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  37th TIAFT Triennial Meeting Cracow

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Opening Lectures

Interpretation of Analytical Results

Drugs of Abuse and Doping

Validation of Analytical Procedures

Analysis of Alternative Materials

Driving under the Influence

Free Topics



Surface Ionization Mass Spectrometry. Selective Determination of Trace Amounts of Opioids in Urine

U. Kh. Rasulev1, Usman Khasanov1, Turgun Kh. Islamov2, Makhamudjan M. Shakhitov3, B. Eshmuratov3
1 Arifov Institute of Electronics, Tashkent, Uzbekistan
2 Republican Criminal Research Center, Tashkent, Uzbekistan
3 Republican Bureau of Forensic Medicine Expertise, Tashkent, Uzbekistan

The volume of illegal turn-over of opioid narcotics, especially heroin, is sharply increasing in Central Asia, where Uzbekistan is used as a transit country. Recently, the use of narcotics has become more frequent. The situation in Uzbekistan strongly requires the development of rapid and sensitive methods of narcotics detection and identification in biological samples.

It is known that, owing to the rapid metabolism of opioids in the body, the establishment of the primary drug is a rather difficult problem in analytical toxicology.

In this paper, for the first time, the results of surface ionization mass spectrometry (SI/MS) of urine samples of the users of poppy juice, poppy "tea", heroin and others opioids are reported.

Urine samples were hydrolyzed with sodium bisulfate and concentrated HCl. After heating and cooling, the 50% solution of trichloracetic acid was added up to its concentration of 7%, which resulted in the precipitation of protein substances. The solution was neutralized with ammonia up to pH 6.0-7.0, saturated with sodium bicarbonate and extracted with a mixture of butanol-chloroform (1:9). After filtration and evaporation the residue was dissolved in chloroform and examined by SI/MS, TLC and GC/MS, the latter with the HP-5890 device.

The SI mass-spectra of the samples were compared with the mass spectra of CAS preparations of opioids. It was found that in the mass spectrum there were base fragments of opioids with m/z=144; 146 a.m.u. and no quasimolecular ions of CAS preparations. However, in the mass spectrum there were quasimolecular ions of metabolites: for heroin there were acetylmorphine, acetylcodeine, norcodeine, normorphine, for poppy juice and poppy "tea" normorphine, papaverine, noscapine and their metabolites.

High selectivity and few lines of SI mass spectra made it possible to identify, with high accuracy, the substance used with a sensitivity exceeding that of the well-known methods of analysis and without chromatographic separation of samples.



Diagnosis of Illicit Drug Overdoses: Difficulties Found

Estela G. P. Marques, Alice M. Castanheira, Paula Proenca, Paula V. Monsanto, Sofia A. Oliveira, Sofia Avila, Duarte N. Vieira
Instituto de Medicina Legal de Coimbra, Largo da Se Nova, 3000-213 Coimbra, Portugal

Drug abuse is becoming a national problem in Portugal. Heroin is the drug most often used, followed by cocaine. This work describes a retrospective study of the 1990s in relation to the deaths of individuals known to be drug addicts. The study was carried out for the central zone, which represents approximately one third of the country. The number of overdoses is much lower than the country average, maybe because this zone is largely rural. Overdose was diagnosed only when the concentration of free morphine in the blood was 0.2 µg/ml or higher, and when no other injuries were found on the body which could be considered as the cause of death. This cut-off was considered in our laboratory as a reference of positive overdose. In the 1990s overdose concentrations ranged from 0.2 µg/ml to 1.5 µg/ml. Morphine was taken as the criterion since in almost 100% of "hard drugs" cases heroin was involved. Only in 17.3% of cases was this in association with cocaine. Cocaine concentrations ranged from 0.13 µg/ml to 2.0 µg/ml in blood, benzoylecgonine (BE) ranged from 0.14 µg/ml to 25.0 µg/ml and ecgonine methyl ester (EME) was always detected. Four cases were attributed to cocaine, one being attributed to the male cocaine "boby packer". This last case presented 16.5 µg/ml for cocaine, 11.6 µg/ml and 1.8 µg/ml for BE and EME respectively, in blood. The cut-off values for cocaine and BE were 0.10 µg/ml. Cases of death by accident where morphine was present are described, but they were not considered as overdoses. Victims who exhibited no other cause of death, but whose morphine blood levels were lower than mentioned cut-off values, were held to have died from drug-related causes. According to the criteria, many of the drug-related deaths could be caused by drugs, since there were many cases of association with alcohol and other psychotropic substances. The findings in blood, urine and/or tissue samples have considerable implications for the interpretation of the test results of drugs of abuse to help determine if there has been absorption of illicit drugs.

This study shows that it is in general almost impossible to make a toxicological evaluation by determining free morphine blood concentrations alone. Other analytical data and additional information, obtained by e.g. hydrolysis of the samples, are important aids in the diagnosis. This allows us to know approximately a recent absorption if the concentration of free-morphine in blood is higher than the concentration of morphine glucuronides. The 6-MAM detection is also known as a heroin biomarker. Analyses of the tissues samples, especially liver and kidney, could be another complementary way to aid in the diagnosis. Some information about the victims, such as whether or not they were drug addicts, the existence of a prick of a needle, the presence of a syringe affixed or alongside the body, a spoon, aluminium foils or a lemon near the body and so on, could also contribute to the diagnosis.

Keywords: Overdose, Hard Drugs, Interpretation of Analytical Results.



Heroin Cases in Routine Forensic Toxicological Examination of Biological Fluids

Aniko Kovacs, Zsuzsanna Miko Hideg and Andras Benko*
National Institute of Forensic Toxicology, Budapest, Hungary

In the first half of 1999, the National Institute of Forensic Toxicology (NIFT) received 856 requests from police investigators to analyse blood and urine samples focusing on illicit drugs. The screen used during standard investigations is usually the FPIA (Axsym, Abbott ). 64 positive opiate cases were detected among them. The confirmation and quantitative analysis were developed by Remedi (BIO-RAD), DAD- HPLC (LC-10, Shimadzu) and GC/MS (QP-5000, Shimadzu). 6-MAM, morphine and codeine were usually detected. However, in a number of cases, some different illicit drugs (amphetamines or THC), were revealed as well.

The conditions and sensitivity of the analytical methods, the concentrations of illicit drugs and their metabolites in the blood and urine samples, and the statistical data will be presented on the poster.

According to our statistical data, covering the last 10 years, narcotic drug consumption has been increasing rapidly in Hungary, because our country is a well-known transit area for the drug smuggling "Balkan Route". Observations of trends over the last few years have shown that Hungarian consumers are gradually turning from soft drugs (marihuana, hashish, ecstasy or speed) to hard narcotics, mainly to heroin. The consumption of cocaine is not significant.

Keywords: Heroin addicts, Complex Forensic Toxicological Analysis, Statistical view.



The Role of Ethanol in Cocaine Concentration in Human Post Mortem Whole Blood

Alice A. da Matta Chasin*1, and A. F. Midio2
1 Medical Legal Institute of Sao Paulo, Brazil 2 Department of Toxicology, University of Sao Paulo, FCF-USP, Av. Professor Lineu Prestes, 580 Sao Paulo, SP, Brazil cep 05508-900

Regardless of its form, the co-ingestion of cocaine (COC) and ethanol is still a very important problem in drug abuse. The formation of the transesterification by-product, cocaethylene (CE), which has lower LD50, has led to the idea that the association of the two drugs is more toxic (lethal) than just a cocaine response by itself and that smaller concentrations of this drug would be present in those related cocaine deaths.

The aim of this study was to evaluate the role of ethanol as an agent of interaction in lethal intoxication and to establish the influence of CE on post mortem whole blood cocaine concentration. Thirty six post mortem cases in which cocaine was the only cause of death were compared to eighteen cases of cocaine/ethanol interaction in terms of COC, benzoylecgonine (BE) and ecgonine methylester (EME) concentrations. Cocaine and cocaethylene, BE, EME concentrations correlated positively, but CE concentrations did not correlate with blood ethanol. When each of the metabolites and the precursor were analyzed by ANOVA, no differences between groups were found. The ratio of BE and COC concentrations (BE/COC) for cocaine individually was statistically greater than BE/COC in the interaction with ethanol. On the other hand, statistics with MANOVA (Wilks l test, a=0,05) showed that there is a significant difference between the two groups when COC and its main products of biotransformation, BE and EME, were focused on variables.

Keywords: Cocaine Concentrations, Cocaethylene, Ethanol Interaction, Metabolites.



Benzodiazepine Findings in Blood and Urine by Gas Chromatography and Immunoassay

Ilpo Rasanen*, Ilkka Ojanpera and Erkki Vuori
Department of Forensic Medicine, P.O. Box 40, FIN-00014 University of Helsinki, Finland

The presence of benzodiazepines is usually tested by screening urine samples by immunochemical techniques and confirming positive findings by GC, HPLC or GC/MS. On many occasions it is necessary also to perform a quantitative benzodiazepine determination in the blood. This study compares GC and immunoassay techniques and blood and urine specimens for benzodiazepine screening in post mortem forensic toxicology.

For the present study, 506 such successive medical examiner's cases were selected where both urine and blood were available. Urine specimens were first analysed by Emit d.a.u. Benzodiazepine Assay (ETSPLUS) using a 200 ng cutoff-value. Blood and enzyme-hydrolysed urine samples (1 ml) were screened for benzodiazepine drugs or metabolites by a dual-column gas chromatographic method using DB-5 and DB-17 capillary columns, EC detectors and advanced software for the interpretation of the results. The GC method allows simultaneous quantitation of the drugs. The urine samples which were negative or invalid by immunoassay but positive by GC were also analysed by ETS after enzyme hydrolysis.

The results are summarised in the following table:

  A B C D E F G H I J K Total positive
Urine (ETS) - + - - + - + + Invalid* Invalid Invalid 153/175**
Urine (GC) - + + + + - - - - + + 200
Blood (GC) - + + - - + - + - - + 185
                        Total no. of cases
No. of cases 283 145 31 16 4 4 2 2 16 1 2 506

- negative for benzodiazepines, + positive for benzodiazepines, *no result obtained by ETS

** after enzyme hydrolysis of urine samples in groups C, D, J and K

The results suggest that benzodiazepines can be detected better by GC in urine (200 positive cases) or blood (185 cases) than by immunoassay in urine (153 cases). The same applies although an enzymic hydrolysis is carried out prior to immunoassay (175 cases).

Keywords: Benzodiazepines, Immunoassay, Gas Chromatography.



Parathion and Acute Intoxication. The Importance of Toxicological Analytic Tests

Estela G. P. Marques*, M. M. Oliveira, Paula V. Monsanto, Paula Proenca, Fernando Castanheira, Duarte N. Vieira
Instituto de Medicina Legal de Coimbra, Largo da Se Nova. 3000-213 Coimbra, Portugal

Parathion is an organophosphate insecticide responsible for a large number of cases of acute poisoning, both fatal and clinical, in central Portugal. This work describes the distribution of this insecticide in blood and various other organs, according to the etiology of the intoxication. As a rule, concentration levels are small, especially in the blood. The partition coefficients relative to the different specimens are given. Concentrations in the stomach are higher, particularly in cases of suicide. Analysis of the small intestine may be important in cases where a certain length of time has elapsed between ingestion of the compound and death. Normally, parathion levels are very small in cases where death has already occurred, except in the stomach, where levels are very high. In these cases, death may have occurred immediately after ingesting the insecticide, which would agree with our findings in our experimental study on rabbits. When results from analysis of the stomach and gastric contents were low, the maximum residual limits established for foods were borne in mind. In studies carried out on some patients admitted to hospital with parathion poisoning, entero-hepatic re-circulation could have been a factor. Eight days after treatment, tests yielded negative results. We would stress the importance of all information (clinical, circumstantial and any other) in the interpretation of the results so as to arrive at a safe diagnosis.

Keywords: Parathion, Organophosphate Insecticide, Interpretation of Analytical results.



Distribution of Hydrogen Sulphide in Rats' Organs and Associated Histological Changes in Experimental Intoxication

Roman Wachowiak1, J. Miekowiak2, Jaroslaw Tobolski 1
1 Dept. of Forensic Medicine, University of Medical Science, Poznan, Poland
2 Dept. of Histology and Embryology University of Medical Science, Poznan, Poland

Chemistry-based diagnosis in some cases of sudden death due to unnatural causes, in cases of anoxemic death (e.g. in sewage collecting reservoirs), requires the ability to estimate hydrogen sulphide levels in autopsy material. The physicochemical properties of hydrogen sulphide (its volatile nature, rapid transformation to inactive sulphates and potential for endogenous formation) should determine the selection of appropriate autopsy material and the optimum timing for performing reliable estimations. In the present study analytical techniques which identify and determine the levels of hydrogen sulphide in biological material were evaluated.

A spectrophotometric method (l = 670 nm) was used, taking advantage of the selective colour reaction of hydrogen sulphide with N,N-dimethyl-p-phenyl-diamine and also gas chromatography (headspace technique), using a thermal conductivity detector (TCD). Retention parameters were established for hydrogen sulphide as related to other volatile toxins (CO, HCN, SO2) using various column fillings (Porapaq Q or Hay Sep Q 80/100 mesh). Statistical analysis of the results, as well as the ranges of calibration, suggest that the spectrophotometric technique is better than gas chromatography. Hydrogen sulphide determinations in biological samples taken from rats lethally intoxicated with gaseous hydrogen sulphide, permitted the selection of the most suitable autopsy material needed for routine toxicological analysis. The results indicated that mean hydrogen sulphide levels in individual tissues (in mg/g) were as follows: blood - below 1, lungs - 1.65, muscle - 3.57, brain - 5.45, liver - 7.38 and kidney - 11.22. Some histological changes (numerous erythrocytes) were observed in the kidneys and lungs of the exposed rats as compared to the controls.



Pyrolysis of Cocaine to Methyl Ecgonidine and Subsequent Metabolic and Hydrolytic Transformation to Ecgonidine

Buddha D. Paul
Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, Armed Forces, Institute of Pathology, Rockville, MD 20850, USA

Methyl ecgonidine is a pyrolytic product of smoking free-base cocaine. Methyl ecgonidine and unconverted cocaine were measured in smoke using an experimental condition similar to that used by "crack" smokers, yielding relative results of 4.4 and 94%, respectively. Randomly selected human urine specimens previously reported to be positive for benzoylecgonine (>100 ng/ml) were tested for methyl ecgonidine. The compound was detected in 17 of 23 specimens. It was postulated that methyl ecgonidine could not be detected in the remaining 6 specimens because the compound was completely metabolized or spontaneously hydrolyzed to ecgonidine. When the urine specimens were tested for ecgonidine, the compound was detected in 22 of 23 specimens indicating metabolic or hydrolytic conversion and that smoking was the major route of cocaine administration. Concentrations of ecgonidine and methyl ecgonidine had a strong correlation using regression analysis (r2 = 0.8662).

Ecgonidine was detectable in some specimens (intercept=174 ng/ml) when the methyl ecgonidine concentrations were below the limit of detection of the procedure (0.7 ng/ml). To demonstrate metabolic conversion, methyl ecgonidine was incubated with human liver homogenate. The metabolic product was ecgonidine. Rate of metabolism was 2.2 mole percent/h/g of liver. Cocaine, benzoylecgonine and ecgonine under the same metabolic conditions did not produce any methyl ecgonidine or ecgonidine. The results suggest that methyl ecgonidine and its metabolic product, ecgonidine, are unique to smoking cocaine. Methyl ecgonidine at urine pH <6.5 was stable at room temperature for at least 24 h. Substantial hydrolysis was observed at urine pH >8.5 (10-23%).

Metabolic and hydrolytic transformation of methyl ecgonidine to ecgonidine suggests that ecgonidine is a better marker for identifying ingestion of cocaine by smoking.



Comparison of GC-MS and LC-MS Methods for Quantification of Morphine and its 3- and 6- Glucuronides, Codeine, Codeine-glucuronide and 6-Monoacetylmorphine in Human Serum for Routine Analysis in Forensic Toxicology

Laurent Rivier*, Agnes Dienes and Patrice Mangin
Institut Universitaire de Medecine Legale, Rue du Bugnon 21, Lausanne, Switzerland

Simultaneous determination of opiates and their glucuronides in body fluids is of great practical interest in the forensic assessment of heroin intoxication. Two methods for quantification of morphine and its 3- and 6-glucuronides, codeine, codeine-glucuronide and 6-monoacetylmorphine (6-MAM) based on GC MS and liquid chromatography atmospheric pressure turboionspray ionisation mass spectrometry (LC-ESI-MS) are compared. The drugs were analysed in human serum and autopsy blood after a solid-phase extraction on a C8 cartridge after a hydrolysis step whenever necessary. The separations were performed on an Ultra-2 GC capillary column or a new ODS column so that the LC-MS analysis time did not exceed 15 min. For the quantitative analysis, deuterated analogues of each compound were used as internal standard (except for 6-MAM). Selected-ion monitoring was applied for quantification. Data obtained from real case analyses indicate excellent correlation between the two methods. Clearly, the LC-MS approach offers simpler handling of the samples and two times shorter overall analytical time. Advantages of LC-MS in obtaining complete and accurate figures for all opiate metabolites in a single run are obvious. Interpretations could thus be based on more precise results than those obtained by GC-MS and new metabolic markers for opiates tolerance and addiction may emerge from further refined knowledge on such metabolic profiles.

Keywords: Opiate Conjugates, Blood, LC-MS.



Detection Times for Opiates in Urine after Various Doses of Heroin Using Different Screening Methods

Michael L. Smith*1, Buddha D. Paul1, Jacqueline Summers1, Karla A. Moore1, Amanda Jenkins2, William D. Darwin2, Edward J. Cone2, Dan Nichols3, and Edwin K. Armitage3
1Division of Forensic Toxicology, Armed Forces Institute of Pathology, Washington, DC, USA 2Addiction Research Center, IRP, NIDA, NIH, Baltimore, MD, USA 3US Army Forensic Toxicology Drug Testing Laboratory, Fort Meade, MD, USA

In October 1998, the USA Federal Workplace Drug Testing Program changed urine screening and confirmation cutoff values for opiate testing from 300 to 2000 ng/ml using morphine as the target compound. A 6-acetylmorphine (6AM) concentration >10 ng/ml was required to prove heroin use. To assess the change in detection times of opiates in urine, 12 male human subjects, following informed consent, were given heroin on a medically supervised, closed clinical ward. Nine subjects took an oral dose of 10 mg heroin HCl; eight subjects received intravenous (IV) doses of 3, 6, and 12 mg heroin HCl; and four subjects smoked (SM) 3.5-13.9 mg doses of heroin. Urine specimens were collected, frozen, thawed and analyzed by the opiate immunoassays by Behring Emit II®300/2000, Roche ONLINE® (OL) 300/2000, Microgenics CEDIA®300 and Abbott AxSYM® (Ax) 300. Total morphine (Tmor) and 6AM concentrations were measured by GCMS to confirm the screening results. Screening detection times of the last positive specimen and confirmation rates are shown in the table.

Route/Dose Emit 300 OL 300 CEDIA 300 Ax 300 Emit 2000 OL 2000
SM/3-7 mg 7-28 hr 7-17 hr 8-28 hr 7-13 hr 2-5 hr 0-2 hr
SM/ 10-14 10-54 10-48 11-54 11-41 0-22 0-11
IV/ 3,6 11-37 10-37 11-49 2-29 2-10 0-6
IV/ 12 20-54 20-54 20-57 16-46 8-13 3-11
Oral 10 10-58 10-58 14-58 11-47 2-9 2-9
Confirmation rate (%)
Tmor>cutoff 98.1 99.1 87.4 99.6 96.5 98.2
6AM>10 ng/ml 12.3 10.9 10.5 14.6 44.2 50.0

The greatest number of screened positive samples were produced by CEDIA 300 > Emit 300 > OL 300 > Ax 300> Emit 2000 > OL 2000. By changing to higher cutoffs, the length of time one could detect a single heroin use was reduced by approximately one day but heroin confirmation rates (based on 6AM confirmation) of screened positive samples increased by over 30%.

Keywords: Heroin, Immunoassays, Detection Time.



Determination of Various Opiates Including Hydrocodone and Hydromorphone in Biosamples by GC-MS after Silylation

Marie Balikova*, Vera Maresova
Institute for Toxicology and Forensic Chemistry, Charles University, 1st Medical Faculty and Hospital, 121 08 Prague 2, Czech Republic

A suitable method for studying metabolism and disposition and for conducting practical toxicological analyses of unspecified opiates in various biosamples is presented. The universal GC-MS in EI mode method can be convenient for trace analyses of these compounds and their metabolites with the assumption that derivatization is included in the sample preparation procedure. Silylation can be a useful reaction for derivatization of various groups in unknown drugs in toxicological samples and is recommended for efficient GC separation of some hydroxy-stereoisomers originating during metabolism. However, silylation of drugs with tautomeric keto/enol groups in molecular structure as in hydrocodone or hydromorphone can be rather difficult and cause analytical problems due to poor reproducibility of the reaction yield. After using N-Methyl-N-trimethylsilyltrifluoracetamide (MSTFA) no underivatized free ketoforms of hydrocodone or hydromorphone were detected. Validations of the assays of various 4.5-epoxymorphinans in serum and urine based on SPE are compared in our contribution.

Keywords: Hydrocodone, Hydromorphone, Silyl Derivitization, GC-MS Toxicological Analyses.



Analysis of Underivatized Amphetamines and Related Phenethylamines with High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectrometry

Maciej J. Bogusz*, Klaus D. Kruger and Rolf-D. Maier
Institute of Forensic Medicine, Aachen University of Technology, Germany

Amphetamine, methamphetamine, illicit designer phenethylamines (MDA, MDE, MDMA, MBDB, BDMPEA), as well as other phenethylamines (benzyl-1-phenylethylamine, cathinone, ephedrine, fenfluramine, norfenfluramine, phentermine, 1-phenylethylamine, phenylpropanolamine, propylhexedrine) were extracted from serum using a solid phase extraction procedure. The extracts were examined with high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS).

The drugs were separated on an ODS column in acetonitrile-50 mM ammonium formate buffer, pH 3.0 (25:75) as a mobile phase. Full scan mass spectra of drugs examined by means of APCI with collision induced dissociation showed protonated molecular ions and fragments typical for particular drugs.

LC-APCI-MS allowed an unequivocal differentiation of all drugs involved. The quantitation was performed using selected ion monitoring of protonated molecular ions and fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 1 to 5 mg/l serum, the recovery ranged from 58 to 96%. A linear response was observed for all drugs in the range from 5 to 500 mg/l.

The method was applied for routine determination of amphetamine, MDMA, MDA and MDE in one run. Solid phase extraction used assured simultaneous isolation of various groups of basic drugs of forensic interest (opiates, cocaines, phenethylamines, benzodiazepines) from biofluids.

Keywords: Amphetamines, Ecstasy, APCI-LC-MS.



Clean-up Process for Mass Spectral Study of Amphetamines in Putrefied Body Materials

Kenji Hara*, Seiichi Kashimura, Masayuki Kashiwagi, Tomoko Hamanaka and Mitsuyoshi Kageura
Department of Forensic Medicine, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan

In forensic cases, clear evidence such as indicating an intact mass spectrum is required for elucidating criminal facts. When specimens are decayed, some interfering objects occur in various circumstances. In our poisoning case we were also required to demonstrate a trace level metabolite of methamphetamine in a cadaver found about 40 days after death. In this screening test by using a simple extractive heptafluoro-n-butyrylation, methamphetamine was indicated as an intact mass spectrum in each body material, but its main metabolite amphetamine was observed as an unclear mass spectrum. For this reason, we attempted to clean-up the specimens before this simple extractive derivatization.

Tissue specimen was homogenized with 0.1M phosphate buffer (pH 6.0), and the homogenate was centrifuged at 15000 rpm for 30 min. After adjustment of pH 12.6, a portion of supernatant was applied to an Extrelut column. After 15 min equilibration, the amines were extracted by passing diethyl ether through the column. The ether was back-extracted with 0.01M HCl, then the aqueous solution was washed with ether. The aqueous solution at pH 12.6 was again applied to an Extrelut column. After equilibration, the analytes were derivatized and extracted by passing n-hexane including heptafluoro-n-butyryl chloride through the column. The extracted solution was condensed under a nitrogen stream at room temperature. The condensed solution was diluted with ethyl acetate, then the objects were analyzed.

By this clean-up, most amino acids, polyamines and organic acids occurring in putrefactive procedure were eliminated. As a result, an intact mass spectrum of amphetamine derivative from each specimen was recorded. However, the apparent recovery of another main metabolite 4-hydroxymethamphetamine decreased.

This clean-up method is very effective in forensic toxicology for the purpose of obtaining criminal evidence.

Keywords: Amphetamine, Putrefied specimen, Clean-up.



Capillary Electrophoretic Enantiomer Separation of Amphetamines after the Administration of l-Deprenyl to Man

Eunmi Kim1, Sunchun Kim1, Heesun Chung*1, Youngchan Yoo 1, Kongju Lee2 and Hwajung Kim2
1National Institute of Scientific Investigation, 331-1 Shinwol-dong, Yangchon-ku, Seoul, Korea
2College of Pharmacy, Ewha Women's University, Daehyun-dong, Seadaemoon-ku, Seoul, Korea

l-Deprenyl is metabolized to desmethylselegiline, l-methamphetamine (l-MA) and l-amphetamine (l-AM) and has recently been marketed in Korea for the treatment of Parkinson's disease. Since l-deprenyl has been introduced into the market, it has become necessary to separate the enantiomer of methamphetamine in order to distinguish it from the illicit drug MA, which is the predominant drug of abuse as a d-enantiomer in Korea.

To investigate illicit drug use, separations of enantiomer of MA and AM were performed by capillary electrophoresis with P/ACE system with diode-array detector. 10 mg of l-deprenyl was administered to five subjects and urine was collected every 3 hours for 24 hours.

l-MA, l-AM, N-desmethylselegiline and l-deprenyl were detected in urine samples and were well resolved from d-isomers, potent central nervous system stimulants in this condition. Urinary recoveries of desmethylselegiline, l-MA, l-AM ranged from 0.97-1.31, 12.26-21.18 and 3.23-8.54% respectively in urine collected for 24 hrs. The ratios of l-AM/l-MA ranged from 0.26-0.43 indicating interspecies differences in metabolism of l-deprenyl in man and rat. The level of l-AM/l-MA is higher in the user of l-deprenyl than the ratio of d-AM/d-MA in MA abusers.



Evaluation of Sample Preparation Techniques Adequate for Multivariate Algorithms Applied for the Identification and Classification of Amphetamine Analogues with GC-FTIR

Mirela Praisler2, Inge Dirinck*2, Willy Lambert, Andreas De Leenheer, and D. Luc Massart3
1Laboratory of Toxicology, Universiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium
2Department of Physics, University of Galati, Domneasca St. 47, 6200 Galati, Romania
3Laboratory of Pharmaceutical and Biomedical Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussel, Belgium

Sample preparation techniques were evaluated for street samples as part of an optimisation strategy in forming the GC-FTIR knowledge base of an expert system based on Soft Independent Modelling of Class Analogy (SIMCA). The system assigns an unknown to one of the modelled classes of compounds by seeking a pattern-to-pattern match, as opposed to the identification of an individual compound performed using a peak-to-peak match. The system is expected to discriminate the class of amphetamines from other toxicologically relevant compounds, and to differentiate between stimulant amphetamine analogues and hallucinogenic amphetamine analogues. We have explored two types of sample preparations, both yielding analytical advantages potentially relevant to the selectivity of the expert system. The derivatization procedure using heptafluorobutyric anhydride (HFBA) yields HFB-derivatives displaying increased infrared sensitivity and spectra with a larger number of absorption bands, associated with the chemical groups added to the molecular structure. On the other hand, the spectra of the underivatized analogues are more selective, FTIR spectra of lower weight molecules being more sensitive to small changes in the molecular structure.

The relevance of these advantages was explored using SIMCA classification. Up to 81.13% of the 159 tested compounds were classified with a significance level of 5%, and the total correct classification rate was 93.93%. The best selectivity in discriminating among phenyl nonsubstituted amphetamine analogues, 3,4-methylenedioxy- amphetamine analogues, and nonamphetamine compounds was obtained with nonderivatized samples. The classification yielded 96.30% true positive (nonderivatized) amphetamines, and only 85.71% true positive derivatized samples. The plots measuring the discrimination power of the wavenumbers clearly show that the spectra of nonderivatized samples exhibit a much larger number of wavenumbers with high discrimination power. They prove that significant changes in band profiles are more valuable information for the knowledge base of the expert system than an increased number of absorption bands with profiles less sensitive to small molecular structural changes.

Keywords: Amphetamines, FTIR spectra, Soft Independent Modelling of Class Analogy.



Comparison between Automated Knowledge-Based Systems Identifying and Classifying Amphetamine Analogues Using Vapor-phase FTIR Spectra and Mass Spectra

Inge Dirinck*1, Mirela Praisler2, Jan Van Bocxlaer, Andreas De Leenheer and D. Luc Massart3
1Laboratory of Toxicology, Universiteit Gent, Harelbekestraat 72, B-9000 Gent, Belgium 2Department of Physics, University of Galati, Domneasca St. 47, 6200 Galati, Romania 3Laboratory of Pharmaceutical and Biomedical Analysis, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussel, Belgium

Many amphetamine analogues have emerged as recreational drugs, so-called "designer drugs", over the last decade. In an attempt to circumvent existing controlled substance laws, clandestine laboratories are synthesizing slightly modified chemical structures by adding or changing substituents at various positions on the amphetamine molecule without significantly altering its psychotropic effect. The unambiguous identification of novel amphetamine analogues is frequently necessary in judicial cases mainly related to deaths from overdose or to illicit drug seizures.

The feasibility of identifying and classifying novel amphetamine analogues according to the substitution pattern of the phenyl ring was explored by principal component analysis (PCA) run for appropriately feature weighed vapor-phase infrared spectra and mass spectra. Automated knowledge-based systems using vapor-phase FTIR spectra (GC-FTIR) and mass spectra (GC-MS) of amphetamines were compared according to their ability to distinguish between closely related amphetamines. Infrared spectra were imported from a laboratory-made vapor-phase FTIR library, which contains 159 reference spectra of commercially available amphetamines, several in-house synthesized amphetamine analogues, and other related drugs of abuse. The mass spectra of the same compounds were imported from general MS libraries (NIST, AAFS, and an in-house MS library).

The appropriately pre-processed FTIR spectral features enabled the automated discrimination and recognition of the subclasses of phenyl nonsubstituted and 3,4-methylenedioxy- amphetamines within high (statistically measured) reliability limits. PCA analysis of the MS data matrix proved that identification and classification of amphetamine analogues according to the substitution patterns of the phenyl ring is much less reliable as the differences in fragmentation patterns are not correlated with this structural feature. Discrimination according to the substitution pattern of the phenyl ring could not be obtained within statistically acceptable reliability limits. On the other hand, the analysis indicated that MS spectra might be more appropriate for the classification of positional isomers.

Keywords: Amphetamines, Vapor-phase FTIR spectra, Mass spectra, Principal component analysis.



Multicenter Study on Reproducibility of Mass Spectra of Toxicologically Relevant Drugs Generated by Atmospheric Pressure Ionization Mass Spectrometer

Maciej J. Bogusz*1, Rolf-Dieter Maier1, Klaus D. Kruger1, Kenneth S. Webb2, Julie Romeril2, Mark L. Miller3
1Institute of Forensic Medicine, Aachen University of Technology, Germany
2Laboratory of the Government Chemist, Teddington, UK
3Forensic Science Research and Teaching Center, FBI Academy, Quantico, USA

The purpose of the study was to examine the intra- and inter-laboratory reproducibility of mass spectra obtained with liquid chromatography-atmospheric pressure mass spectrometry (LC-API-MS) both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) modes. As test substances toxicologically relevant drugs of different polarity (morphine-6-glucuronide, 6-monoacetylmorphine, codeine, LSD, MDMA) were selected. The study was performed in two laboratories using identical instruments and in one using a slightly different instrument. Basic instrument settings and mobile phase were identical in all laboratories.

Mass spectra of drugs were taken at four levels of collision energy and using mobile phase of different composition (four concentration levels of acetonitrile and of ammonium formate buffer).

The experiments demonstrated that mass spectra of given drugs, obtained in identical conditions with identical instruments, may show very different degrees of fragmentation. Mass spectra obtained with different instruments differed profoundly not only in the degree of fragmentation, but also different fragments and adducts were observed.

Short-term intralaboratory reproducibility of mass spectra was satisfactory. On the other hand, the long-term experiments showed different degrees of fragmentation of APCI-generated mass spectra at nominally identical fragmentation energy. Mass spectra obtained in ESI mode were also reproducible in long-term studies.

The changes in the composition of the mobile phase (concentration of organic modifier or buffer molarity) did not affect the reproducibility of fragmentation to any relevant degree.

The study showed that the interlaboratory exchange and use of an API-generated mass spectrum library is hardly feasible at the moment, even under very carefully standardized conditions.

Keywords: LC-API-MS, Interlaboratory Reproducibility, Mass Spectra.



Microextraction in Bioanalysis of Drugs

Mette Krogh*
National Institute of Forensic Toxicology, P.O. Box 495 Sentrum, N-0105 Oslo, Norway

The primary goal of traditional sample preparation techniques such as liquid-liquid extraction and solid-phase extraction is to extract the analytes quantitatively from the analytical matrices. The enrichments obtained by these methods are usually in the range of 1-5. The solvent must be evaporated and the analytes reconstituted in a smaller volume of solvent in order to achieve higher enrichment. In microextraction (ME) the analyte is extracted from a large volume of sample solution (1-2 ml) and into a small volume of acceptor solution (1-50 ml). Enrichment factors of 50-100 can be obtained from ME when 1 ml of sample is extracted with 1-10 ml acceptor solvent as well as excellent sample clean-up. ME is well suited for analysis by capillary electrophoresis (CE) and capillary gas chromatography (GC). The potential of membrane based ME for the extraction of drugs from urine and plasma will be demonstrated.

Keywords: Microextraction, Sample Preparation, Drugs.



Determination of GHB by Head-Space GC/MS in Forensic Samples

Sys Stybe Johansen* and Soren Felby
Institute of Forensic Medicine, Frederik V's vej 11, University of Copenhagen, DK-2100 Copenhagen, Denmark

A fast, simple and selective method for determination of gammahydroxybutyrat (GHB) in whole blood and urine is developed. GHB is a new club drug known as 'easy lay' which is also used by body-builders.

GHB is converted to its lactonic form gammabutyrolactone (GBL) under acidic conditions and high temperatures. This reaction takes place in a headspace vial, storing the semi-volatile product GBL. Therefore a simple and selective sample preparation technique such as headspace (HS) sampling is ideal.

The highest recovery of GBL using HS sampling is observed under acidic saturated conditions achieved by addition of 0.5 g NaHSO4 to the 1 ml sample. The optimised HS conditions are determined to be a sampling temperature of 120°C, an equilibrium time of 20 min with the use of cryofocusing. Analysis is done by HS-GC with flame ionisation detector or coupled to a mass spectrometer.

Other sampling procedures such as solid phase microextraction (SPME) have been investigated but the sensitivity is not yet adequate.

The method is evaluated for application to whole blood and urine. Under optimised conditions the transformation rate of GHB to GBL in whole blood is about 80% (RSD: 8%). The detection limit of GHB/GBL is low ppm. The method is linear using FID or MS over a range of several factors in whole blood and urine. The reproducibility is below 10%.

Keywords: GHB, HS-GC/MS, Forensic Sample.



The Determination of GHB in Biological and Adulterated Samples without Forming Gamma-Butyrolactone

Thomas F. August*, Chester J. Kitchen and Michael Telepchak
United Chemical Technologies Inc. 2731 Bartram Street, Bristol, Pa 19007 USA

GHB (Liquid Ecstasy, Scoop, Easy Lay) has been popularized as the "sex drug" of the 1990's. It is used as a social drug in rave parties and other gatherings.

It produces an euphoric condition that increases sexual awareness, releases inhibitions and has been implicated in a number of sexual assault cases especially "date rape". GHB is only clandestinely manufactured since the FDA banned its use and distribution in 1990. Currently numerous states have placed GHB as a banned drug substance.

The small polar nature of the molecule and the lack of an UV chromatophore complicate the chromatographic and spectrophotometric analysis of GHB.

Chemically GHB is unstable and readily forms Gamma-butyrolactone when heated in acid conditions. Most analytical methods are based upon the interconversion to the lactone and chemical derivatization to form the TMS derivative.

The following paper reports on a method for the quantitative determination of GHB in human urine, blood and adulterated liquid sample using a combined solid phase extraction (Clean ScreenO GHB - Patent pending) with a liquid/liquid cleanup step and chemical derivatization without forming the lactone.

This method is quick, sensitive and easily performed. The limit of detection for GHB is 5 mg/l. The validation of this method (i.e. linearity, intraday and interday variability etc) will be presented as well as the collaborative analysis of actual clinical samples.

Keywords: GHB, Date Rape Drugs, Solid Phase Extraction.



Pharmacokinetic Study of Dextromethorphan with Urinary Excretion

Heesun Chung*, Wonkyung Yang, Hwakyung Choi, Wontack Jin, Sihnyoung Sihn and Youngchan Yoo
National Institute of Scientific Investigation, 331-1 Shinwol-dong, Yangchon-ku, Seoul, Korea

Because the abuse of Dextromethorphan, a non-narcotic antitussive agent, is very common and characteristic in Korea, the metabolic phenotype of dextromethorphan is studied to understand the disposition of the drug in the Korean population. Firstly, the pharmacokinetic study with urinary excretion in man was performed to obtain the urinary excretion rate constant (K) of the unchanged drug and elimination half-life (t1/2) of the drug. Secondly, the pharmacokinetics of dextromethorphan in rat was studied.

After a single 30 mg dextromethorphan oral administration to volunteers, dextromethorphan and free dextrorphan concentrations in urine were measured using solid-phase extraction by GC and GC/MS. Urine was collected every 2 hours for 8hrs, 3-4 times in 24 hrs and then every 12 hours for 72 hrs.

The excretion rate constant of dextromerhophan was 0.056/hr. The elimination half-life of dextromethorphan in the body was 6.58 hrs. Urinary recovery of dextromethorphan ranged from 0.23-0.55% in five subjects over 24 hrs. The metabolic ratio of free dextrorphan to dextromethorphan varied between subjects. The ratio between dextrorphan/dextromethorphan ranged from 1.6 to 37.3.

The plasma concentration of dextromethorphan was determined in five rats after administration of 10 mg/kg of dextromethorphan intravenously. The first order elimination rate constant of dextromethorphan by two compartment model was 0.036 and the value of Vd (l/kg) was 0.66.

Keywords: Dextromethorphan, Urinary Excretion, Pharmacokinetics.



Surface Ionization Mass Spectrometry. Analysis of Post-Mortems for Fatal Poisoning by Chlorpromazine

U. Kh. Rasulev1, Usman Khasanov1, Turgun Kh. Islamov2, Makhamudjan M. Shakhitov3, B. Eshmuratov3
1 Arifov Institute of Electronics, Tashkent, Uzbekistan
2 Republican Criminal Research Center, Tashkent, Uzbekistan
3 Republican Bureau of Forensic Medicine Expertise, Tashkent, Uzbekistan

In this paper the results of the investigation of post-mortems (stomach, intestine and kidney) are presented for chlorpromazine poisoning, using methods of surface ionization mass spectrometry (SI/MS) [1] as well as chemical, optical, TLC and GC/MS methods widely used in analytical toxicology.

Grinding post-mortems were soured by 10% HCl solution up to pH 2.0-3.0, they were extracted by acetonitrile. Acetonitrile extractions were filtrated into 2.5% aqueous solution of NaSO4 and soured by HCl up to pH 2.0-3.0. Extraction was performed by ether portions. Filtrated and dried residue was studied.

In the experiment the mass spectrometer of a modernized MI-1201 B type was used for SI studies. An oxidized tungsten band served as a thermoemitter. The Knudsen cell evaporator was fed by dozed chloroform solution of the residue. To choose the optimal mode of ion emission, the emitter temperature was scanned over the range of 700-1200 K.

The SI mass spectra obtained show the high efficiency of the ionization of chlorpromazine and its metabolites: monodesmethyl-, sulphoxid- and hydroxichlorpromazine. The high selectivity of SI to nitrogen bases and few lines of the SI mass spectra make it possible to identify, with a high accuracy, the samples without their chromatographic separation.

To demonstrate the high analytical possibilities of SI/MS, the results of the sample analysis by optical, TLC and GC/MS (Hewlett-Packard HP-5890) methods are presented.

1. U. Kh. Rasulev and E. Ya. Zandberg. Surface ionization of organic compounds. Progress in Surface Science, 28, 181-412 (1998).



Urinary Excretion of Diazepam Metabolites in Healthy Volunteers Taking a Single Dose of Diazepam, and in Drug Users

Anne Smith-Kielland*, Bjorn Skuterud, Kirsten M. Olsen, Anne Christine P. Jorgensen, Jorg Morland
National Institute of Forensic Toxicology, PO. Box 495 Sentrum, N-0105 Oslo, Norway

Detailed studies with regard to urinary excretion profiles of diazepam, a commonly used benzodiazepine, are scarce. In the present study diazepam excretion was studied in (1): healthy volunteers and in (2): drug users starting to serve their prison sentence. Informed consent was obtained.

(1): Six participants were each given 10 mg diazepam. Every urinary void was collected; the volume was measured and aliquots were taken for analysis until at least 6 consecutive negative screening results were found. Three subjects repeated the experiment one year later. (2): Participants reported their drug intake. Urine specimens were collected 1-5 times daily until found negative as judged from several consecutive negative screening results. Samples were screened by EMIT dau/EMIT II (cut-off 300 ng/ml and 200 ng/ml, respectively). Confirmation analysis was performed by HPLC, measuring acid hydrolysis products: 2-methylamino-5-chloro-benzophenone (MACB) and 2-aminochloro-benzophenone (ACB). Creatinine was measured in all urine specimens.

Results: (1): Volunteers with EMIT positive specimens for the shortest and longest time are reported. Shortest: A peak urinary concentration of 1.2 and 1.5 µmol/l (MACB, ACB) was found after 1.4 days. The last confirmed and EMIT positive specimens were found after 2.4 and 2.5 days, respectively. Longest: One female had a peak urinary concentration of 3.0 and 1.1 µmol/l (MACB, ACB) after 3.8 days. The last confirmation and EMIT positive specimens were observed after 8.4 and 10.3 days. Retests after a year showed that intra individual differences could be substantial. (2): Ten out of 22 drug users reported diazepam intake and had benzodiazepine positive urines. The highest values observed were 29.1 and 21.9 µmol/l (MACB, ACB), after a reported intake of 20 'tablets' of diazepam three days before the collection of the first urine specimen. A urinary specimen was screening and confirmation positive 11 days after intake.

After a single dose of diazepam large intra - and inter individual differences in urinary excretion profiles of metabolites are seen. This has to be taken into account when interpreting urinary testing results. High concentrations of urinary diazepam metabolites could be observed after intake in drug users.

Keywords: Benzodiazepines, Diazepam Metabolites, Urinary Excretion.



Screening for the Presence of para-Methylthioamphetamine in Urine by some Commercial Immunoassays and Confirmation by GC/MS

Ingrid J. Bosman*1, Douwe de Boer2 and Robert A. A. Maes1
1Department of Human Toxicology, Utrecht Institute of Pharmaceutical Sciences, Utrecht, The Netherlands
2 Laboratorio de Analises de Dopagem e Bioquimica, Lisboa, Portugal

Para-methylthioamphetamine (MTA) is a relatively new sulphur containing phenylalkylamine designer drug. It is present in so-called S-5 tablets which are sold in smart Dutch shops. In the Netherlands, at least one intoxication after the intake of one S-5 tablet was reported. MTA is also thought to be responsible for two deaths in the United Kingdom.

In this study, we evaluate the detection of MTA in urine specimens using commercial amphethamine-like immunoassays and the confirmation of the designer by GC/MS.

In order to test an enzymatic (EMIT) and fluorescence polarisation immunoassay (FPIA), blank urine was spiked with various concentrations of MTA. For comparison and determination of the cross-reactivity, amphetamine, dexamphetamine and methamphetamine were tested as well. The curves of log-concentration of MTA versus absorbance show that MTA has an acceptable immunoreactivity for the antibodies of both the EMIT monoclonal assay and the EMIT polyclonal assay. In a similar way, the curves of log-concentration versus net polarisation demonstrate that MTA also cross-reacts with the antibodies in the FPIA procedure.

For confirmation, MTA was analysed by GC/MS using different derivatisation methods. Respective electron ionisation mass spectra and retention indices of N-TFA and N-HFB derivatives of MTA are discussed.

From the obtained results, it can be concluded that the presence of MTA in urine can be detected by the respective commercial immunoassays and confirmed by GC/MS.

Keywords: para-Mmethylthioamphetamine, Immunoassay, GC/MS.



Mutagenic Effects of the Food Colour Erythosine in Rats

Hamdy A. Mekkawy*1, A. A. Massoud2, A. M. El-Zawahry3
1The National Center for Social and Criminological Research, P.O. Zamalek, P.C.11561, Cairo, Egypt
2Faculty of Sciences, Tanta University, Tanta, Egypt
3NODCAR, Pyramids Ave, P.O.29, Cairo, Egypt

Following treatment of male rats with a diet supplemented by the synthetic food colour erythrosine (0.08 and 0.4 g/kg diet), daily for 30 days. Changes in mutagenic activities, as an index for evaluation of possible toxic effects, were monitored by measuring chromosomal aberrations, nucleic acids and total protein concentrations of liver and brain. The present study found that erythrosine induced chromosomal aberrations. Chromosomal aberrations of bone marrow cells were centromeric attenuation, centric fusion, deletions, 'ringshapedness', stickiness, end to end association and polyploidy. Biochemical assays revealed that the nucleic acids and the total protein had increased markedly during the various periods of treatment. Results indicated that two doses of erythrosine were found to be mutagenic agents and a high dose of erythrosine was more effective than a lower one.

Keywords: Erythrosine, Mutagenic, Rats.



Coma after Intake of Gamma-hydroxybutyric acid (GHB): Two Case Reports

Peter X. Iten*1, Andrea Oestreich1, Regina Lips2, and Michael Brabetz2
1 Institute of Legal Medicine, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich 2 Waid Hospital, Tiechestrasse 99, CH-8037 Zurich, Switzerland

The abuse of Gamma-hydroxybutyric acid (GHB, "Liquid ecstasy") in discos and among bodybuilders was first registered in the early nineties in the USA and later on in Europe.

We are presenting both clinical findings and chemical laboratory results in an attempt to evaluate the symptoms and findings most valuable in the diagnosis of GHB intoxication. In two separate cases young males were hospitalised from a disco situation after GHB intake. After several hours of deep coma they awoke rapidly and were fully orientated.

In one case the residue of a colourless and odourless liquid was recovered, in which we determined a GHB content of 0.5 g/ml. Themaximum GHB dose swallowed was calculated to be 17.5 g. Doses as small as approximately 5 - 6 g can lead to coma.

In the second case we quantified in blood and urine GHB concentrations of 12 and 1,200 µg/ml, respectively. In this case, blood and urine were taken about 8 hours after the intake of the GHB liquid. GHB analysis was conducted by a modified method of Louagie et al. (Clin. Toxicol., 35, 591-594, 1997). Deuterated GHB was added as an internal standard to blood and urine samples or to the GHB containing fluids. Extraction was done with methanol. After centrifugation the supernatant was taken to dryness, silylated with MSTFA and analysed using GC/MS.

Keywords: Gamma-hydroxybutyric acid (GHB), Effects, Analysis, Case reports.



Direct Extraction of Benzodiazepine Metabolites with Supercritical Fluid from Whole Blood

Kenichi Takaichi, Shuji Saitoh, Yoshio Kumooka, and Noriko Tsunoda
National Research Institute of Police Science, Kashiwa-no-ha, 6-3-1, Kashiwa, Chiba 277-0882, Japan

Our target is to develop a simple and a speedy extraction method of drug from biological sample. Supercritical fluid extraction (SFE) is well known as a speedy and contaminant-free extraction method. So we applied the advantages of the SFE, which has replaced a conventional extraction method, that is, liquid-liquid extraction (LLE) and solid phase extraction (SPE).

In previous papers, we reported that SFE methods could be combined with freeze-drying or filling materials to reduce the problems of the LLE and the SPE. The SFE method with the filler can directly extract drugs from biological fluid samples without the need for deproteinization. However, the recovery of the drug metabolite has not yet been considered.

In this report, an evaluation has been made of whether or not the drug metabolite can be extracted from whole blood with the SFE method combined with filler. The metabolite of the drug is generally a polar compound having a strong polar group such as hydroxyl (OH), desalkylamino (NH), and carboxyl (COOH) group. Therefore, the metabolite of the drug is adsorbed easily by the filler and might not be extracted. Oxazepam was chosen as a typical metabolite of the drug (Diazepam).

Quantitative analysis of the extract was carried out with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS).

The metabolite of diazepam can be extracted as long as conditions are satisfactory in the SFE using molecular sieves 5A as a filler. The extraction conditions of the SFE are pressure, temperature, composition and flow rate of supercritical fluid, kind and amount of filler, and other factors.

Keywords: SFE, Benzodiazepines, Biological samples.



Application of Gas Chromatography to the Identification of Petroleum Products in Environmental Contamination Cases

Zofia Chlobowska, Ewa Chudzikiewicz, Czeslawa Swiegoda, Wojciech Piekoszewski*
Institute of Forensic Research, Cracow, Poland

Petroleum products are a menace to the environment not only as air-polluting car exhaust gases, but also as various sewage effluents (e.g. from car repair garages and car washers) or outflows of fuels and lubricants from leaking tanks leading to contamination of soil and water.

The gas chromatography method was used to identify the petroleum products, thus often indicating the source of contamination. In the case of contamination of soil and water, and in the case of materials secured after fires and biological material taken from persons exposed to the influence of these products, the headspace phase of the investigated materials and their n-pentane extracts were analysed.

The investigations were performed using the FISONS series 8000 gas chromatograph equipped with a flame-ionization detector and DB-1 30 m x 0.25 mm column (0.25 µm film thickness) with temperature programme from 40 °C to 220 °C, 20 °C/min temperature increase. The detector was at a temperature of 300 °C, injector 250 °C. The carrier gas was helium, at a flow rate of 1.1 ml/min, with split of 1:100.

Chromatographic analysis of petroleum products such as gasolines, kerosene, diesel fuel and heating oil was carried out in established conditions, and the obtained data were used to set up a data base of chromatograms. This data base facilitated subsequent identification of these kinds of compounds isolated from materials delivered as evidence to the Institute of Forensic Research.

This paper presents expertises concerning:

  • soil and potable water intake samples contaminated with aviation fuel,
  • well water sample contaminated with a mixture of automotive gasoline and diesel fuel,
  • fish stored in a reservoir contaminated with kerosene.
Keywords: Petroleum Products, Gas Chromatography.



Comparison of an Enzymatic Alcohol Dehydrogenase Assay and Alcohol Headspace GC-FID Method Using Statistical Analysis on Real Forensic Blood and Urine Samples

Katrien Arys*, J. Van Bocxlaer, Willy Lambert, C. Van Peteghem and Andreas De Leenheer
Laboratorium voor Toxicologie, Universiteit Gent, Faculteit Farmaceutische Wetenschappen, Harelbekestraat 72, B-9000 Gent, Belgium

At our laboratory, two independent measurement techniques, an enzymatic alcohol dehydrogenase assay (Syva-EmitO) and a headspace GC-FID method, are used to determine alcohol levels in forensic samples. Over the last five years, many samples have been analyzed with both methods, enabling a statistically based comparison of both methods' performance for blood and urine. The statistical procedure we used was a two-sided paired sample t-test, because there was a one-to-one correspondence between the members of the sample populations. Alternatively, regression analysis was used.

The samples were selected equally divided over the range of the headspace calibration curve (0.5 - 3 g ethanol/l). The assumptions of normal distribution of all sample populations and homogeneity of variance were checked with the appropriate statistical tests (Kolmogorov-Smirnov test of normality, and Levene test statistic, respectively).

From the statistical test performed we conclude that at the chosen significance level (a = 0.05), the Emit and headspace method for the analysis of ethanol in blood and urine were not equivalent. For both matrices analyzed, a bias was detected in one or both of the methods. Of great diagnostic value was the Bland and Altman plot, where the difference between the emit value and the headspace value was plotted against the mean value. When evaluating these plots, for blood as well as for urine, a proportional error was detected, The emit values were higher than the headspace values in the higher concentration range (> 2.5 g/l). This may be due to a calibration error in the emit method, where a one-point calibration was performed at the 1g/l level. Additionally, some of the higher emit results obtained for the blood samples can be (partially/completely) attributed to an earlier published interference of LDH in the emit assay. This can be the cause of the deviation found in some of the real forensic samples analyzed in our study.

Keywords: Statistical analysis, Emit, Headspace, Alcohol.



Death Caused by Acute Formaldehyde Poisoning during Haemodialysis Treatment in Hospital

Vincent K. K. Mok*, David T. W. Chan
Government Laboratory, Homantin Government Offices, 88, Chung Hau St., Homantin, Kowloon, Hong Kong Simultaneous Determination of Triazolam and its Metabolites in Human Blood and Urine by GC-MS Keiko Kudo*, Noriaki Ikeda and Yukiko Hino Department of Forensic Medicine Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

A sensitive and reliable method was developed for the simultaneous determination of triazolam and its major metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam in human whole blood and urine. The drugs, effectively extracted using a 3-step solvent extraction procedure followed by tert.-butyldimethylsilyl derivatization were subjected to gas chromatography-mass spectrometry, with the negative ion chemical ionization mode. Estazolam was the internal standard used. The calibration curve for each compound was linear in the concentration range from 0.5-100 ng/g, and the lower limit of detection was 0.1 ng/g for whole blood and 0.2 ng/g for urine. The accuracy and precision of the method were evaluated in every sample at the concentration 10 ng/g. The coefficient of variation ranged from 2.7-10.8%. A practical application is also presented.

Key words: Triazolam, Alpha-hydroxytriazolam, 4-Hydroxytriazolam, GC-MS.



Analysis of Meconium for Amphetamines and Opiates. Our First Experience

Eva Novakova
Institute for Toxicology and Forensic Chemistry, Charles University and University General Hospital Na bojisti 3, 121 08 Prague 2, Czech Republic

Peri- and postnatal complications in infants born to drug dependent mothers have recently arisen in the Czech Republic due to an increase of drug abuse in the last ten years. Meconium is a widely accepted specimen for the determination of fetal drug exposure, therefore we started with its analysis for methamphetamine and opiates, which are the most frequently abused drugs in our country.

Drug free meconium was spiked with methamphetamine or morphine in different concentrations (4 m g/g - 0.5 m g/g) for the isolation study and the determination of detection limits for the EMIT screening on ACA Du Pont analyzer. The extraction of homogenized meconium with various organic solvents was compared with SPE on Chem-Elut or Tox-Elut columns. HPTLC mainly in 2-dimensional modification and GC/ECD or NPD on capillary columns HP-17 and HP-5 were used as the confirmation methods. GC/MS was used only in a few cases for technical reasons. The lowest concentration tested for both drugs was 0.5 m g/g meconium. The detection limit of EMIT had to be decreased in comparison with that used for urine. 0.5 m g morphine/g and 1 m g methamphetamine/g were confirmed by HPTLC, 0.5 m g methamphetamine/g by GC/ECD/NPD. Concentrations lower than 0.5 m g/g have not been tested yet.

Up to the end of 1998 we analyzed meconium in 20 cases of suspected drug abuse during pregnancy, 8 of them being positive. Methamphetamine was found 3 times (in one case together with amphetamine), morphine - 5 times. Medium to strong abstinence syndrome has developed in 2 infants with positive morphine. In 8 cases the findings in meconium and urine of newborns were compared. In 5 positive morphine cases the urine was positive 3 times, whereas out of 3 positive methamphetamine cases urine was positive 2 times. A possible interpretation is discussed.

We conclude that the analysis of meconium can serve as a diagnostic tool of fetal drug exposure requested by the medical community.

Keywords: Meconium Analysis, Methamphetamine, Opiates.



Involvement of Drugs in Accident Causation

Jim Gerostamoulos*, Helen Batziris (Hons) and Olaf H. Drummer
Victorian Institute of Forensic Medicine and Department of Forensic Medicine, Monash University, 57-83 Kavanagh Street, Southbank, 3006 Melbourne, Australia

Responsibility analysis has been used to assess the contribution of drugs in traffic accidents involving fatally injured drivers in Australia in 1995 and 1996. These results have been compared with those of a similar study conducted in drivers killed from 1990-1993. The prevalence of drugs in drivers killed in 1995-96 averaged 27%, an increase of 5% from 1990-93. The prevalence in individual states across Australia ranged from 11 to over 41%. The most prevalent drug was cannabis, which increased in prevalence from 11% to 13% in this period. There were lesser changes for the other major drug groups: opioids, benzodiazepines and amphetamine-like stimulants.

The incidence of alcohol (BAC 0.01%) averaged 32% in 1995-96 cases, a decrease of 4% from 1990-93.

Risk analysis showed no significant increase in risk for drivers positive for cannabis and opioids alone, but when combined with other psychoactive drugs, a significant increase in risk occurs (3.5-fold). Drivers positive for alcohol over 0.05% gave an increase in risk of 9-fold over drug-free drivers. Users of amphetamines and related stimulants and benzodiazepines show a trend to a higher risk, but this was not significant. Of interest was the significant increase in risk for users of psychotropic drugs (3.4-fold). THC positive culpable drivers had an average blood concentration of 15 ng/ml compared to 3.5 ng/ml in non-culpable drivers. These data suggest that campaigns to reduce drug use are best targeted to drivers likely to drive shortly after consuming cannabis, using non-prescribed doses of drugs, those who mix psychoactive drugs, and those combining drugs with alcohol.

Keywords: Drugs, Driving and Causation.



Risks for Driving Under the Influence of Clomethiazole

Emma Logemann
Institute of Forensic Medicine, University of D-79104 Freiburg/Brsg., Germany

According to the WHO definition any substance which impairs one or more functions of the central nervous system in living organisms is a drug. In view of problems concerning traffic medicine the most important drugs considered are alcohol, addictive drugs and pharmaceuticals. In a case history the risks of driving a motor vehicle under the influence of clomethiazole (syn. chlormethiazole; main indication: treatment of alcohol withdrawal syndrome) is demonstrated: A 53year old male car driver caused an accident by deviating from the lane in a traffic circle, but he continued his trip without stopping. The police could follow the winding lines of the car for about 2 000 m, ending in the garage of the car owner, since the oil tank of the car and the entrance of the garage were damaged. Toxicological analysis (GC-ECD) of a blood sample of the car owner indicated 3 300 ng clomethiazole/ml serum (CLARKE: therapeutic concentration range: 100 to 2 800 ng clomethiazole/ml plasma). Neither alcohol (head space GC; ADH) nor any other drug (GC-MS, HPLC) could be detected in this blood sample. Policemen reported that one day after this event the car owner committed suicide by swallowing at least 60 tablets of Distraneurin (R) (clomethiazole). In this case no post mortem blood sample was taken and no dissection of the corpse was made.

1. M. Faller-Marquardt, E. Logemann, D. Ropohl. Risks for motor vehicle drivers following out-patient treatment of alcohol withdrawal symptoms with chlormethioazole. Blutalkohol. 36, 44-50 (1999).

2. R. Iffland. Zum Nachweis therapeutischer Konzentrationen Chlormethiazol im Blut. Rechtsmedizin, 80, 27-33 (1977).

3. Clarke's Isolation and identification of drugs, 2nd edit., Moffat, A.C., et al. (eds), pp. 448-449, The Pharmaceutical Press, London (1986).

Keywords: Clomethiazole, Chlormethiazole, Motor Vehicle Driving.



Fatal Traffic Accidents Influenced by Drugs in South Moravia in 1998

Helena Samkova*, Andrea Brzobohata, Miroslav Hirt
Institute of Forensic Medicine, Brno, Czech Republic

Three cases of fatal injuries to men are reported: a 25-year-old man run over by a train, a 35-year-old pedestrian knocked down by a car and a 21-year-old driver of a car. Toxicological analysis of the autopsy material revealed the presence of methamphetamine in all cases. Further addictive drugs detected were: cannabinoids in two cases and methylendioxymethamphetamine and biperiden in one case.

The identification was performed by GC-MS after extractive derivatisation with pentafluorobenzoyl chloride (PFBCI). Blood levels of methamphetamine (0.1; 0.2; and 1.5 mg/l), and urine levels of methamphetamine (0.7; 2.7 and 106.0 mg/l) were determined by gas chromatography. Ethanol was positive in one case.

Our results suggest that toxicological analysis in traffic accidents has become very important and it will possibly be necessary to perform it routinely.



Drugs of Abuse: On-Site Saliva Sampling and Analysis

Jorg Zimmermann*1,2, Hans-Jurgen Duchstein1, Kai Bierans2, Thomas Wuske3, Rainer Polzius3, Andreas Manns2
1University of Hamburg, Institute of Pharmacy, D-20146 Hamburg, Germany 2Drager Sicherheitstechnik GmbH, D-23560 Lubeck, Germany 3Dragerwerk AG, D-23542 Lubeck, Germany

For the past 20 years, there has been a burgeoning interest in the use of saliva in pharmacokinetic studies of drugs in general and in therapeutic drug monitoring in a variety of clinical situations, including, in recent years, the measurement of drugs of abuse. Particular interest has been expressed by law enforcement agencies for roadside testing of potentially intoxicated drivers.

There are different methods available for collecting whole saliva e.g. draining, spitting, suctions and the swab method or by using osmotic collection devices. With the swab method, saliva is collected (absorbed) by a swab, cotton roll or gauze sponge placed in the mouth and removed after a defined collection period.

Saliva drug testing can reveal the presence of a pharmacologically active drug in an individual at the time of testing. Significant correlations have been found between saliva concentrations of drugs of abuse and behavioral and physiological effects. Results indicated that saliva testing can provide valuable information for diagnosis, treatment and forensic investigations, e.g. testing individuals suspected of drug abuse at the roadside. One of the advantages of being able to test a saliva specimen is the opportunity to conduct studies in a non-medical setting, employing personnel with only rudimentary training. It is important for the success of such studies that the method of collection of saliva is as simple as possible and provides a specimen of sufficient quantity and quality. Till now, only a few on-site saliva drug testing devices have been available.

Our goal is the development and assessment of new applications by combining new sampling devices and methods with known immunochemical drug detecting test systems, e.g. lateral flow ELISA systems. The development and application assessment of these methods is focused primarily on ease-of-use on site, performance time, and reliability of results. We based development on swab saliva collecting devices, using amphetamine as the model analyte for drugs of abuse.

Data describing the measurement strategy, new concepts for saliva-sampling devices and on-site applicability in combination with immunochemical screening for drugs of abuse will be presented.

Keywords: Drugs of Abuse, Roadside Testing, Saliva Sampling.



Identification of Basic Drugs in Forensic Samples by Capillary Gas Chromatography

Irene Breum Muller
Institute of Forensic Medicine, University of Copenhagen, 11 Frederik V's vej, DK-2100 Copenhagen, Denmark

Numerous drugs are basic and a broad screening method for these drugs is of toxicological interest. Currently in use is an older version of the Hewlett Packard Chemstation containing a special design program with retention index search. This program has to be exchanged before year 2000. Therefore a new HP GC-6890 with retention time locking system has been bought. Using this system it is possible to use the same GC-conditions on every GC in the laboratory and thereby compare results e.g. of different NPD-detectors. This poster describes how the screening is performed using an automated library search.

A basic extract is injected on a HP GC. The analysis uses NPD-detection and a retention time locked library to find and identify basic drugs in the extract. Once the screen is completed, positive hits are verified by their MS spectra using an ion-trap GC-MS. The retention time locked library contains approximately 200 basic drugs. At present the system is at the experimental stage.

Examples of applications using the retention time locked library will be presented in the poster.

Keywords: Basic Drugs, Retention Time Locking, RT Library.



The Decision to Use Modern Gas Chromatography/Mass Spectrometry Methods in Forensic Examinations

Alexandr Tsymbal
Research Institute of Forensic Expertises of the Justice Ministry of Ukraine

The possibilities of gas chromatography in expert practice are expanding with the introduction of progressive gas-chromatographic equipment - application of new modes of realisation of chromatographic analysis with the use of temperature programmed injectors and electronic flow rate controllers, and also modern mass-spectral methods of detecting.

Temperature-programmed injectors and electronic regulation of gas-carrier velocity allow us to increase the detection limit of substances, to reduce their mass discrimination during evaporation in injector and input in a column, and to optimise the process of chromatographic separation.

The mass-spectral detector allows identification of substances according to their mass-spectra (including isomers), reveal trace amounts of substances (the screening of substances), conduct quantitative analysis of substances even in the case of bad chromatographic separation - by peaks superposition of different substances. The use of the modern chromatograph with the mass-spectral detector QP (Shimadzu) has allowed us:

  • to identify trace amounts of drugs, even psychotropic ones;
  • to establish the method of their manufacture and storage conditions (solvents, precursors and ingredients on one chromatogram);
  • to identify the contents of toxic volatile substances in various matrices (including in fats, drinks) with applications both static, and dynamic headspace methods of analysis; to establish the nature of substances of unknown origin.

The Direct Input in the mass-spectral detector QP also allows identification of nonvolatile substances after thin layer chromatography.

The software package "Expert" enables reconstruction of the structure of a substance which is absent from commercial libraries of mass-spectra.

Keywords: Gas chromatography, Mass-spectrometry, Progressive gas-chromatographic equipment.



Investigation of Biological Markers of Chronic Alcoholism

Erzsebet Jeszenszky*1, Tibor Varga1, Peter Freudenstein2, Wolfgang Bonte2
1Albert Szent-Gyorgyi Medical University, Department of Forensic Medecine, Szeged, Hungary
2Heinrich Heine University, Department of Forensic Medicine, Dusseldorf, Germany

Alcohol is a prominent risk factor in traffic accidents. Investigations have shown that both in Germany and in Hungary a significant number of chronic alcoholics take part in traffic and form a special risk-group.

Diagnosing chronic alcoholism at present is almost exclusively based on psychiatric and psychological tests. It is necessary to work out an objective laboratory investigation method, by means of which chronic alcoholics can be identified in time and, if necessary, sanctioned effectively. Besides the high concentration of blood alcohol, the so-called biochemical markers of chronic alcoholism are taken into account, one of which is the increased methanol level of the blood.

The methanol level of the blood and the methanol content of consumed beverages are related to each other. There are significant regional differences in alcohol-consuming habits and in the quality of alcoholic beverages.

In our work we collected blood samples from chronic alcoholics in the Szeged region, Hungary. The samples were analysed with head-space gas chromatography. The following biochemical markers of alcoholism were measured: ethanol, methanol, acetone, 2-propanol and 1-propanol. Our data were compared with similar data from Dusseldorf and Vienna.

The result of the investigation contributes to the unambiguous evaluation of methanol as a biochemical marker of chronic alcoholism. The results can be used in Hungary for the modification of traffic safety regulations.

Keywords: Chronic Alcoholism, Biological Markers, Methanol.



Methadone Substitution Therapy Program and Toxicological Studies

Halina Matsumoto*, Elzbieta Wozny, Anna Dziklinska, Malgorzata Abramowska
The University Medical School of Warsaw, I Department of Psychiatry, Laboratory of Psychopharmacology, Nowowiejska 27, 00-665 Warsaw, Poland

The purpose of this paper was to present the significance of toxicological studies assessing the effectiveness of methadone substitution therapy. The research was carried out using urine samples, collected many times from 189 patients dependent on opioids. These patients were treated by methadone (20-130 mg/day) at the Nowowiejski Hospital in Warsaw from 1993-1998. The tests were carried out by fluorescence polarisation immunoassay (FPIA) on TDx apparatus, produced by Abbott. Results higher than the cut-off level were accepted as positives. The Methadone Program lasted 6 years. During that time, 11424 analyses were performed. Of this number 3411 (or 30 per cent) were analyses of opioids, 2451 - of benzodiazepines (21%), 2581 - of barbiturates (23%), 2746 - of amphetamines (24%),and 235 of cannabinoids (2%).The results of the toxicological studies indicate a decrease in the percentage of positive results in the first 5 years of the execution of the Program. But in the last year they have been less satisfactory. The authors discuss a possible reason for this outcome. The obtained findings show that the FPIA method is very useful in the objective assessment of the efficacy of methadone treatment, on condition that one is aware of the possibilities and limitations of this method. There is, however, an urgent need to use reference methods, in clinical practice, too.

Keywords: Methadone Treatment Efficacy, Drug of Abuse Testing, Interpretation of Results.



Salicylamide in Urine after Intake of Acetylsalicylic Acid due to Degradation of Salicylic Acid Conjugates

Kurt Besserer 1, Detlef Tiess 2, Maria Kala *3
1Formerly Institute of Forensic Medicine, University in Tubingen, Germany
2Formerly Institute of Forensic Medicine, University in Rostock, Germany
3Institute of Forensic Research, Westerplatte 9, PL-31-033 Cracow, Poland

In addition to well-known acetylsalicylic acid (ASA) metabolites, salicylamide (SA) was detected in original or acidified urine samples taken from healthy persons after intake of ASA.

SA was found in diethyl ether extracts of urine after samples alkalisation by ammonia solution. SA was identified by means of thin layer chromatography (TLC), high performance chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Broader examinations showed that SA was detected in urine samples only after its alkalisation by ammonia solution.

Using dimethyl-, diethyl- or dibutylamine for alkalisation in urine samples corresponding salicylic acid dialkylamides were detected.

Analytical results lead to the conclusion that ester type salicylic acid glucuronide conjugates undergo degradation by hydrolytical amination to corresponding amides. This gives the possibility of a specific demonstration of the presence of ester type glucuronide conjugates.

Further study will show if this reaction can be transferred to other active substances or other decomposition products with carboxyl groups.

Keywords: Salicylamide, Salicylic acid, Degradation of Glucuronide.



A Simultaneous Determination of Naltrexone and 6-b-Naltrexol in Serum by HPLC

Katja Sarkka, Kari Ariniemi and Pirjo Lillsunde*
Laboratory of Substance Abuse, National Public Health Institute Mannerheimintie 166, Fin-00300 Helsinki, Finland

Liquid-liquid extraction with butylacetate from basic solutions (pH 9) was chosen as the extraction method for the serum samples. Nalorphine was used as an internal standard. Analytes were back-extracted from organic solvent into perchloroacid.

The acid extract was chromatographed by HPLC with reverse-phase ODS-column and electrochemical detector. The mobile phase was NaH2PO4-solution with acetonitrile as an organic modifier and octanesulphonic acid and tetraethylammonium-hydrogensulphate as ion-pair reagents. The identification of drugs was based on retention times, which were checked with calibrating standards before each determination.

The recovery of the extraction method was 48% for naltrexone and 75% for 6-b-naltrexol. The reliability of the developed determination method was tested on the basis of linearity, accuracy and intra-day and inter-day assay precision. The limit of quantitation was 5.0 ng/ml for naltrexone and 1.0 ng/ml for 6-b-naltrexol. Accuracy and precision were tested at two concentration levels. Analysed concentrations of naltrexone differed from theoretical concentrations 0.7-2.3% and those of 6-b-naltrexol 2.6%. The relative standard deviation of the intra-day assay was 0.9-5.7% for naltrexone and 0.8-4.2% for 6-b-naltrexol and inter-day 5.7 and 4.2% respectively. On the basis of the validation results the developed method is found suitable for determination of naltrexone and 6-b-naltrexol in human serum.



Propofol Metabolism after Excessive Abuse

Achim Schmoldt*, S. Iwersen-Bergmann, H. M. Rosner
Department of Legal Medicine, Butenfeld 34, University of Hamburg, D-22529 Hamburg, Germany

A male nurse was found dead in his flat with several empty ampoules of propofol together with 20 ml syringes which were apparently repeatedly used for injections. Toxicological GC/MS analysis of urine extracts showed high concentrations of substances with similar mass spectra to propofol. Thus we tried to identify the propofol metabolites. The neutral and acidic extracts of the urine before and after acid hydrolyzation (100°C/30min) were analyzed by GC/MS and GC/MS-MS (EI and CI-mode).

In addition to the well-known conjugates of propofol and the quinol/quinone several alcohols, aldehydes and lactones were found.

In addition to conjugation and p-hydroxylation, propofol can be oxidized on the side chain by cytochrome P450 (at least at high concentrations) followed by dehydrogenation, finally yielding carbonic acids.

Keywords: Propofol, Abuse, Fatality, Oxidative Metabolism, GC/MS/MS.



Fatal Intoxication by Cibenzoline (CIPRALAN)

N. Richard, Marie H. Ghysel*, M. Savart
Laboratoire de Police Scientifique, 7 Boulevard Vauban, 59800 Lille, France

A woman, 35 years old with an alcohol habit, was found dead 3 hours after dinner with her family. She had been treated with Cibenzoline, an antiarrythmic drug.

Toxicological analysis was performed on blood, gastric content and bile by head space gas chromatography, gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography with diode array detector (HPLC-DAD).

Cibenzoline was quantified by HPLC after alkaline extraction (pH 9) by chloroform/isopropanol (9/1, V/V) with di-p-methylcibenzoline as the internal standard. A C18 Nova Pack Waters column was used with: acetonitrile, methanol, ammonium actate 7.5 g/l (30/30/40/, V/V/V) as the mobile phase. UV detection at 220 nm was used for quantification.

Blood, bile and gastric content cibenzoline levels were respectively 22.8; 76.1 and 31.4 mg/l. Blood alcohol concentratio was 2.9 g/l and no other toxic compounds were detected.

Cibenzoline is an antiarrythmic drug. Therapeutic concentrations are usually between 0,3 and 1 mg/l. Up to now 5 fatal intoxications have been described; blood levels were 3.4; 5.3; 8.2; 7.5 and 12 mg/l.

Keywords: Cibenzoline, Death, Toxicological Analysis.



Analysis of Estazolam in Post-mortem Material

Ewa Pufal*1, Marzena Sykutera1, Grzegorz Lis1, Gertrud Rohcholz2, Karol Sliwka1
1Katedra i Zaklad Medycyny Sldowej, Akademia Medyczna w Bydgoszczy, Bydgoszcz, Poland
2Institut fur Rechtsmedizin, Kiel, Germany

Modern techniques provide an opportunity to detect low levels of Estazolam in body fluids. However, only a few published methods have provided routine screening and subsequent confirmative analysis in whole blood or organ material, which are the most common samples received by forensic toxicology laboratories. This analysis still poses problems due to the interference resulting from co-extracted components of biological material.

In this study, an evaluation of suitable extraction and clean up techniques for Estazolam in whole blood and organ material was made. Solvent extraction methods were compared with solid-phase extraction using Extrelut or modified silica adsorbents. For end-step detection method TLC, UV-spectrophotometry, high pressure liquid chromatography (HPLC) with UV detector and gas chromatography-mass spectrometry (GC/MSD) were evaluated.

Keywords: Estazolam, Determination, Post-mortem material.



Stability of Citalopram, Fluoxetine, Paroxetine and their Metabolites in Plasma and Whole Blood from Spiked and Real Samples

Lena Kristoffersen*1, Mette Krogh1, Jorgen G. Bramness1 and Elsa Lundanes2
1National Institute of Forensic Toxicology, P. O. Box 495 Sentrum, N-0105 Oslo, Norway
2Department of Chemistry, University of Oslo, P. O. Box 1033 Blindern, N-0315 Oslo, Norway

The objective of this study was to determine the stability of citalopram, fluoxetine, paroxetine and their metabolites in sodium fluoride whole blood and sodium-citrated plasma from spiked and real samples under different storage conditions. Simultaneous determination of the analytes was performed by solid phase extraction followed by reversed phase high performance liquid chromatography using ultraviolet and fluorescence detection (1).

Spiked whole blood and plasma samples stored at room temperature in dark and light, and at 4°C were analyzed 0, 1, 2, 3 and 7 days after the preparation. The samples were also stored at -20°C and -78°C and analyzed after 0, 15, 30, 58 and 90 days. The short-term stability tests revealed that the stability of the analytes in spiked whole blood and plasma samples were different, and generally, the analytes were more stable in plasma than in whole blood. Furthermore, generally no difference was found between the spiked samples stored in light and dark conditions. The long-term stability results will be presented.

Real plasma and whole blood samples were obtained from patients who were treated with a therapeutic dose of either Seroxat (Novo Nordisk, Copenhagen, Denmark), Fontex (Eli Lilly, Indianapolis, USA) or Cipramil (H. Lundbeck, Copenhagen, Denmark). The stability under different storage conditions and successive thawing-freezing procedures will be reported.

1. L. Kristoffersen, A. Bugge, E. Lundanes and L. Slordal. Simultaneous Determination of Citalopram, Fluoxetine, Paroxetine and their Metabolites in Plasma and Whole Blood by High Performance Liquid Chromatography with Ultraviolet and Fluorescence Detection. J. Chromatogr. B. In press.

Keywords: Selective Serotonin Reuptake Inhibitors, Drug Stability, Real and Spiked Samples.



Magic Mushrooms: Case Reports, Screening and Detection of Psilocin in Mushrooms and in Urine

Werner Bernhard1, Beat Aebi1, Justus Beike2, Michael Frost2 and Herbert Honegger3
1Institute of Legal Medicine, University of Bern, Buehlstrasse 20, CH-3012 Bern, Switzerland
2Institute of Legal Medicine, University of Munster, Von-Esmarch-Str. 62, D-48149 Munster, Germany
3Easy - Link AG, Scientific Services, Post Box 354, CH-4501 Solothurn, Switzerland

In 1998 the number of Magic Mushroom seizures in Switzerland and Germany increased significantly. Magic Mushrooms are imported on a kilogram scale, mainly from Holland. The biggest seizure in 1998 by the Bern Police was 7931 g dried Magic Mushrooms in the possession of a Hashish importer and dealer, see case B. Magic mushrooms were found vacuum sealed in bags and smaller portions were also found packed in re-sealable plastic bags (minigrip). The police in Germany also seized tea bags containing Magic Mushrooms. The screening for Psilocin in dry mushrooms is easily performed by Ion Mobility Spectrometry (IMS). A few milligrams of the powdered sample can be directly introduced into the ion mobility spectrometer on a membrane filter. Psilocin traces are detected by IMS in approx. 4 seconds. Confirmation and determination of Psilocin and Psilocybin is straightforward and can be done either by Capillary Electrophoresis (CE), by HPLC, or by Instrumental Thin Layer Chromatography (TLC). Results of recent case work are given follow:







(CHF per g)


Copelandia Cyanescens

Stropharia Cubensis







20 to 60








*Hemp shop, respectively street drug price 1998/99

The confiscated tea bags contained approximately 4 g of drug material per bag ("Hawaii Tea"). To assess the quantity of Psilocin and Psilocybin liberated in an "average" tea a confiscated tea bag was placed in 250ml of boiling water and left to stand for 8 minutes.

Quantification was done by HPLC-UV and gave 2.25 mg Psilocin and 0 mg Psilocybin in the infusion for the Hawaii tea (A) and 0.45 mg Psilocin and 4.40 mg Psilocybin in the infusion "Mix Tea" (B).

Immuno-chemical test kit: We present the first results of the development of an immuno-chemical test kit for the urine screening: BALB/c mice were immunized with a BSA - Psilocin conjugate with 4 boosters over a period of 3 month. After subcloning and testing for specificity, 2 clones were selected which were specific to Psilocin and did not exhibit any cross - reactivity to the BSA carrier protein. Both clones had similar displacement at a given concentration of Psilocin and Psilocybin as determined by ELISA; however the cross- reactivity to Psilocybin is somewhat reduced.


Clone A

Clone B

50% displacement (ELISA)

50 ng/ml

45 ng/ml

Cross-reactivity to Psilocybin

(Psilocin = 100%)



N,N- Dimethyltryptamine



Our work indicates that the two clones selected are suitable for employment in assays for the detection of Psilocin in urine as well as Psilocybin present in mushroom extracts.

Keywords: Magic Mushroom, Psilocin, Psilocybin, Immuno-Assay, Ion Mobility Spectrometry (IMS), HPLC.



Street Drugs in Denmark 1995-1998

Kirsten W. Simonsen*1, E. Kaa1, E. Nielsen2, D. Nielsen3
1 University Institute of Forensic Medicine, DK-8240 Aarhus, Denmark
2 University Institute of Forensic Medicine, DK-2100 Copenhagen, Denmark
3University Institute of Forensic Medicine, DK-5000 Odense, Denmark

In 1995 a pilot investigation of drugs for illicit sale on the street was introduced by the Danish National Board of Health. The investigation has been performed every year since January 1st 1995 and is continuing in 1999.

The aim of the examination is to get insight into the supply of illicit drugs in different parts of Denmark.

The study shows data from 1995 to 1998. Deviations between years and different parts of Denmark with a view to type and purity will be given.

At present we do not have all the data from 1998 but in 1997 the police seized 217 samples. The most common drugs seized were: heroin base: 89, heroin hydrochloride: 41, amphetamine sulphate: 56 and cocaine chloride: 19. Two of the seized samples were designer drugs (MDMA, MBDB).

Keywords: Street Drugs, Heroin, Amphetamine, Cocaine.



The Application of Spectral and X-ray Diffraction Methods in the Analysis of Street Drugs

Bretislav Smysl*1, Dagmar Krausova2, Jan Hlavac 2
1 Institute of Forensic Medicine and Medical Law of Palacky University, Olomouc, Czech Republic
2 Departments of Analytical and Inorganic Chemistry of Palacky University, Olomouc, Czech Republic

The aim of this paper is to verify the availability of Infrared-Spectroscopy and X-Ray Diffraction for the analysis of so called 'street drugs'.

The growing trend in the abuse of drugs of addiction, which we recently observed, is also reflected in the increasing demands on the work of the forensic toxicologists.

It is necessary, due to the requirements of the police, to analyse the samples of substances which have been taken from the drug addict and/or from the dealers - so called "street drugs".

The essential question is whether the sample is a drug at all, and in the case of a positive results, which drug it is and of what quality. This information is of decisive importance for the possible punishment.

For the identification of pure substances, the method of the Infrared Spectroscopy is convenient. For the analysis of mixtures of substances it is better to use the X-Ray-Diffraction.

Using Infrared-Spectroscopy, samples of amphetamines, opiates etc. were identified. Hence, X-Ray Diffraction was used especially in the identification of mixtures.

The use of both methods is manifested on the cases from routine work.

Keywords: Street Drugs, Analysis, Spectral Methods.



Chemical Programmes in Thanatometric Laboratory

Volodymyr Bilkun
Zaporozhye Medical University, Ukraine

We have in our Thanatometric Laboratory several chemical Programmes. The Special Department of Necrochemistry have been constructing instruments for estimation of the postmortem interval at the death scene and in morgue by chemical methods.

We have been elaborating Complex-Programme "Thanexplos" (Death and Explosion) with 3 Subprogrammes: "Chemistry of Explosion", "Chemistry of Shot", "Chemistry of Fire". The General Programme "Thanrisk" (Death and Risk) has Subprogrammes - "Toxithan" and "Thanecol".

We need cooperation and collaboration among scientists in Forensic Medicine and Toxicology.

Key Words: Thanatometria, Necrochemistry, Estimation time of death, Chemistry of Explosion and Shot.



Three Cases of Thinner-Intoxication and Estimating the Lethal Concentration of Toluene by Toxicokinetics

Tatsuya Hobara, M. Okuda, C. Yamada, H. Kobayashi, M. Gotoh, K. Oki and H. Segawa
Department of Public Health Yamaguchi University School of Medicine, 1144 Kugushi, Ube 755-8505, Japan

Acute intoxication due to inhaling thinner happens occasionally in many painting situations, and some accidental deaths are caused by this poisoning every year in Japan.

The concentration of thinner in ambient air that can cause death is unknown. To prevent accidental death, it is very important to determine the potentially fatal level of thinner concentration in the air. Between 4 January 1966 and 21 April 1977, we encountered three patients who suffered severe acute thinner intoxication. One died and the other two recovered with no after-effects. During their treatment, we measured the concentration of toluene in the blood and those of toluene and hippuric acid in the urine of these patients. By extrapolation from the results of our previous toxicokinetics research on toluene poisoning in anaesthetised dogs, we estimated the fatal levels of toluene concentration in ambient air over certain exposure periods. The fatal concentration of toluene was estimated to be approximately 1800 to 2000 ppm over a one-hour exposure.

Keywords: Toluene, Fatal Concentration, Toxicokinetics.



Validation and Quality Assurance of a Broad Scale Gas Chromatographic Screening Method for Drugs

Merja Gergov*, Jari Nokua, Ilpo Rasanen and Ilkka Ojanpera
Department of Forensic Medicine, University of Helsinki, P.O.Box 40, FIN-00014, Helsinki, Finland

In post mortem forensic toxicology, the laboratory should be able to detect a broad range of potentially toxic xenobiotic compounds that may have had an influence on the death. Consequently, various drug screening procedures play an important role in the investigations. The broadness of the screening as well as the quantitative accuracy are important parts of the quality policy for drug screening.

The present study describes the validation and quality assurance procedures of a gas chromatographic (GC) screening method for basic drugs in autopsy blood. The method was accredited in 1997 by FINAS. The method involves liquid-liquid extraction and automated dual-column GC screening with simultaneous quantification. The identification of 95 basic drugs is based on retention index monitoring and the quantification is carried out using a single internal standard.

The validation of the method includes the determination of injection repeatablity, stability of calibration, linearity, accuracy, precision, limit of quantitation and uncertainty of measurement. The preventive maintenance of the GC instrument is carried out weekly, and the qualitative and quantitative calibration with all compounds at 1-3 concentration levels is performed monthly. The performance of the instrument and column is checked daily with a control sample before each sequence of samples. The internal quality control consists of the analysis of a control sample once a week. This sample has a selection of four representative drugs, which are changed annually. The external quality control involves participation in two Nordic interlaboratory test comparisons twice a year. The validation procedures and quality control results will be presented and discussed.

Keywords: Drug Screening, Validation, Quality Assurance.



Human Liver Carboxylesterases: Purification of these Two Enzymes, Properties in Relation to Cocaine and Alcohol Metabolism, and Specificities

Karine Berthoin*1,2, Francois Widmer3, Christian Giroud2, Patrice Mangin2 and Luc Barret1
1Laboratoire de Medecine Legale et Toxicologie, UFR de Medecine, Grenoble, France 2Laboratoire de Toxicologie Analytique, Institut Universitaire de Medecine Legale, Lausanne, Switzerland 3Institut de Biologie et Physiologie Vegetales, Lausanne, Switzerland

With respect to cocaine metabolism, two human liver carboxylesterases, designated hCE-1 and hCE-2, catalyze hydrolysis of the methyl and benzoylester linkages of cocaine, respectively. In the presence of ethanol, hCE-1 also catalyzes the ethyl transesterification of cocaine forming cocaethylene. As far as we know, few data are available concerning other pathways for the mutual metabolism of cocaine and alcohol.

The purpose of this work was: 1) to Investigate cocaine metabolism in association with alcohol intake thoroughly. Carboxylesterases specificities with cocaine and cocaethylene metabolites were determined by incubating one of the two purified enzymes with benzoylecgonine, ecgonine methylester, ecgonine ethylester, norcocaine and norcocaethylene; 2) to determine each enzyme's affinity for xenobiotics and different endogenous substrates. Affinity for xenobiotics with a chemical structure related to cocaine was tested with scopolamine and other similar molecules. hCE-1 capacity to esterify fatty acids of various chain length and polarity was also determined.

The first step consisted in isolating carboxylesterases I and II from human liver obtained by autopsy. The whole purification procedure was performed with a SMART System® from Pharmacia. This procedure required three steps: 1) Ions exchange chromatography (Mono Q HR 5/5 column); 2) Affinity chromatography on Concanavalin A Sepharose 4B (Mono Q HR 5/5 column); 3) Gel filtration (Superdex 200 PC 3.2/30 column). Enzymatic activities were measured by Reverse-Phase High-Performance Liquid Chromatography/ Diode Array Detector.

Based on partial results, benzoylesterase specific activity (ecgonine methylester forming) was 0,26 U/mg at the first step of the purification using cocaine as the substrate. Methylesterase specific activity (benzoylecgonine forming) was 0,78 U/mg at the second step.

Keywords: Cocaine, Ethanol, Cocaethylene, Carboxylesterases, Human, Liver.



Paraoxanase and Acetylcholinesterase Activities in Human Exposure to Organophosphorous Compounds

Serap A. Akgur*1, P. Uzturk1, E. Y. Sozmen2, Y. Delen2, T. Tanyalcyn2, B. Ege1
1Ege Unyversity, Department of Forensic Medicine, Turkey
2Ege Unyversity, Department of Biochemistry, Turkey

Different kinds of organophoshorous compounds (OPc) are used in the agricultural industry, in Turkey. Deliberate or accidental intoxication or occupational exposure are common in developing countries. Serum paraoxonase (PON) hydrolyses the toxic metabolites of a variety of Opc's. It is known that there are competing reactions for -oxon; one is the direct interaction with cholinesterase, and the other enzymatic hydrolysis by PON. In recent years some studies have shown that PON activity might be more important in individuals who are exposed to Opc's.

Cholinesterase and PON activity in serum were measured spectrophotometrically in 18 agricultural workers who were chronically exposed to azinphos metyl, chlorpyrophos and malathion etc. during cereal spraying, transportation and storage. The individuals were classified for PON phenotypes using the antimode 60% stimulation as the dividing point between non-salt stimulated A type (homozygotes for the low activity allele) and salt stimulated AB (heterozygotes) and B types (homozygotes for the high activity allele).

There was a positive correlation between acetylcholinesterase (AChE) activities and percent of PON stimulation (r=0.6493, p=0.009). The individuals with phenotype A had lowest activities (35.2+/-8.2 Rap.U/ml).

This study suggests that individuals with phenotype A also had low AChE activities and thus might be more sensitive to OPc toxicity.

Keywords: Paraoxonase, Cholinesterase, Organophosphorous.



Distribution of Heavy Metals in Normal Korean Tissues

Youngchan Yoo1, Sangki Lee*1, Jayeol Yang1, Sangwhan In1, Kiuk Kim1, Sunchun Kim1, Taejung Kwon1, Youngchang Ko1, Kyuhyuck Chung2
1 National Institute of Scientific Investigation, 331-1 Shinwol-dong, Yangchon-ku, Seoul, Korea
2 College of Pharmacy, Sungkyunkwan University, 300 Chunchun-dong, Jangan-ku, Suwon, Korea

To obtain the usual value of heavy metals in the normal human body, the amounts of 16 metals (Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Se, Si, Sn, V and Zn) were determined in 90 Korean cadavers.

A portion of the tissues (0.1~2.0 g) was digested with concentrated nitric acid and hydrogen peroxide in a sealed teflon vessel, diluted with distilled water and heavy metals were determined by inductively coupled argon plasma atomic emission spectrometer (ICP-AES).

Correlation coefficients (r) of standards solutions (0.025 ~ 2.0 ppm) were more than 0.999 and recoveries of heavy metals ranged from 81 to 106.3%.

From the results, Cd, Hg, Pb and Zn were found in large quantities in the metabolic organs, whereas the concentration of Al, Cr, and Si were greatest in the tissues exposed to the exterior. Almost every heavy metal was deposited in the hair and nail.

High correlations between essential elements such as Se and Zn, and toxic elements such as Hg and Cd were observed in human organs. These results might be a reflection of the protective effects of essential elements against toxic elements. For example, the correlation between Zn and Cd in the kidney was significant (r=0.799).

Keywords: Heavy Metals, Distribution, Korean.



Aluminum Phosphide Fatalities, New Local Experience

Hasn A. Abder-Rahman, Abdelkader H. Battah*, Y. M. Ibraheem, M. S. Shomaf and N. El-Bataineh
Faculty of Medicine, University of Jordan, Jordan

Aluminum phosphide (AlP) pesticide is a highly toxic, low cost, and easily accessible rodenticidal agent. Its toxicity results from the liberation of phosphine gas upon exposure to moisture, which leads to multisystem involvement, resulting in serious consequences. The highly toxic parathion insecticide was a common cause of mortality in pesticide fatalities, prior to its banning. Its toxicity was familiar to the public as well as to physicians. Recently, ten fatalities due to AlP were encountered within a three-month period during spring.

A1P was used as a rodenticide in the vicinity of grain stores. Individuals' ages ranged from 1-34 years. The circumstances of death were accidental in six cases, suicidal in two and possibly homicidal in two cases.

Retrospectively, the clinical manifestations, scene investigation, autopsy, histological and toxicological findings supported the diagnosis of AlP intoxication. Immediate recognition was difficult due to physicians' unfamiliarity with this agent. The occurrence of these fatalities might suggest changes of pesticide poisoning patterns. This should raise the attention of physicians to the problem of AlP poisoning and also necessitates awareness on the part of the public to the hazards of this poison. Education, proper handling, strict observation and abiding by the regulations controlling this material are good protective measures against AlP poisoning.

Keywords: Phosphide, Fatalities.



Hepatotoxicity of Paracetamol and Trichloroethylene Combination in Rats

Barbara Zielinska-Psuja*1, Joanna Kowalowka-Zawieja1, Andrzej Plewka2, Jerzy Orlowski1, Marcin Kaminski2
1Department of Toxicology, Karol Marcinkowski University of Medical Sciences, Poznan, Poland
2Department of Histology and Embryology, Silesian School of Medicine, Katowice-Ligota, Poland

Paracetamol (APAP) is a readily available drug that is often used in intentional intoxication. Paracetamol overdosage can cause liver damage and even acute liver failure. Trichloroethylene (TCE) is one of the most important organic solvents used in aerosol sprays, fabric protectors, and metal degreasers. Many life situations may be associated with exposure to both these xenobiotics.

The aim of the study was to evaluate the hepatotoxic effects of paracetamol given alone (500 mg/kg b.w.) or in combination with trichloroethylene (250 mg/kg b.w.). Both compounds were administered orally through a stomach tube.

Rats were examined 4, 6, 12, 24, 48 and 72 hrs after intoxication. To assess the function of the liver, the following activities of plasma enzymes were determined: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) In addition, we measured the levels of liver protein, cytochrome P-450 and cytochrome b5 and the activities of NADPH- cytochrome P-450 reductase and NADH-cytochrome b5 reductase.

When given alone, both APAP and TCE increased AST and LDH activities 12 h after intoxication. In contrast, APAP combined with TCE increased LDH activity only; however, the effect was weaker than those observed when APAP and TCE were given separately. Forty eight hours after intoxication all enzyme activities were lower than the control values.

The changes in enzymes activities within 24 h after APAP and TCE administration were accompanied by a decrease in content of cytochrome b5 and a lowering of activity of both reductases. APAP increased, whereas TCE decreased cytochrome P-450 content.

The increased AST and LDH activities and changes in the cytochrome P-450 dependent monooxygenase system suggest subclinical liver damage caused by the simultaneous action of APAP and TCE. The study has shown a significant negative correlation between cytochrome P-450 content and LDH and AST activities.

Keywords: Paracetamol, Trichloroethylene, Hepatotoxicity.



Experimental Data Regarding the Effects of Amiodarone on Pregnant Rats

Razvan Gligor*1, Virginia Gligor2
1UMF Timisoara, Romania
2University of V. Goldis, Arad, Romania

The present study was designed to evaluate the effects of amiodarone, an antianginal and antiarrhythmic agent on pregnant rats.

The drug was administered at dosages of 10 mg/kg/day and 90 mg/kg/day, intaperitoneally, once, to the pregnant female rats (on the 10th and the 12th day of gestation). Clinical signs, body weight and feed consumption were recorded daily.

Evaluations were made for number of newborn cubs and number of cubs born dead, number of cubs born with malformations and number of cubs dead after birth (during the first 30 days); cubs were also examined for body weight. An increased frequency of deaths amongst newborn cubs and a decrease in their body weights were observed in rats to which doses of 90 mg/kg/day were administered. In the same way, it has been shown that the offspring nursing by lactating rats, treated during gestation with amiodarone, were less viable and have gained reduced body-weight.

Keywords: Amiodarone, Embriogenesis, Malformations.



Electrophoretic Analyses of Serum Proteins of Birds and Mammals

E. G. E. Helal1, S. A. M Zahkouk2, Hamdy A. Mekkawy3
1Faculty of Sciences, Al - Azhar University (Girls)
2Faculty of Sciences, Al - Azhar University (Boys)
3The National Center for Social and Criminological Research, Zamalek, P.O., P.C. 11561, Cairo, Egypt

The electrophoretic patterns of the blood serum of birds such as chickens and mammals such as Guinea pigs, cats, dogs and rabbits were checked. Various protein, glycoprotein and lipoprotein bands were separated by both native and SDS methods. Chickens showed 14 bands, while pigeons showed 12 bands by the native electrophoretic method for total proteins. On the other hand, both cats and dogs showed 7 bands, while Guinea pigs and rabbits showed 10 and 13 bands respectively. Total proteins by SDS methods for birds achieved 8 and 14 bands for pigeons and chickens respectively. The SDS method revealed 13 bands for Guinea pigs and rabbits, and 10 and 18 bands for cats and dogs respectively. Native glycoprotein serum showed 8 bands for both pigeons and chickens. Rabbits and Guinea pigs showed 8 bands while white cats showed 10 and dogs showed 7 bands. SDS glycoproteins were significantly different between pigeons and chickens showing 4 and 10 bands respectively. The serum of Guinea pigs showed 8 bands but that of dogs and cats showed 11 and 16 bands respectively.

Prestaining lipoprotein showed 2 bands for cats, dogs and rabbits. Four bands were separated for both chickens and Guinea pigs and 5 bands for pigeons. The basic data reported here represents a Fingerprint specific to each animal useful in classification and evolution studies and it may be of great importance in forensic science.

Keywords: Electrophoretic, Birds, Mammals.



A Silent Killer of Intelligence: Lead Poisoning in Children in the USA, Thailand, Laos, Mexico, El Salvador, Nigeria and Jamaica

Karl Verebey*, Raul Rudelli and Philip Fischer
Leadtech Corporation, One Marine Pl., N. Bergen, NJ, USA
Mayo Clinic, Rochester, MN, USA

Elevated blood lead concentration (BLC > 100.0 µg/l) without overt physical symptoms of lead poisoning decreases the intelligence quotient (IQ) of young children from birth to six years of age. During the neurodevelopmental period dendrite networking occurs which is blocked by lead if present in the bodies of children. Other than lower IQ, lead causes attention deficit and hyperactivity disorders which prevents the education and future employability of affected children. Legal proceedings are rampant in the USA against negligent landlords of apartments painted with old peeling lead containing paints which correlates with resident children having low intelligence and attention deficit-hyperactivity disorders. Early diagnosis allows treatment of the children using environmental intervention, nutrition and chelation therapies. The only diagnostic technique of low level lead exposure is screening for BLC. A practical atomic absorption based analytical method requiring two drops of finger stick blood onto a filter paper collector was utilized to identify various locations in the world, where elevated lead concentration in the environment may cause serious intellectual damage to developing children. The dried blood samples on the filter paper collectors are stable for at least six months. In 1996-98 Leadtech screened 220,000 children for blood lead in the USA and spot check screenings were performed in Thailand (n=189), Laos (n=179), Mexico (n=100), El Salvador (n=301), Nigeria (n=632) and Jamaica (n=111). Average BLCs in order of increasing lead concentration were: Thailand 43.0 µg/l, USA 44.5 µg/l, El Salvador 63.5 µg/l, Laos 66.5 µg/l, Mexico 79.7 µg/l and Nigeria 97.3 µg/l. The% of subjects >200.0 µg/l were in ascending order: Thailand 0%, Laos 1%, USA 2.2%, Mexico 3%, El Salvador 6% and Nigeria 8.22%. Children in the Jamaican study had extremely high BLC (> 600.0 µg/l). These children lived in an area where an old ore mine operated in the past. Thus, this practical method helps to identify very quickly areas of concern and allows environmental and medical intervention of affected children.