and 2 for 2D6*5. These preliminary 

results indicate that the multi-center study is proceeding on schedule, albeit with the expected degree of 

logistic and technical difficulty. 

Key words: methadone, pharmacogenomics, death 

Page 396

P62 

2,4-DINITROPHENOL (DNP): AN UNUSUAL FATALITY 

lain M. Mclntyre*, Ray D. Gary, James L. Sherrard, Dawn R. Parker, Julio Estrada and 

Robert E. Whitmore 

San Diego County Medical Examiners Office, 5555 Overland Ave., Bldg. 14, San Diego, CA 92123 

The decedent was a single 28-year-old male college student who resided with a roommate. His roommate 

found the decedent, with a fever, in a tub of ice. The roommate transported him to a local fire department, 

from where he was then transported to a local hospital. The decedent was conscious upon admission, but 

was soon pronounced dead despite resuscitative efforts. The decedent's roommate informed hospital staff 

that the decedent had been taking bodybuilding supplements. Bottles of medications and other materials 

found at the scene were brought to the Medical Examiner's Office. These included steroids, ephedrine, 

thyroid hormones, a yellow powder and a syringe. 

Autopsy showed a young muscular jaundiced Caucasian male with left ventricular hypertrophy of 1.6 cm, 

multiorgan congestion, and severe pulmonary edema. The liver was of a soft mottled purple and brown 

appearance. There was 150 mL of blood in the stomach, signs of hemorrhagic gastritis, and evidence of 

CPR (fractured sternum and anterior rib fractures). Autopsy also showed severe cerebral edema with uncal 

herniation and other evidence of medical therapy. Microscopically, the liver showed severe sinusoidal 

congestion and subtle centrilobular hepatocyte necrosis. No other significant macroscopic or microscopic 

findings were noted. 

Routine toxicological screens (alcohols, drugs of abuse, basic drugs, acidic drugs) were conducted on the 

antemortem specimens obtained from the admission. The only therapeutic drugs detected were 

diphenhydramine (0.13 mgIL) and lidocaine «0.05 mg/L). Specific analyses for steroids (postmortem 

urine) and ephedrine (antemortem blood) were negative. Analysis of the yellow powder by gas 

chromatography/mass spectrometry identified the powder as 2,4-dinitrophenol (DNP). Subsequent analysis 

of the postmortem urine (Toxi-Lab, confirmed by gas chromatography/mass spectrometry), then detected 

the presence of DNP. The acid drug screen (HPLe) also detected an unknown compound in the 

antemortem blood that was later identified as DNP. DNP was then quantitated by HPLC in the antemortem 

blood and in the following postmortem specimens: blood (peripheral), serum (peripheral), liver, urine, bile 

and gastric contents. 

DNP is primarily used commercially in the production of dyes, wood preservatives, photographic 

developers, explosives and pesticides. In the 1930's DNP was used extensively as a diet pill. DNP 

effectively increases the basal metabolic rate by inhibiting the synthesis of A TP in the mitochondria. This 

action also results in increased sweating, breathing rate, heart rate, weight loss and a marked increase in 

body temperature. In 1938 the U.S. Food and Drug Administration banned the use of DNP due to its 

harmful effects, which included cataracts, liver damage, and in some cases death. 

This is the first report of a fatality associated solely with DNP that describes the detection ofthe compound 

in antemortem blood and postmortem tissue concentration distribution. 

Keywords: 2,4-Dinitrophenol, postmortem, fatality 

Page 397

P63 

GHB CONCENTRATIONS IN A V ARlETY OF ALCOHOLIC AND NONALCOHOLIC 

BEVERAGES AND LIQUID FOOD PRODUCTS 

Ines B. Collison"', Kristin Houg, Debra Eck, Robin Kott. Forensic Science Services, Orange County 

Sheriff/Coroner Department, 320 North Flower St. Santa Ana CA 92703 

Casework indicated the presence of small amounts of GHB in samples of Cabemet Sauvignon and Steel 

Reserve beer, which according to previous reports could be expected, at least in wine. Due to these results a 

variety of samples of different alcoholic, nonalcoholic beverages, and fermented liquid food products were 

analyzed for the presence of GHB following the method previously described by Couper and Logan* using 

smaller volumes of sample (100-200 mcL). The limit of detection was 0.25 mgIL. A total of 108 samples 

were tested in duplicate. 

A number of the samples analyzed were positive for GHB although at very low concentrations. In general, 

the concentration ofGHB was higher in red wines (2.00-23.00mglL; n=l1) than in white wines (0.65-9.53 

mglL; n=l3). Of all the different kinds of vinegar tested (n=5) only white vinegar was negative for GHB 

while rice wine, red wine, balsamic and apple cider vinegar were positive (0.83-11.25 mgIL). The GHB 

concentration in beer «025-2.10 mgIL; n=29) was lower than it was in wine. For the different kinds and 

brands of beer tested, there appears to be a correlation between the GHB concentration and the ethanol 

content rather than with the type of beer analyzed. Although the sample size was small (n=3), the home 

brewed beers which typically have a higher alcoholic content also had the higher GHB concentration (1.71 

2.1 0 mg/L). The GHB concentration was either not detected or < 0.50 mgIL in the distilled alcohol samples 

tested (n=23). The GHB concentration in the liqueurs tested varied from not detected to 4.20 mgIL (n=II). 

A variety of other non distilled alcoholic drinks, such as sherry, vermouth, and champagne, showed 

intermediate concentrations of GHB (1.88-6.68 mglL; n=5). Neither of the two malt beverages contained 

GHB above our cutoff. GHB was detected in grape juice (n=2) but not in apple juice. GHB was in noni 

juice (Morinda citrifolia, (0.73 mglL)·, but not in the tea from the noni leaves. NoGHB was detected in the 

teriyaki sauce tested (n=I). Thethree different kinds of soy sauce tested were positive for GHB (2.79-18.10 

mglL). 

We conclude that although some of these products contain GHB,. a minimum of 210 L of these liquids 

would need to be consumed in order to ingest a 5 g dose of GHB. 

Couper and Logan *(2000) JAnalTox 24: 1-7 

Key Words: GHB, alcoholic beverages, food products. 

Page 398

P64 

THE USE OF CLUSTER ANALYSIS TO ESTABLISH A REFERENCE RANGE FOR BETA 

HYDROXYBUTYRATE CONCENTRATIONS IN POSTMORTEM BLOOD AND ITS 

APPLICATION TO THE INVESTIGATION OF SUDDEN UNEXPECTED DEATH IN PROBLEM 

DRINKERS 

A Robert W Forrest* & Daniel Cooper. Forensic Pathology Unit, University of Sheffield, Medico-legal 

Centre, Watery Street, Sheffield S3 7ES, UK 

Alcoholic ketoacidosis is a relatively common cause of death in problem drinkers. (l) It should be 

considered in every case of sudden unexpected death in a problem drinker, particularly if hepatic steatosis 

(a fatty liver) is present and "routine" toxicology screening is unrevealing. An important clue may be a 

trace of acetone apparent on the ethanol assay chromatogram in association with a low or zero alcohol 

concentration. If the toxicologist or pathologist have any degree of suspicion that the death might be 

associated with alcoholic ketoacidosis, then beta-hydroxybutyrate should be measured in post mortem 

blood. 

Whilst the diagnosis of alcoholic ketoacidosis is straightforward when the beta-hydroxy butyrate 

concentration is grossly elevated in post mortem blood, the interpretation of the borderline result may be 

difficult. In life, clinical laboratories typically quote the upper limit of normal for the fasting plasma betahydroxybutyrate 

concentration as from 300 to 600 micromoles per litre. Unfortunately, whilst in principle it 

should be a simple matter to establish a reference range for the beta-hydroxy butyrate concentration in postmortem 

blood, in practice, for those working in England and Wales, this is fraught with difficulty. Most 

post-mortem examinations are non-consensual, carried out under the direction of the coroner. In the 

analysis of samples collected during such post-mortems, it is only permitted to carry out analyses directed 

at establishing who the deceased was and where and when he came to his death without the specific 

permission of the coroner and the relatives of the deceased. Whilst a "black letter" interpretation of the 

present law is arguably more liberal, those who have retained specimens obtained at a coroner's postmortem 

for research or teaching have been subject to considerable public criticism in the UK. (2). The 

practicalities of obtaining permission from the relatives of the deceased who is about to be subject to a 

coronial post mortem examination are daunting. If legislation currently before the UK Parliament becomes 

law in its present form, then the researcher who analyses a post mortem blood sample without permission 

will be liable to prosecution with a possible penalty of up to 3 years in prison on conviction. (3) 

Consequently it is necessary to make the best possible use of the data obtained when investigating any 

particular case to inform the interpretation of future cases. One approach that we have found useful is to use 

cluster analysis of all of the data we have obtained in the investigation of possible cases ofalcoholic ketosis 

to delineate normal, equivocal and elevated beta-hydroxybutyrate concentrations in post-mortem blood. We 

further believe that the technique may be of more general application in the analysis of post-mortem 

toxicology data. To illustrate the technique we present the analysis of our data in relation to the 

investigation of alcoholic ketoacidosis and, more briefly, possible overdoses of diphenhydramine and 

tramadol. 

References: 

I. Pounder DJ, Stevenson RJ, Taylor KK. Alcoholic ketoacidosis at autopsy. Journal of Forensic 

Sciences 1998;43(4):812-6. 

2. Metters J. Isaacs Report. London; 2003. http://www.publications.doh.gov.uklcmolisaacsreport/ 

3. Human Tissue Bill. 

2003/4.http://www.publications.parliament.uklpalcm200304/cmbills/049!2004049.htm 

Keywords: alcoholic ketoacidosis, beta-hydroxy butyrate, cluster analysis 

Page 399

P65 

POSTMORTEM BLOOD ALCOHOL RELIABILITY: FEMORAL SAMPLING 

A.E. Hodda*, Forensic Toxicology, DALIICPMR, Lidcombe, Australia 

P. Ellis, Dept. Forensic Medicine, ICPMR, Westrnead, Australia 

The analysis of blood for alcohol is one of the more fundamental analysis undertaken by a forensic 

toxicology laboratory. The sources of error in the laboratory are well documented and international 

standards of laboratory practice (AS/ISO 17025) have been developed to safeguard the analytical 

processes, for any analysis. Discrepancies in the alcohol levels in post mortem specimens from a recent 

. vehicle accident death resulted in a detailed review of all aspects of the post mortem process. In this case 

two femoral blood samples gave alcohol levels of 0.3 IOg/IOOmL but a vitreous humor sample had no 

detectable alcohol. 

A number of papers have been published on the importance of the source of blood samples used in 

quantitative alcohol and drug analysis. Errors due to contamination, dilution and "post mortem 

redistribution" are cited. The conclusion is that peripheral samples should be used and best source is the 

femoral vessels. The recommended procedure for post mortem femoral blood sampling is reviewed. The 

sampling point is normally within the pelvic girdle and it is clear that poor adherence to correct procedures 

can result in adventitious contamination or a non-representative blood sample. Traumatic deaths present the 

greatest risk of contamination. The Laboratory receives approximately 3000 toxicology cases per year and 

a review of 2 years showed the median volume of preserved blood submitted from coronial cases was 

20mL. 

Mathematical modelling demonstrated that 200j.IL (4 drops) of an alcoholic spirit in the pelvic cavity could 

result in blood alcohol levels of 0.300g/l OOmL, if allowed to contaminate a 20mL blood sample. Several 

tables are presented of various collected blood volumes and potential contamination factors. 

The reliability and predictability of comparing blood and vitreous humor alcohol .levels was also tested. A 

set of33 blood alcohol levels greater than 0.200g/100mL were compared to their respective vitreous humor 

alcohol levels. The regression equation was y=1.2734x-O.0451, the R2 was 0.9488. This result was similar 

to other reports and demonstrated the value of this comparison as well as the improbable results for the 

reviewed case. 

The review did not categorically identify the source of error in this case but the work has highlighted the 

ease of introducing significant errors in blood alcohol analysis. It has also supported the strong correlation 

between blood alcohol and vitreous humor. This observation makes this specimen particularly useful in 

contentious toxicology cases and should be taken in matters involving severe trauma. The growing 

convention in forensic toxicology is to use femoral blood samples for quantitative analysis, is endorsed but 

correct sampling procedures must be followed by pathologists. 

Keywords: Blood alcohol, error, femoral, vitreous humor. 

Page 400

SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

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