La toxicologie forensique, une discipline scientifique en plein essor [Forensic toxicology, a growing scientific discipline]

La recherche de substances ayant pu jouer un rôle direct ou indirect dans la cause de la mort ou ayant pu modifier le comportement d'un individu constitue la mission première de la toxicologie forensique. L'augmentation de la consommation de substances illégales et de médicaments dans les sociétés modernes a fait connaître à la toxicologie forensique un essor très important ces dernières décennies. D'autre part, les développements technologiques analytiques ont permis d'obtenir des outils très sensibles et spécifiques pour la recherche et le dosage d'une multitude de substances dans une grande variété d'échantillons biologiques, parfois présentes à des concentrations très faibles, conséquence d'une dose équivalente à la prise d'une seule unité galénique. Forensic toxicology has to bring evidence of substances that could have been involved directly or indirectly in the cause of death or that could influence the behaviour of somebody. The increase of the consumption of illegal and legal drugs in modern societies during last decades gave a boost to forensic toxicology. Moreover, improvement with analytical technology gave tools with high degrees of sensitivity and specificity for the screening and quantification of a large amount of substances in various biological specimens, even with very low concentration resulting of a single dose of medication.

Development and validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347-->163 (for the quantification) and m/z 347-->119 (for the identification) for EtG, and m/z 352-->163 for EtG-d(5) used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.

Pharmacogenetics and forensic toxicology

Large inter-individual variability in drug response and toxicity, as well as in drug concentrations after application of the same dosage, can be of genetic, physiological, pathophysiological, or environmental origin. Absorption, distribution and metabolism of a drug and interactions with its target often are determined by genetic differences. Pharmacokinetic and pharmacodynamic variations can appear at the level of drug metabolizing enzymes (e.g., the cytochrome P450 system), drug transporters, drug targets or other biomarker genes. Pharmacogenetics or toxicogenetics can therefore be relevant in forensic toxicology. This review presents relevant aspects together with some examples from daily routines.

In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

An investigation into the suitability of time-of-flight mass spectrometry in forensic toxicology

Liquid chromatography coupled with mass spectrometry is one of the most powerful tools in the toxicologist’s arsenal to detect a wide variety of compounds from many different matrices. However, the huge number of potentially abused substances and new substances especially designed as intoxicants poses a problem in a forensic toxicology setting. Most methods are targeted and designed to cover a very specific drug or group of drugs while many other substances remain undetected. High resolution mass spectrometry, more specifically time-of-flight mass spectrometry, represents an extremely powerful tool in analysing a multitude of compounds not only simultaneously but also retroactively. The data obtained through the time-of-flight instrument contains all compounds made available from sample extraction and chromatography, which can be processed at a later time with an improved library to detect previously unrecognised compounds without having to analyse the respective sample again. The aim of this project was to determine the utility and limitations of time-of-flight mass spectrometry as a general and easily expandable screening method. The resolution of time-of-flight mass spectrometry allows for the separation of compounds with the same nominal mass but distinct exact masses without the need to separate them chromatographically. To simulate the wide variety of potentially encountered drugs in such a general screening method, seven drugs (morphine, cocaine, zolpidem, diazepam, amphetamine, MDEA and THC) were chosen to represent this variety in terms of mass, properties and functional groups. Consequently, several liquid-liquid and solid phase extractions were applied to urine samples to determine the most general suitable and unspecific extraction. Chromatography was optimised by investigating the parameters pH, concentration, organic solvent and gradient of the mobile phase to improve data obtained by the time-of-flight instrument. The resulting method was validated as a qualitative confirmation/identification method. Data processing was automated using the software TargetAnalysis, which provides excellent analyte recognition according to retention time, exact mass and isotope pattern. The recognition of isotope patterns allows excellent recognition of analytes even in interference rich mass spectra and proved to be a good positive indicator. Finally, the validated method was applied to samples received from the A& E Department of Glasgow Royal Infirmary in suspected drug abuse cases and samples received from the Scottish Prison Service, which we received from their own prevalence study targeting drugs of abuse in the prison population. The obtained data was processed with a library established in the course of this work.

Fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry for forensic analysis of cannabinoids in whole blood.

The present work describes a fast gas chromatography/negative-ion chemical ionization tandem mass spectrometric assay (Fast GC/NICI-MS/MS) for analysis of tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in whole blood. The cannabinoids were extracted from 500 microL of whole blood by a simple liquid-liquid extraction (LLE) and then derivatized by using trifluoroacetic anhydride (TFAA) and hexafluoro-2-propanol (HFIP) as fluorinated agents. Mass spectrometric detection of the analytes was performed in the selected reaction-monitoring mode on a triple quadrupole instrument after negative-ion chemical ionization. The assay was found to be linear in the concentration range of 0.5-20 ng/mL for THC and THC-OH, and of 2.5-100 ng/mL for THC-COOH. Repeatability and intermediate precision were found less than 12% for all concentrations tested. Under standard chromatographic conditions, the run cycle time would have been 15 min. By using fast conditions of separation, the assay analysis time has been reduced to 5 min, without compromising the chromatographic resolution. Finally, a simple approach for estimating the uncertainty measurement is presented.

Identificação química da clorofenilpiperazina (CPP) em comprimidos apreendidos

Designer drug is a term used to describe psychoactive drugs of abuse which are usually synthesized by modifying the molecular structures of existing drugs of abuse. The term gained widespread popularity when MDMA (ecstasy) experienced a popularity boom in the mid 1980´s. In Brazil, designer drugs seizures have increased in the last few years, and actually tablets with unknown psychoactive compounds began to be forwarded to the Forensic Laboratories. This work describes the analytical assays that were performed to identify the chlorophenylpiperazine, a psychoactive substance first time identified in seized tablets in Sao Paulo state.

Cannabinoid contents in cannabis products seized in Sao Paulo, Brazil, 2006-2007

A rapid and simple method was optimized for determination of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), cannabidiol (CBD), and cannabinol (CBN) contents in cannabis products by gas chromatography with flame-ionization detection (GC-FID), using diazepam as internal standard. All parameters of validation of the method such as linearity, intraassay precision, and limits of detection and quantification of the analytes were satisfactory. Using the described method, cannabinoid contents of 55 cannabis product samples seized in Sao Paulo City, Brazil, in 2006 and 2007 were measured. Delta(9)-THC content in marijuana and hashish samples varied between 0.08% and 5.5%, with an average of 2.5%. The phenotypic ratio showed that the products were able to be designated as ""drug type.""

Desenvolvimento e procedimentos de validação de uma metodologia analítica por GC/MS/MS para a determinação de antidepressivos em sangue total

A depressão é uma das doenças de foro psiquiátrico que mais prevalece na nossa sociedade, subsistindo evidências epidemiológicas que indicam um aumento substancial da sua incidência nos últimos anos. Esta evidência é consubstanciada pelo aumento significativo do consumo de antidepressivos em Portugal. Este cenário pressupõe a necessidade de uma metodologia que permita analisar, com rigor e numa perspectiva de rotina, os antidepressivos que podem ser encontrados em amostras de sangue. No contexto do Serviço de Toxicologia Forense do Instituto Nacional de Medicina Legal, Delegação do Norte, torna-se necessário o desenvolvimento de uma metodologia analítica para a determinação simultânea de 15 antidepressivos em sangue total e a sua validação relativamente a vários parâmetros analíticos. Os antidepressivos considerados foram Amitriptilina, Citalopram, Clomipramina, N-Desmetilclomipramina, Dotiepina, Fluoxetina, Imipramina, Maprotilina, Mianserina, Mirtazapina, Nortriptilina, Paroxetina, Sertralina, Trimipramina e Venlafaxina. A técnica utilizada para este efeito foi o GC/MS/MS, aplicando um procedimento extractivo prévio apropriado, baseado em procedimentos convencionais de extracção em fase sólida. A escolha desta técnica teve por base a possibilidade de identificar inequivocamente os compostos presentes na amostra, independentemente da complexidade da matriz, e de originar metodologias com uma sensibilidade elevada e com limites de detecção muito baixos. Os parâmetros analíticos considerados para validação da metodologia estabelecida foram selectividade/especificidade e capacidade de identificação; limites de detecção e de quantificação; linearidade e gama de trabalho; eficiência de extracção; arrastamento; exactidão (precisão, veracidade e incerteza de medição) e robustez. Com excepção da exactidão, um parâmetro que carece ainda de estudos complementares, todos os parâmetros estudados foram validados de acordo com os requisitos internos do Serviço. De uma forma geral, os resultados obtidos com o método desenvolvido revelaram-se selectivos e apresentaram respostas analíticas tanto para concentrações de antidepressivos em níveis terapêuticos como para níveis letais destas drogas. Os procedimentos extractivos revelaram-se eficazes e não foram verificados fenómenos de arrastamento em concentrações mais elevadas. O método foi ainda considerado robusto.

Long-term effects of cannabis on brain structure.

The dose-dependent toxicity of the main psychoactive component of cannabis in brain regions rich in cannabinoid CB1 receptors is well known in animal studies. However, research in humans does not show common findings across studies regarding the brain regions that are affected after long-term exposure to cannabis. In the present study, we investigate (using Voxel-based Morphometry) gray matter changes in a group of regular cannabis smokers in comparison with a group of occasional smokers matched by the years of cannabis use. We provide evidence that regular cannabis use is associated with gray matter volume reduction in the medial temporal cortex, temporal pole, parahippocampal gyrus, insula, and orbitofrontal cortex; these regions are rich in cannabinoid CB1 receptors and functionally associated with motivational, emotional, and affective processing. Furthermore, these changes correlate with the frequency of cannabis use in the 3 months before inclusion in the study. The age of onset of drug use also influences the magnitude of these changes. Significant gray matter volume reduction could result either from heavy consumption unrelated to the age of onset or instead from recreational cannabis use initiated at an adolescent age. In contrast, the larger gray matter volume detected in the cerebellum of regular smokers without any correlation with the monthly consumption of cannabis may be related to developmental (ontogenic) processes that occur in adolescence.

Analyse des cannabinoïdes par spectrométrie de masse en mode tandem = Analysis of cannabinoids by tandem mass spectrometry

Depuis quelques années, la spectrométrie de masse en tandem (MS/MS) ne cesse de gagner du terrain comme méthode d'analyse en toxicologie forensique, notamment pour le dosage des cannabinoïdes. Couplée à la chromatographie liquide (LC) ou gazeuse (GC), elle permet l'identification fiable et le dosage rapide du THC, de son précurseur acide, et de ses principaux métabolites, y compris les glucuronides. Au cours de ces dix dernières années, un nombre significatif de publications sont parues sur ce sujet. L'objectif de cet article est de passer en revue les analyses par spectrométrie de masse en tandem des cannabinoïdes dans diverses matrices biologiques. In recent years, tandem mass spectrometry (MS/MS) is gaining ground as a reference method of analysis in clinical and forensic toxicology, especially for the determination of cannabinoids. Coupled to liquid chromatography (LC) or gas chromatography (GC), it allows the definitive identification and rapid determination of THC, its acid precursor, and its major metabolites, including the glucuronides. During the past decade, several methods of analysis of cannabinoids in different matrices have appeared on this subject. The aim of this paper is to review the analysis of cannabinoids by tandem mass spectrometry methods in various biological matrices

Accuracy profile validation of a new method for carbon monoxide measurement in the human blood using headspace-gas chromatography-mass spectrometry (HS-GC-MS).

The aim of our study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas ((13)CO) formed by the reaction of labeled formic acid formic acid (H(13)COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of (13)CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10 μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.

Driving under the influence of cocaine or therapeutic administration of cocaine?

Introduction: In forensic toxicology, cocaine is better known for its powerful stimulating effects of nervous system and its high potential for recreational abuse, than for his therapeutic use. However, cocaine is still use as a topical anesthetic and peripheral vasoconstrictor in surgeries of eye, ear, nose and throat. Last decade, an increase of the presence of cocaine and metabolites in blood samples of drivers suspected to drive under the influence of drugs (DUID) was observed in Switzerland (Augsburger et al., Forensic Sci Int 153 (2005) 11-15; Senna et al., Forensic Sci Int 198 (2010) 11-16). Observed blood concentration ranges of cocaine and benzoylecgonine were 10-925 μg/L and 20-5200 μg/L, respectively. Since 2005, zero-tolerance approach was introduced in the Swiss legislation for different substances, especially cocaine (analytical cutoff: 15 μg/L). Thus, the interpretation often amounts to determine if the concentration is situated above or under the limit. However, it is important for the interpretation to take into account the context and to be critical with the obtained results, at the risk of ending in erroneous conclusions. Methods: Systematical toxicological analyses were performed on blood and urine, if available, for 5 DUID cases, as already published (Augsburger et al., Forensic Sci Int 153 (2005)). Positive results were confirmed and drugs were quantified in biological samples by GCMS, GC-MS/MS or LC-MS/MS. Results: Administration of cocaine after traffic accident was identified in five cases. All people were admitted to the emergency room because of severe trauma. Maxillofacial surgery was done shortly after admission to the emergency room, involving use of nasal application of cocaine (swab). For all cases, use of cocaine swab was not mentioned in the document filled by the police and by medical staff requested for blood and urine sampling. The information was obtained retrospectively after consultation of the medical records, without precise indication of the application time or dose. Case 1. A 83-year old man (pedestrian) was hit by a car. Blood (+11h after the accident): cocaine (16 μg/L), benzoylecgonine (370 μg/L). Urine: cocaine (1700 μg/L), benzoylecgonine (560 μg/L). Case 2. A 84-year old woman (pedestrian) was hit by a car. Blood (+1.5h after the accident): cocaine (230 μg/L), benzoylecgonine (370 μg/L). Urine was not available. Hair (+4 months after the accident): segment 1 (0-2 cm), cocaine not detected; segment 2 (2-4 cm), cocaine: <0.5 ng/mg. Case 3. A 66-year old man was involved in a car/car accident. He died 2 hours and 5 minutes after the crash. Blood (+1.5h after the accident): cocaine and metabolites not detected. Blood (+2h after the accident): cocaine (1750 μg/L), benzoylecgonine (460 μg/L). Blood (post-mortem): cocaine (370 μg/L), benzoylecgonine (200 μg/L). Urine (+1.5h after the accident): cocaine not detected. Case 4. A 57-year old woman on a motor scooter was hit by a car. She died 2 hours and 10 minutes after the crash. Blood (+0.5h after the accident): cocaine and metabolites not detected. Urine (post-mortem): cocaine (<20 μg/L), benzoylecgonine (120 μg/L). Case 5. A 30-year old man was involved in a car accident. Blood (+4h after the accident): cocaine (29 μg/L), benzoylecgonine (< 20 μg/L). Urine (+4h after the accident): cocaine and metabolites not detected. Ethanol (1,32 g/kg) and cannabinoids (THC (2,0 μg/L), THCCOOH (38 μg/L)) were also detected in blood. Conclusion: To our knowledge, this is the first description of DUID cases involving therapeutic use of cocaine after an accident. These results indicate that even if a per se law is effective for prosecution case of DUID, a critical interpretation of the results is always needed, especially if a medical intervention occurs after an accident.