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BACKGROUND AND AIMS Short screenings for alcohol use disorder (AUD) are crucial for public health purposes, but current self-reported measures have several pitfalls and may be unreliable. The main aim of our study was to provide empirical evidence on the psychometric performance of self-reports currently used. Our research questions were: Compared with a gold standard clinical interview, how accurate are 1) self-reported AUD, 2) self-reported alcohol use over time, and 3) biomarkers of alcohol use among Swiss men? Finally, we aimed to identify an alternative screening tool. DESIGN A single-center study with a cross-sectional design and a stratified sample selection. SETTING Lausanne University Hospital (Switzerland) from October 2017 through June 2018. PARTICIPANTS We selected participants from the French-speaking participants of the ongoing Cohort Study on Substance Use and Risk Factors (n=233). The sample included young men aged on average 27.0 years. MEASUREMENTS We used the Diagnostic Interview for Genetic Studies as the gold standard for DSM-5 AUD. The self-reported measures included 11 criteria for AUD, nine alcohol-related consequences, and previous twelve months' alcohol use. We also assessed biomarkers of chronic excessive drinking (ethyl glucuronide and phosphatidylethanol). FINDINGS None of the self-reported measures/biomarkers taken alone displayed both sensitivity and specificity close to 100% with respect to the gold standard (e.g., self-reported AUD: sensitivity=92.3%, specificity=45.8%). The best model combined eight self-reported criteria of AUD and four alcohol-related consequences. Using a cut-off of three, this screening tool yielded acceptable sensitivity (83.3%) and specificity (78.7%). CONCLUSIONS Neither self-reported alcohol use disorder (AUD) nor heavy alcohol use appear to be adequate to screen for AUD among young men from the Swiss population. The best screening alternative for AUD among young Swiss men appears to be a combination of eight symptoms of AUD and four alcohol-related consequences.

Background There is a need for empirical studies assessing the psychometric properties of self-reported alcohol use as measures of excessive chronic drinking (ECD) compared to those of objective measures, such as ethyl glucuronide (EtG). Objectives To test the quality of self-reported measures of alcohol use and of risky single-occasion drinking (RSOD) to detect ECD assessed by EtG. Methods A total of 227 samples of hair from young Swiss men were used for the determination of EtG. Self-reported measures of alcohol use (previous twelve-month and previous-week alcohol use) and RSOD were assessed. Using EtG ( Results A total of 23.3% of participants presented with ECD. Previous twelve-month alcohol use with a cut-off of >15 drinks per week (sensitivity = 75.5%, specificity = 78.7%) and weekly RSOD (sensitivity = 75.5%, specificity = 70.1%) yielded acceptable psychometric properties. No cut-off for previous-week alcohol use gave acceptable results. In the multivariate logistic regression, after controlling for the previous twelve months of alcohol use, RSOD was still significantly associated with EtG (p = .016). Conclusion Self-reported measures of the previous twelve months of alcohol use and RSOD were acceptable measures of ECD for population-based screening. Self-reported RSOD appeared to be an interesting screening measure, in addition to the previous twelve months of alcohol use, to understand ECD among young people.

The consensus on alcohol markers in hair was revised for the fourth time by an expert group of the Society of Hair Testing based on current state of research. This revision was adopted by the members of the Society during the business meeting in Brisbane on August 29th 2016. For both markers, ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs), two cut-off values for discrimination between teetotalers or occasional low amount consumption and moderate alcohol drinking (low cut-off), and between non-excessive (abstinence up to moderate alcohol intake) and chronic excessive drinking (high cut-off value) were critically examined. For the current revision, the cut-off values for EtG (7pg/mg and 30pg/mg, respectively) remained unchanged despite different findings or discussions published in the meantime. This was mainly due to the lack of broader data collections from new studies with great numbers of volunteers following thorough study concepts. In contrast, an essential change of the consensus was accepted for the FAEEs, where the concentration of ethyl palmitate (E16:0) can be used autonomously for interpretation instead of the concentration sum (ΣFAEE) of the four esters ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate, as previously applied. After evaluation of the data from seven laboratories, the E16:0 cut-off for abstinence assessment was defined at 0.12ng/mg for the 0-3cm segment and at 0.15ng/mg for the 0-6cm segment. The cut-off for chronic excessive drinking was fixed at 0.35ng/mg for the 0-3cm segment and at 0.45ng/mg for the 0-6cm segment. The use of E16:0 with these cut-offs in place of ΣFAEE for alcohol intake assessment produces only a minor loss in discrimination power, leads to no essential difference in the interpretation concerning chronic excessive alcohol consumption and is suitable to confirm EtG results in abstinence assessment if ethanol containing hair sprays or lotions are excluded.

Alcohol use disorder (AUD) is frequently comorbid with other psychiatric disorders. However, few studies investigated the psychometric properties of AUD screening tools in presence of co-occurring disorders. This study examined the diagnostic accuracy of a short AUD screening tool among young adults, in the presence of high vs. low or moderate symptomatology of other common psychiatric disorders. Data were collected among young Swiss men (n = 233) between 2016 and 2018. Measures included a diagnostic interview for AUD and screening tools for AUD and other psychiatric disorders (attention deficit hyperactivity disorder, antisocial personality disorder, bipolar disorder, borderline personality disorder, major depressive disorder, and social anxiety disorder). We computed receiver operating characteristic curves to test whether the AUD screening tool was an accurate indicator of AUD for groups with high vs. low or moderate symptomatology of each psychiatric disorder. The results showed that the optimal cut-off score was ≥3 (the original cut-off of the scale) for participants with a low or moderate symptomatology and ≥4 for participants with a high symptomatology. Our findings highlighted the urgent need for an integrated approach to screening. Psychiatric comorbidities should be included in the screen for AUD to obtain accurate results.

Estimating alcohol consumption using biomarkers raises interpretation problems. The biomarkers currently used in clinical settings have limited performances to identify unhealthy alcohol use (e.g. CDT, AST, ALT). New direct biomarkers, ethylglucuronide (EtG) and phosphatydilethanol (PEth) are available and offer better sensitivity and specificity compared to indirect biomarkers. In forensic medicine, EtG and PEth are replacing indirect biomarkers. However, in clinical routine practice these markers are usually not considered. Still, for specific purposes such in pre-liver transplant evaluations, direct markers may help specialists in the decision process.Estimer la consommation d’alcool en se basant sur des tests biologiques pose des problèmes d’interprétation. Les marqueurs actuellement utilisés en clinique (CDT et transaminases) présentent des performances limitées pour l’identification du mésusage d’alcool. Des nouveaux marqueurs directs, l’éthylglucuronide (EtG) et le phosphatidyléthanol (PEth), sont à disposition et offrent de meilleures performances en termes de sensibilité et spécificité que les marqueurs indirects. Ils sont principalement utilisés dans le cadre de suivis médico-légaux et remplacent les marqueurs indirects. En pratique clinique, l’EtG et le PEth ne sont que peu utilisés. On voit apparaître l’utilisation de ces tests dans le cadre d’évaluations spécifiques, par exemple avant transplantation hépatique.

Background: Hyphenation of liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) offers the potential to develop broad-spectrum screening procedures from low volumes of biological matrices. In parallel, dried blood spot (DBS) has become a valuable tool in the bioanalysis landscape to overcome conventional blood collection issues. Herein, we demonstrated the applicability of DBS as micro-sampling procedure for broad-spectrum toxicological screening. Methods: A method was developed on a HRMS system in data dependant acquisition (DDA) mode using an extensive inclusion list to promote collection of relevant data. 104 real toxicology cases were analysed, and the results were cross-validated with one published and one commercial screening procedures. Quantitative MRM analyses were also performed on identified substances on a triple quadrupole instrument as a complementary confirmation procedure. Results: The method showed limits of identification (LOIs) in appropriateness with therapeutic ranges for all the classes of interest. Applying the three screening approaches on 104 real cases, 271 identifications were performed including 14 and 6 classes of prescribed and illicit drugs, respectively. Among the detected substances, 23% were only detected by the proposed method. Based on confirmatory analyses, we demonstrated that the use of blood micro-samples did not impair the sensitivity allowing more identifications in the low concentration ranges. Conclusion: A LC-HRMS assay was successfully developed for toxicological screening of blood microsamples demonstrating a high identification power at low concentration ranges. The validation procedure and the analysis of real cases demonstrated the potential of this assay by supplementing screening approaches of reference.

IntroductionShort screenings of alcohol-related dependence are needed for population-based assessments. A clinical interview constitutes a reliable diagnosis often seen as gold standard, but it is costly and time consuming and as such, not suitable for population-based assessments. Therefore, self-reported questionnaires are needed (eg, alcohol use disorder (AUD) as in the Diagnostic and Statistic Manual of Mental Disorders (DSM) 5), but their reliability is questionable. Recent studies called for more evidence-based measurements for population-based screening (eg, heavy alcohol use over time (HAU)). This study aims to test the reliability of different self-reported measures of alcohol use.Methods and analysisBased on stratified random selection, 280 participants will be recruited from the French-speaking subgroup of the Swiss National Science Foundation-supported Cohort Study on Substance Use and Risk Factors (C-SURF). This cohort is a population-based sample of young Swiss men in their mid-20s (n=2668). The sample size calculation is based on a proportion non-inferiority test (alpha=5%, power=80%, margin of equivalence=10%, difference in sensitivity between self-reported AUD and HAU=5%, correlation between AUD and HAU=0.35, and drop-outs=15%). Assessment will include a clinical interview as the gold standard of alcohol-related dependence, self-reported alcohol measures (HAU, AUD and drinking patterns), biomarkers as gold standards of chronic excessive drinking, and health outcomes. To assess the validity of the self-reported alcohol measures, sensitivity analyses will be run. The associations between alcohol-related measures and health outcomes will be tested. A non-response analysis will be run using the previous waves of the C-SURF study using logistic regressions.Ethics and disseminationThe study protocol has been approved by the Human Research Ethics Committee of the Canton of Vaud, Switzerland (no. 2017–00776). The results will be submitted for publication in peer-reviewed journals and presented at national and international conferences.

The aim of this study was to evaluate the reliability of insect larvae as samples for toxicological investigations. For this purpose, larvae of Lucilia sericata were reared on samples of minced pig liver treated with different concentrations of codeine: therapeutic, toxic, and potentially lethal doses. Codeine was detected in all tested larvae, confirming the reliability of these specimens for qualitative toxicology analysis. Furthermore, concentrations measured in larvae were correlated with levels in liver tissue. These observations bring new elements regarding the potential use of opiates concentrations in larvae for estimation of drug levels in human tissues. Morphine and norcodeine, two codeine metabolites, have been also detected at different concentrations depending on the concentration of codeine in pig liver and depending on the substance itself. The effects of codeine on the development of L. sericata were also investigated. Results showed that a 29-h interval bias on the evaluation of the larval stage duration calculated from the larvae weight has to be considered if codeine was present in the larvae substrate. Similarly, a 21-h interval bias on the total duration of development, from egg to imago, has to be considered if codeine was present in the larvae substrate.

Objective There is a need of empirical evidence of the relationship between heavy alcohol use and alcohol markers. More precisely, associations of these markers with patterns of alcohol use, such as risky single occasion drinking (i.e., drinking 6 drinks or more on a single occasion) have been scarcely investigated. This study aimed to derive psychometric properties of alcohol markers in comparison with different measures of alcohol use and to test whether it allows detecting excessive alcohol use. Methods Participants (n = 233, male, around 20 years old) were recruited based on a stratified random selection of the Swiss Cohort Study on Substance Use and Risk Factors. Inclusion criterion was an Audit score ≥ 13. Assessments included a clinical interview based on the Diagnostic Interview for Genetic Studies (DIGS) and self-reported alcohol measures [1] . Hair (n = 230) and capillary blood (n = 233) on Hemaxis® DBS device were collected for ethyl glucuronide (EtG) and phosphatidylethanol (PEth) determination, respectively. EtG was extracted from washed hair by micropulverized extraction and SPE purification. The extracts were submitted to LC-MS/MS analysis (6500 Qtrap Sciex). Chromatographic separation was achieved using a Merck Chromolith® RP-C18e column (150 × 3 mm). The PEth homologs 16:0/18:1 and 16:0/18:2 concentrations were determined by LC-MS/MS (5000 Qtrap Sciex) according to a validated method [2] . For the interpretation, only the PEth homolog 16:0/18:1 were used. Statistical data treatment (ROC-analysis) was carried out using R software. Results EtG could be detected in 151 of 230 samples with concentrations ≥ 4.0 pg/mg (LOQ). The maximum measured concentration was 690 pg/mg. Twenty-five percent, 50% (median) and 75% percentiles were 6.8, 15, and 34 pg/mg, respectively. In 53 cases, EtG concentrations were ≥ 30 pg/mg, suggesting excessive alcohol consumption. The area under the ROC curve for EtG ≥ 30 pg/mg vs. PEth was 0.8322. In cases where PEth ≥ 210 ng/mL, more than 75% presented also EtG ≥ 30 pg/mg. PEth 16:0/18:1 detected in 218 of 233 samples with concentrations ≥ 10 ng/mL (LOQ). The maximum measured concentration was 1990 ng/mL. Twenty-five percent, 50% (median) and 75% percentiles were 71, 182, and 360 ng/ml, respectively. In 99 cases, PEth 16:0/18:1 concentrations ≥ 210 ng/mL, suggesting excessive alcohol consumption, were found. Compared to self-reported data (last week total alcohol intake diary), area under the ROC curve for PEth ≥ 210 ng/mL were 0.8174. For detecting a risky single occasion drinking (≥ 5 drinks at one occasion), the area under the ROC curve for PEth were 0.7955. Conclusion EtG and PEth measurement allowed detecting excessive alcohol consumption with high specificity and sensitivity. Both marker are complementary and do not necessarily provide the same information because of their different detection window and change of consumption during the evaluation period. The results support the statement in the SoHT consensus for the use of alcohol markers: “It is not advisable to use the results of hair testing for alcohol markers in isolation” [3] .

The increasing request for hair ethyl glucuronide (HEtG) in alcohol consumption monitoring according to cut-off levels set by the Society of Hair Testing (SoHT) has triggered a proficiency testing program based on interlaboratory comparisons (ILC). Here, the outcome of nine consecutive ILC rounds organised by the SoHT on the determination of HEtG between 2011 and 2017 is summarised regarding interlaboratory reproducibility and the influence of procedural variants. Test samples prepared from cut hair (1mm) with authentic (in-vivo incorporated) and soaked (in-vitro incorporated) HEtG concentrations up to 80pg/mg were provided for 27-35 participating laboratories. Laboratory results were evaluated according to ISO 5725-5 and provided robust averages and relative reproducibility standard deviations typically between 20 and 35% in reasonable accordance with the prediction of the Horwitz model. Evaluation of results regarding the analytical techniques revealed no significant differences between gas and liquid chromatographic methods In contrast, a detailed evaluation of different sample preparations revealed significantly higher average values in case when pulverised hair is tested compared to cut hair. This observation was reinforced over the different ILC rounds and can be attributed to the increased acceptance and routine of hair pulverisation among laboratories. Further, the reproducibility standard deviations among laboratories performing pulverisation were on average in very good agreement with the prediction of the Horwitz model. Use of sonication showed no effect on the HEtG extraction yield.

Besides the psychoactive Delta(9)-tetrahydrocannabinol (THC), hashish and marijuana as well as cannabis-based medicine extracts contain varying amounts of cannabidiol (CBD) and of the degradation product cannabinol (CBN). The additional determination of these compounds is interesting from forensic and medical points of view because it can be used for further proof of cannabis exposure and because CBD is known to modify the effects of THC. Therefore, a method for the simultaneous quantitative determination of THC, its metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), CBD and CBN from plasma was developed. The method was based on automatic solid-phase extraction with C(18) ec columns, derivatization with N,O-bistrimethylsilyltrifluoroacetamide (BSTFA), and gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) with deuterated standards. The limits of detection were between 0.15 and 0.29 ng/mL for THC, 11-OH-THC, THC-COOH, and CBD and 1.1 ng/mL for CBN. The method was applied in a prospective pharmacokinetic study after single oral administration of 10 mg THC alone or together with 5.4 mg CBD in cannabis extract. The maximum plasma concentrations after cannabis extract administration ranged between 1.2 and 10.3 ng/mL (mean 4.05 ng/mL) for THC, 1.8 and 12.3 ng/mL (mean 4.9 ng/mL) for 11-OH-THC, 19 and 71 ng/mL (mean 35 ng/mL) for THC-COOH, and 0.2 and 2.6 ng/mL (mean 0.95 ng/mg) for CBD. The peak concentrations (mean values) of THC, 11-OH-THC, THC-COOH, and CBD were observed at 56, 82, 115, and 60 min, respectively, after intake. CBN was not detected. Caused by the strong first-pass metabolism, the concentrations of the metabolites were increased during the first hours after drug administration when compared to literature data for smoking. Therefore, the concentration ratio 11-OH-THC/THC was discussed as a criterion for distinguishing oral from inhalative cannabis consumption.

Cutting-edge omic strategies are bringing new opportunities to many fields of investigation from the biomedical to the biomedicolegal. Comprehension of molecular mechanisms associated with pathologies such as acute or chronic toxicity or sudden cardiac death and the discovery of associated biomarkers represent key elements in the development of novel routes for a better understanding of complex phenomena. Omics and mining of complex generated data have reached a state of maturity. Recent innovations, notably for mass spectrometry with a remarkable gain in sensitivity and selectivity, and the development of next-generation sequencing technologies, have considerably increased the power of these approaches. In spite of ongoing progress in the omic methodologies, their application can already provide informative results leading to more accurate interpretations and evidences. Beyond the remarkable potential in forensic toxicology such as the research of new biomarkers, we predict that these technologies will reinforce translational research between forensic and clinical disciplines.

Introduction Les vaporisateurs electroniques portables permettent de vapoter du cannabis pur, sans adjonction de tabac et sans combustion nocive. Objectif Les performances de deux vaporisateurs commerciaux (DaVinci™ Ascent, Organicix, LLC, Nevada, Etats-Unis et Mighty Medic™, Storz & Bickel GmbH & Co, Tuttlingen, RFA) ont ete testees et comparees. Le premier chauffe la chambre de remplissage par conduction, le second combine conduction et convection avec un generateur d’air chaud. Methodes Plusieurs systemes de collecte des aerosols issus de la vaporisation du cannabis (sommites fleuries de la variete Bedrobinol® (12,6 % THC, < 0,1 % CBD) et granules de la variete Bediol®, cultivar L. « Elida », Bedrocan, Pays-Bas, 6,3 % de THC et 8 % de CBD total) ont ete testes avec une machine a fumer. Le dispositif de collecte des aerosols est constitue d’une partie frontale d’echantillonnage branchee en serie avec une partie de garde arriere. Plusieurs materiaux adsorbants ont ete evalues : charbon actif, XAD-2 et Sep-Pak® C18ec. Des flacons laveurs avec fritte, remplis de solvants organiques (methanol, ethanol, heptane/acetate d’ethyle et methanol/isopropanol) montes en serie ou servant de flacon de garde a une cartouche d’adsorbant solide ont aussi ete testes. La duree, l’intervalle de temps entre deux bouffees et le volume de chaque bouffee ont ete respectivement de 5–6 s, 25 s, 70–250–600 mL. Entre les bouffees, un flux residuel de 1–3 mL/s est maintenu pour mimer un effet de cheminee a travers le vaporisateur. Le nombre de bouffees a ete incremente de 1 a 2 puis 5, 10, 20 et 30 bouffees. Le cannabis (150 a 350 mg) a ete moulu dans un moulin a herbe puis introduit dans la chambre de remplissage. Des que la temperature de vaporisation est atteinte, la pompe a membrane (debit maximal 30 L/min) est enclenchee. Le residu de vaporisation est recupere, pese, extrait et analyse. Les cannabinoides pieges dans le liquide des flacons laveurs sont analyses directement. Les cartouches sont eluees 4× avec un melange methanol/dichloromethane 9:1 v/v. Les parties internes des vaporisateurs et les tuyaux de raccordement ont ete laves avec du methanol et analyses. Les cannabinoides acides (THC-A et CBD-A) et neutres (THC, CBD et CBN) ont ete doses par HPLC-DAD. Resultats Les cannabinoides sont presents dans les varietes testees sous la forme de leurs precurseurs acides. Les concentrations indiquees par Bedrocan sont en accord avec celles mesurees. Lorsque la temperature de vaporisation est de 190 °C, une tres faible proportion de cannabinoides acides (< 2 %) reste mesurable dans le residu apres 1–2 bouffees. Apres 5 bouffees (2,5 min, 190 °C) ou des la premiere bouffee a des temperatures superieures (210–220 °C), le CBD-A et le THC-A sont completement decarboxyles en leurs molecules filles. A 190 °C, le rendement de vaporisation des cannabinoides est tres faible. Pour comparer les 2 vaporisateurs, ceux-ci ont donc ete regles a leur temperature maximale et charges avec une dose identique de 150 mg de cannabis de la variete Bediol®. Le nombre de bouffees a ete incremente de 2 a 20. Apres 10 bouffees, le residu chauffe recupere dans le Mighty Medic™ ne contient presque plus de cannabinoides alors que le residu repris du DaVinci™ Ascent contient encore environ 1/3 des cannabinoides de la dose initiale. Les quantites retenues dans les 4 cartouches Sep-Pak® branchees en parallele et connectees au Mighty Medic™ sont environ 2 fois superieures (6 mg THC et 9 mg CBD) a celles trappees avec le DaVinci™ Ascent. Conclusion Le Mighty Medic™ semble plus performant que le DaVinci™ Ascent avec moins de pertes dans les parties internes de l’appareil et une proportion delivree de cannabinoides plus importante, suggerant que cet appareil est mieux a meme de satisfaire les attentes des consommateurs, que l’inhalation soit faite pour des raisons therapeutiques ou recreatives.

Objectif Evaluer la possibilite de differencier la prise de cannabis legal, defini selon la loi suisse comme contenant moins de 1 % de tetrahydrocannabinol (THC) et pouvant etre riche en cannabidiol (CBD), de la prise de cannabis illegal, defini en Suisse comme contenant un taux de THC egal ou superieur a 1 %, independamment de la teneur en CBD, a partir des concentrations sanguines de THC, 11-OH-THC, THC-COOH et CBD, et urinaires de THC-COOH et de CBD. Methodes Au total, 284 echantillons de sang et 130 echantillons d’urine provenant de personnes suspectees de conduire sous influence et indiquant une prise de cannabis illicite, ainsi que 13 echantillons de sang et 11 echantillons d’urine preleves chez des personnes consommant du cannabis legal, riche en CBD, suivies en addictologie, ont ete inclus dans l’etude. Les analyses ont ete effectuees par des methodes validees, basees sur l’utilisation de la GC-MS et de la LC-MS/MS, avec une limite de quantification fixee a 1 ng/mL pour le THC, le 11-OH-THC et le CBD, et a 5 ng/mL pour le THC-COOH, dans le sang et l’urine. Resultats Parmi les consommateurs de cannabis illicite, 220 presentaient des concentrations sanguines de THC comprises entre 1,0 et 35 ng/mL, toujours superieures aux concentrations de CBD. Cette derniere substance n’a ete mise en evidence que dans 105 echantillons de sang (37 %). Parmi les consommateurs de cannabis legal, le CBD a ete observe dans tous les echantillons de sang, alors que pour 12 cas (92 %), la concentration de CBD etait comprise entre 2,1 et 24 ng/mL, toujours superieure a la concentration de THC. Des resultats similaires ont ete obtenus dans l’urine, entre le THC-COOH et le CBD. Les medianes, ainsi que les ecarts interquartiles, des concentrations des cannabinoides mesurees dans le sang et l’urine de consommateurs de cannabis illicite (THC ≥ 1 %) et de consommateurs de cannabis legal (THC Tableau 1 . En appliquant la legislation suisse en matiere de circulation routiere, 2 consommateurs de cannabis legal presentaient des concentrations de THC superieures a 1,5 ng/mL (valeur limite selon la legislation suisse), respectivement 1,7 et 2,8 ng/mL. Conclusion Parmi les echantillons analyses, les rapports des concentrations sanguines de cannabinoides ont permis de confirmer le caractere illicite ou legal, selon la legislation suisse, du cannabis consomme. En outre, du fait de la presence de THC dans le cannabis legal et selon la dose consommee, il est possible que la concentration sanguine de THC soit superieure a la valeur limite definissant une infraction en matiere de circulation routiere.

Objectif Developpement, validation et implementation en routine d’une methode de criblage de drogues et medicaments a partir d’un micro-prelevement de sang sur papier buvard (DBS) en utilisant la spectrometrie de masse haute resolution (HRMS) couplee a la chromatographie liquide ultra haute-performance (UHPLC). Methodes Une methode UHPLC-HRMS a ete developpee pour l’analyse de DBS. Pour ce faire, deux spots de 10 μL de sang complet ont ete dopes avec 142 standards representatifs des substances trouvees dans des cas cliniques et de suspicion de consommation de drogues et medicaments au volant (MDV). La methode est basee sur une acquisition en mode full-scan en ionisation positive suivi d’une fragmentation en donnees dependantes (DDA) basee sur une liste d’inclusion de 1008 composes, incluant notamment des nouvelles substances psychoactives (NPS). Au cours de la meme analyse, une acquisition ciblee (Tsim) en ionisation negative est realisee sur l’ion [M-H]− du 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH). La methode a ete evaluee par validation croisee en comparant les substances detectees dans 94 cas MDV avec les resultats de deux autres approches actuellement utilisees en routine [1] , [2] pour repondre aux demandes cliniques et forensiques. Resultats La methode developpee a permis la detection de l’ensemble des 142 substances representatives. Les limites d’identification (LOIs) ont ete determinees sur un echantillon de 30 substances. Celles-ci ont ete mesurees comme etant egales ou inferieures a 20 ng/mL, malgre le faible volume de sang utilise. La methode a egalement ete evaluee grâce a l’utilisation de controles de qualite externes. La comparaison de 94 cas entre la methode HRMS developpee, une methode ciblee commerciale pour la toxicologie clinique (Bruker Toxtyper™) [1] appliquee apres extraction liquide-liquide sur 0,5 ml de sang complet, ainsi qu’une methode ciblee developpee sur un systeme QTrap pour l’analyse de DBS a permis de confirmer l’interet de l’approche HRMS. Au total, 271 identifications de 79 substances differentes ont ete effectuees. La methode HRMS developpee dans ce projet a montre des meilleurs resultats, en identifiant 98,9 % de la totalite des substances contre 71,6 % pour l’approche Toxtyper et 56,8 % pour l’approche QTrap. Le THC-COOH a ete detecte dans 50 % des cas mesures et est le principal responsable de ce gain de performance obtenue grâce a l’approche HRMS, confirmant ainsi la necessite d’implementer cette substance dans les approches de criblage. Conclusion Le recent developpement des appareils HRMS amenant un gain de sensibilite permet d’utiliser le DBS comme micro-prelevement pour le screening toxicologique large echelle.

Objectif Demontrer la faisabilite de l’analyse du phosphatidylethanol (PEth) a partir d’un micro-echantillon de sang capillaire collecte sur papier filtre (dried blood spots [DBS]). Description Le PEth est un metabolite de l’ethanol issu d’une voie non-oxydative mineure. Par rapport a des marqueurs indirects comme la CDT et la ɣ-GT, le PEth est un marqueur sanguin direct de la consommation d’alcool offrant de meilleures performances en termes de sensibilite et de specificite [1]. En raison de sa longue demi-vie (4 jours), il est possible de tracer la consommation d’un individu jusqu’a 3 semaines avant le prelevement. Le PEth n’est toutefois pas stable une fois collecte si le tube de sang n’est pas conserve au congelateur [2], ainsi l’optimisation du pre-analytique s’est revele etre un facteur determinant pour le rendu de resultats fiables. L’utilisation de papier filtre associe a un micro-prelevement de sang capillaire a ete evaluee pour la quantification du PEth. Methode Les echantillons sanguins sont preleves a l’aide du dispositif microfluidique HemaXis™ (DBS System) afin de standardiser le volume de sang depose sur le papier filtre (10,5 μL). Pour l’analyse, les echantillons DBS sont extraits selon la procedure in vial[3] et analyses par une methode LC-MS/MS validee au laboratoire. La repetabilite du dispositif de prelevement HemaXis™ a ete evaluee sur la base d’echantillons de sang blanc fortifies avec le PEth a deux niveaux de concentration (40 et 300 ng/mL). En parallele, la stabilite du PEth a ete investiguee en comparant trois conditions de stockage (sang liquide conserve a −20 °C, lyophilisats conserves a 4 °C et DBS conserves a temperature ambiante) sur une periode de 6 mois. Resultats Les concentrations moyennes de PEth mesurees pour les deux niveaux de concentration et pour chaque temps (t1 = 14 j, t2 = 47 j, t3 = 88 j, t4 = 196 j) donnent des biais compris entre −13,2 et 5,6 % pour le sang liquide stocke a −20 °C ; −20,8 et 2,9 % pour les lyophilisats a 4 °C et −9,4 et 9,1 % pour les DBS a temperature ambiante. Les repetabilites observees pour les trois conditions, respectivement 2,8 % pour le sang liquide (n = 5) ; 2,8 % pour le lyophilisat (n = 5) et 3,4 % pour HemaXis™ (n = 10) sont statistiquement similaires. Conclusion L’utilisation du dispositif HemaXis™ permet de controler le volume de sang collecte au bout du doigt avec le meme niveau de precision qu’une micropipette de laboratoire. De plus, le format DBS sur papier filtre permet de stabiliser le PEth sur une periode d’au moins 6 mois sans avoir besoin de recourir a une chaine du froid. Considerant ces resultats, l’analyse du PEth a ete introduite en routine au sein du laboratoire en collaboration avec differents medecins addictologues de Suisse Romande. L’optimisation du pre-analytique permet ainsi d’effectuer les prelevements DBS directement au cabinet des cliniciens avant qu’ils ne soient envoyes par poste dans une simple enveloppe au laboratoire.

Objective Nowadays, alcohol consumption has still been a public health concern that frequently has forensic consequences. The measurement of markers of alcohol consumption or alcohol abuse is thus important to assess alcohol consumption or to confirm alcohol abstinence during the weeks or the months before the sampling, which can be decisive and guide drastically a court decision. Direct alcohol markers, such as ethanol, ethyl glucuronide (EtG) and ethyl sulfate (EtS) in serum, EtG and fatty acid ethyl esters (FAEE) in hair, or phosphatidylethanol (PEth) in blood, are available markers with high specificity and sensitivity [1] . In some particular situations, elevation of the concentration of the marker is not the consequence of alcohol consumption by the patient, but the results of alcohol or marker exposition before sampling [2] . Thus, this could conduct to erroneous interpretation even if the result is not an analytical false-positive case. Recently, we have been confronted to several situations involving presence of PEth in blood of patients denying any alcohol consumption for a couple of months, which led us to look for a possible pitfall. Methods Dried blood spots (10.5 μL) were obtained from three volunteers (two females and one male) after cleaning their hands with soap and water or disinfectant solution containing ethanol (70% or 98%) using Hemaxis® (DBS System SA) kit. The PEth homolog 16:0/18:1 concentration was determined by LC-MS/MS (API 5000 Sciex) according to validated and published method [3] . Results The first volunteer (woman) declared to be teetotaler. PEth blood concentrations after washing her hands with Sterilium® gel [ethanol 85% (w/w)] and ethanol solution [ethanol 98% (v/v)] were 130 ng/mL and 350 ng/mL, respectively. The second volunteer (woman) declared to consume alcohol Conclusion An abnormal elevation of PEth concentration in blood was observed after using ethanolic solution for hand cleaning. Thus, ethyl alcohol disinfection have to be avoid before blood sampling for DBS. If not, a rapid formation of PEth could happen during blood sampling, involving a risk of erroneous interpretation concerning alcohol consumption.

Passive exposure to cannabis smoke may induce effects on behavior and psychomotor skills, and have legal consequences, including the risk of being falsely considered as a cannabis user. This can become a concern, especially in occupational contexts or when driving vehicles. In order to enable a differentiation between a passive and an active exposure to cannabis and to limit the likeliness to be detected positive following passive exposure, this review identified specific biomarkers of passive exposure in urine, blood, oral fluid, hair, and sebum. Out of 958 papers identified on passive exposure to cannabis, 21 were selected. Although positive tests had been observed in all matrices following extremely high passive exposure, some distinctive features were observed in each matrix compared to cannabis active use. More specifically, in everyday life conditions, 11-nor-delta-9-THC-carboxylic acid (THC-COOH) urinary level should be detected below the positivity threshold used to confirm active smoking of cannabis, especially after normalization to creatinine level. Measuring delta-9-tetrahydrocannabinol (THC) and THC-COOH in blood is an appropriate alternative for appraising passive exposure as low and very low concentrations of THC and THC-COOH, respectively, should be measured. In hair, oral fluid (OF) and sweat/sebum emulsion, no THCCOOH should be detected. Its presence in hair argues for regular cannabis consumption and in OF or sweat for recent consumption. The experts should recommend to persons who have to demonstrate abstinence from cannabis to avoid heavily smoky and unventilated environments.

The importance of direct and indirect alcohol markers to evaluate alcohol consumption in clinical and forensic settings is increasingly recognized. While some markers are used to prove abstinence from ethanol, other markers are suitable for detection of alcohol misuse. Phosphatidyl ethanol (PEth) is ranked among the latter. There is only little information about the correlation between PEth and other currently used markers (ethyl glucuronide, ethyl sulfate, carbohydrate deficient transferrin, gamma-glutamyl transpeptidase, and methanol) and about their decline during detoxification. To get more information, 18 alcohol-dependent patients in withdrawal therapy were monitored for These parameters in blood and urine for up to 19 days. There was no correlation between the different markers. PEth showed a rapid decrease at the beginning of the intervention, a slow decline after the first few days, and could still be detected after 19 days of abstinence from ethanol.

Postmortem angiography is becoming increasingly essential in forensic pathology as an adjunct to conventional autopsy. Despite the numerous advantages of this technique, some questions have been raised regarding the influence of the contrast agent injected on the results of toxicological and biochemical analyses. The aim of this study was to investigate the effect of the injection of the contrast agent Angiofil®, mixed with paraffin oil, on the results of postmortem biochemical investigations performed on vitreous humor. Postmortem biochemical investigations were performed on vitreous samples collected from bodies that had undergone postmortem angiography (n = 50) and from a control group (n = 50). Two vitreous samples were analyzed for each group and the results compared. Glucose, urea, creatinine, 3-β-hydroxybutyrate, sodium and chloride were tested. Different values were observed between the first and second samples in each group. However, these differences were not clinically relevant, suggesting that the injection of this contrast agent mixture does not modify the concentration of the analyzed substances in the vitreous humor.

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Tolperisone (Mydocalm) is a centrally acting muscle relaxant with few sedative side effects that is used for the treatment of chronic pain conditions. We describe three cases of suicidal tolperisone poisoning in three healthy young subjects in the years 2006, 2008 and 2009. In all cases, macroscopic and microscopic autopsy findings did not reveal the cause of death. Systematic toxicological analysis (STA) including immunological tests, screening for volatile substances and blood, urine and gastric content screening by GC-MS and HPLC-DAD demonstrated the presence of tolperisone in all cases. In addition to tolperisone, only the analgesics paracetamol (acetaminophen), ibuprofen and naproxen could be detected. The blood ethanol concentrations were all lower than 0.10 g/kg. Tolperisone was extracted by liquid-liquid extraction using n-chlorobutane as the extraction solvent. The quantification was performed by GC-NPD analysis of blood, urine and gastric content. Tolperisone concentrations of 7.0 mg/l, 14 mg/l and 19 mg/l were found in the blood of the deceased. In the absence of other autopsy findings, the deaths in these three cases were finally explained as a result of lethal tolperisone ingestion. To the best of our knowledge, these three cases are the first reported cases of suicidal tolperisone poisonings.

In this report, the authors present a case of unusual, accidental methadone intoxication in a 40-year-old man, who had inhaled methadone powder. The drug dealer was a pharmacy technician; methadone had been stolen from a pharmacy and sold as cocaine. After having inhaled methadone powder, he suffered cardiopulmonary arrest. He was admitted to hospital where he died after 24 h of intensive care. The autopsy revealed congestion of internal organs and cerebral and pulmonary edema. Microscopically, the heart showed no changes. The toxicological analyses performed on blood and urine taken at the hospital revealed methadone, cannabinoids, and ethanol. The blood methadone concentration was 290 μg/L. The urine methadone concentration was 160 μg/L. Midazolam and lidocaine, which were administered to the patient at the hospital, were also detected in the blood. The cause of death was determined to be methadone intoxication. The literature has been reviewed and discussed. To date, and to our knowledge, only very few cases of accidental death resulting from methadone inhalation have been described up to the case presented herein. Language: en

Da exzessiver Alkoholkonsum erhöhte individuelle Risiken mit sich bringt, ist es unverzichtbar, einen Risikokonsum genauestens erfassen zu können. Ethylglukuronid (EtG) ist ein spezifischer Marker eines Alkoholkonsums und kann im Blut, im Urin oder in den Haaren gemessen werden. Die Bestimmung von Ethylglukuronid in Urin oder Blut kann zum Beispiel genutzt werden, um einen kürzlich zurückliegenden Alkoholkonsum eines Fahrzeuglenkers nachzuweisen, auch wenn Ethanol selbst nicht mehr nachweisbar ist. Die erfolgte Kommerzialisierung eines spezifischen immunonologischen Tests erlaubt nun auch einen einfachen, schnellen und empfindlichen Vortest auf EtG. Darüber hinaus gestattet die EtG-Bestimmung in den Haaren die Evaluierung des Alkoholkonsums während eines länger zurückliegenden Zeitraums. Die EtG-Konzentration in den Haaren korreliert mit der konsumierten Alkoholmenge. Die EtG-Analyse kann daher zur Abstinenzkontrolle, zur Rückfalldiagnostik oder zur Aufdeckung von Risikokonsumenten eingesetzt werden. Bis heute existiert jedoch noch kein allgemein anerkannter Grenzwert zur Erfassung von Risikokonsumenten. Ein aktuell von der Schweizerischen Stiftung für Alkoholforschung finanziertes Projekt am Universitätszentrum für Rechtsmedizin der Romandie hat zum Ziel, diesen Grenzwert zu ermitteln. Zurzeit ist es daher empfehlenswert, Ethylglukuronid zusätzlich zu den traditionellen Blutmarkern zu bestimmen.

This study examines cases of chronic drug users who died suddenly after drug administration. Victims were young subjects, aged from 19 to 35 from Switzerland and known to the police as long-term drug users. The circumstances of death suggested the occurrence of a sudden, unexpected death. Some victims were undergoing methadone treatment. In each case, a forensic autopsy and toxicological analyses were performed at the Institute of Forensic Medicine in Lausanne in Switzerland between 2002 and 2004, including hair analysis as a means to establish chronic drug use in general, and cocaine use in particular. The conduction system was examined histologically and cases showing potentially lethal changes were chosen for this report. The most frequent lesions found were severe thickening of the atrioventricular node artery, intranodal and perinodal fibrosis, and microscopic foci of chronic inflammatory infiltration. The authors conclude that pathological lesions in the conduction tissue may play a role in the occurrence of death attributed to intoxication consecutive to cocaine ingestion.

Background: Fatty acid ethyl esters (FAEEs) are products of nonoxidative ethanol metabolism. After incorporation in hair, they should be suitable long-term markers of alcohol abuse. Methods: Hair samples from 19 alcoholics in a treatment program, 10 fatalities with verified excessive alcohol consumption, 13 moderate social drinkers who consumed up to 20 g ethanol/day, and 5 strict teetotalers were analyzed in 1–12 segments for four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) by external degreasing with n-heptane, extraction with a dimethyl sulfoxide-n-heptane mixture, headspace solid-phase microextraction of the extracts, and gas chromatography-mass spectrometry with deuterated internal standards. The n-heptane washings were analyzed in the same way for FAEEs from the hair surface. Results: The sum of the four ester concentrations in hair calculated for the proximal 0–6 cm segment was 2.5–13.5 ng/mg (mean, 6.8 ng/mg) for the fatalities, 0.92–11.6 ng/mg (mean, 4.0 ng/mg) for 17 of the alcoholics in treatment, 0.20–0.85 ng/mg (mean, 0.41 ng/mg) for the moderate social drinkers, and 0.06–0.37 ng/mg (mean, 0.16 ng/mg) for the teetotalers. In almost all cases the segmental concentrations increased from proximal to distal. There was no agreement between the self-reported drinking histories of the participants and the FAEE concentrations along the hair length. Ethyl oleate was the dominant ester in all samples. Conclusions: FAEEs are deposited in hair mainly from sebum. Despite large individual differences, FAEE hair concentrations can be used as markers for excessive alcohol consumption with relatively high accuracy.

Fatty acid ethyl esters (FAEE) are products of the nonoxidative ethanol metabolism, which are known to be detectable in blood only about 24h after the last alcohol intake. After deposition in hair they should be suitable long-term markers of chronically elevated alcohol consumption. Therefore, a method for the analysis of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate from hair was developed based on the extraction of the hair sample by a dimethylsulphoxide (DMSO)/n-hexane mixture, separation and evaporation of the n-hexane phase and application of headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) to the extract. For use as internal standards, the corresponding D(5)-ethyl esters were prepared. The HS-SPME/GC-MS measurements were automatically performed using a multi-purpose sampler. The detection limits of the FAEE were between 0.01 and 0.04ng/mg and the reproducibility was between 3.5 and 16%. By application of the method to hair samples of 21 fatalities with known heavy alcohol abuse 0.045-2.4ng/mg ethyl myristate, 0.35-13.5ng/mg ethyl palmitate, 0.25-7.7ng/mg ethyl oleate and 0.05-3.85ng/mg ethyl stearate were measured. For social drinkers (30-60g ethanol per week), the concentrations were about one order of magnitude smaller. For 10 teetotalers negative results or traces of ethyl palmitate were found. It was shown by supplementary investigations in single cases that FAEE are also present in sebum, that there is no strong difference in their concentrations between pubic, chest and scalp hair, and that they are detectable in hair segments after a 2 months period of abstinence. From the results follows that the measurement of FAEE concentrations in hair is a useful way for a retrospective detection of alcohol abuse.

The local anesthetic lidocaine was determined in hair by hydrolysis of the samples with 4% NaOH in the presence of excessive Na2SO4 and subsequent headspace solid-phase microextraction with a 65-pro Carbowax/divinylbenzene fiber, and gas chromatography-mass spectrometry measurement with etidocaine as the internal standard. The calibration curve was linear between 0.1 and 1000 ng/mg. The detection and quantitation limits were 0.1 and 0.4 ng/mg, respectively. The method was applied to hair samples of 49 drug fatalities, and positive results were obtained in 32 cases with lidocaine concentrations between 0.4 and 400 ng/mg and 675 ng/mg in one extreme case. For comparison, morphine, 6-acetylmorphine, codeine, dihydrocodeine, methadone, cocaine, and benzoylecgonine were also determined by usual methods. From segmental investigations in four of the cases and from comparison with the hair concentrations of the other drugs, it follows that lidocaine was consumed for a longer period of time as an adulterant of cocaine and heroin preparations. case of death after repeated use of this anesthetic during episodes of self mutilation. Such high concentrations suggest a very efficient deposition of this drug into hair.

Headspace solid phase microextraction (HS-SPME) has advantages of high purity of the extract, avoidance of organic solvents and simple technical manipulation and can be used in combination with gas chromatography–mass spectrometry (GC-MS) in the hair analysis of a number of drugs. HS-SPME coupled with the hydrolysis of the hair matrix by 4% sodium hydroxide in the presence of excess sodium sulphate and of a suitable internal standard proved to be a convenient one-step method for the measurement of many lipophilic basic drugs such as nicotine, amphetamine derivatives, local anaesthetics, phencyclidine, ketamine, methadone, diphenhydramine, tramadol, tricyclic antidepressants and phenothiazines. Detection limits were between 0.05 and 1.0 ng/mg. From spiked 10-mg hair samples absolute recoveries between 0.04 and 5.7% were found. These recoveries decreased considerably if larger sample amounts were used, perhaps due to increased drug solubility in the aqueous phase or to elevated viscosity in the presence of dissolved hair proteins. Because of the phenolic hydroxyl group a change of pH after alkaline hair digestion (by adding excess orthophosphoric acid) was necessary for the detection of Δ 9 -tetrahydrocannabinol (Δ 9 -THC), cannabinol (CBN) and cannabidiol (CBD) by HS-SPME. Nevertheless, the detection limits were such that only CBN could be detected in hair of a consumer. Clomethiazole, a compound hydrolysed in alkali, was measured by HS-SPME after extraction with aqueous buffer. The detection limit was 0.5 ng/mg. Cocaine could not be detected by HS-SPME. The application of HS-SPME to hair samples from several forensic and clinical cases is described.

The analysis of suitable ethanol markers in hair would be an advantageous tool for chronic alcohol abuse control because of the wide diagnostic window allowed by this specimen and the possibility of segmental investigation. Between the markers practically used or thoroughly investigated in blood or urine, ethylglucuronide, fatty acid ethylesters, phosphatidylethanol, acetaldehyde adducts to protein and 5-hydroxytryptophol can be regarded as possible candidates also in hair, but preliminary data were found in the literature only for ethylglucuronide and acetaldehyde modified proteins. By using headspace gas chromatography and headspace solid phase microextraction in combination with gas chromatography-mass spectrometry (SPME-GC/MS), in alkaline hydrolysates of hair it was possible to determine between 17 and 135 ng/mg of ethanol beside acetone and several other volatile compounds with slightly higher ethanol values for alcoholics than for social drinkers and teetotalers. A part of this is ethanol only absorbed in the hair matrix from the surrounding environment and consequently is not applicable as a diagnostic criterion. By extraction with aqueous buffer, methanol or a methanol/chloroform mixture and subsequent alkaline hydrolysis it was found that another part is generated from ethylesters, which are preferentially deposited in the lipid fraction of hair. In a specific search for ethylesters of 17 carboxylic acids by GC/MS-SIM in most cases ethyl 4-hydroxybenzoate (0.1 to 5.9 ng/mg, a preservative in hair cosmetics) and in four cases traces of indolylacetic acid ethylester were found. Furthermore, diethyl phthalate (a softening agent, present also in many cosmetic products) was identified in the hair of alcoholics as well as of children. As potential markers of alcohol intake, ethyl palmitate, ethyl stearate and ethyl oleate were detected in hair samples of alcoholics by headspace SPME-GC/MS of the chloroform/methanol extracts.

Sensing the chemical warnings present in the environment is essential for species survival. In mammals, this form of danger communication occurs via the release of natural predator scents that can involuntarily warn the prey or by the production of alarm pheromones by the stressed prey alerting its conspecifics. Although we previously identified the olfactory Grueneberg ganglion as the sensory organ through which mammalian alarm pheromones signal a threatening situation, the chemical nature of these cues remains elusive. We here identify, through chemical analysis in combination with a series of physiological and behavioral tests, the chemical structure of a mouse alarm pheromone. To successfully recognize the volatile cues that signal danger, we based our selection on their activation of the mouse olfactory Grueneberg ganglion and the concomitant display of innate fear reactions. Interestingly, we found that the chemical structure of the identified mouse alarm pheromone has similar features as the sulfur-containing volatiles that are released by predating carnivores. Our findings thus not only reveal a chemical Leitmotiv that underlies signaling of fear, but also point to a double role for the olfactory Grueneberg ganglion in intraspecies as well as interspecies communication of danger.

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC–MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200 μL of urine in less than 30 min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5 min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC–MS run to 10 min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC–MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC–COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

Background: Ethyl glucuronide (EtG) in hair has emerged as a useful biomarker for detecting alcohol abuse and monitoring abstinence. However, there is a need to establish a reliable cutoff value for the detection of chronic and excessive alcohol consumption. Methods: One hundred and twenty-five subjects were classified as teetotalers, low-risk drinkers, at-risk drinkers, or heavy drinkers. The gold standard for subjects' classifications was based on a prospective daily alcohol self-monitoring log. Subjects were followed for a 3-month period. The EtG diagnostic performance was evaluated and compared with carbohydrate-deficient transferring (CDT) and the activities of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl-transferase (γGT). Results: A cutoff of >9pg/mg EtG in hair, suggesting an alcohol consumption of >20/30g (at-risk drinkers), and a cutoff of >25pg/mg, suggesting a consumption of >60g (heavy drinkers), were determined by receiver operating characteristic analysis. The EtG diagnostic performance was significantly better (P

The aim of this work is to present some practical, postmortem biochemistry applications to illustrate the usefulness of this discipline and reassert the importance of carrying out biochemical investigations as an integral part of the autopsy process. Five case reports are presented pertaining to diabetic ketoacidosis in an adult who was not known to suffer from diabetes and in presence of multiple psychotropic substances; fatal flecainide intoxication in a poor metabolizer also presenting an impaired renal function; diabetic ketoacidosis showing severe postmortem changes; primary aldosteronism presented with intracranial hemorrhage and hypothermia showing severe postmortem changes. The cases herein presented can be considered representative examples of the importance of postmortem biochemistry investigations, which may provide significant information useful in determining the cause of death in routine forensic casework or contribute to understanding the pathophysiological mechanisms involved in the death process.

In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples. The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC-MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140pg/mg. In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter.

In this report, the authors present a case of unusual, accidental methadone intoxication in a 40-year-old man, who had inhaled methadone powder. The drug dealer was a pharmacy technician; methadone had been stolen from a pharmacy and sold as cocaine. After having inhaled methadone powder, he suffered cardiopulmonary arrest. He was admitted to hospital where he died after 24 h of intensive care. The autopsy revealed congestion of internal organs and cerebral and pulmonary edema. Microscopically, the heart showed no changes. The toxicological analyses performed on blood and urine taken at the hospital revealed methadone, cannabinoids, and ethanol. The blood methadone concentration was 290 μg/L. The urine methadone concentration was 160 μg/L. Midazolam and lidocaine, which were administered to the patient at the hospital, were also detected in the blood. The cause of death was determined to be methadone intoxication. The literature has been reviewed and discussed. To date, and to our knowledge, only very few cases of accidental death resulting from methadone inhalation have been described up to the case presented herein.

Ethyl glucuronide (EtG) is a minor and specific metabolite of ethanol. It is incorporated into growing hair, allowing a retrospective detection of alcohol consumption. However, the suitability of quantitative EtG measurements in hair to determine the quantity of alcohol consumed has not clearly been demonstrated yet. The purpose of this study was to evaluate the influence of ethanol dose and hair pigmentation on the incorporation of EtG into rat hair. Ethanol and EtG kinetics in blood were investigated after a single administration of ethanol. Eighteen rats were divided into four groups receiving 0 (control group), 1, 2, or 3g ethanol/kg body weight. Ethanol was administered on 4 consecutive days per week for 3 weeks by intragastric route. Twenty-eight days after the initial ethanol administration, newly grown hair was shaved. Pigmented and nonpigmented hair were analyzed separately by gas chromatography coupled to tandem mass spectrometry. Blood samples were collected within 12h after the ethanol administration. EtG and ethanol blood levels were measured by liquid chromatography coupled to tandem mass spectrometry and headspace gas chromatography-flame ionization detector, respectively. No statistically significant difference was observed in EtG concentrations between pigmented and nonpigmented hair (Spearman's rho=0.95). Thus, EtG incorporation into rat hair was not affected by hair pigmentation. Higher doses of ethanol resulted in greater blood ethanol area under the curve of concentration versus time (AUC) and in greater blood EtG AUC. A positive correlation was found between blood ethanol AUC and blood EtG AUC (Spearman's rho=0.84). Increased ethanol administration was associated with an increased EtG concentration in hair. Blood ethanol AUC was correlated with EtG concentration in hair (Pearson's r=0.89). EtG concentration in rat hair appeared to reflect the EtG concentration in blood. Ethanol was metabolized at a median rate of 0.22 g/kg/h, and the median elimination half-life of EtG was 1.21 h. This study supports that the bloodstream is likely to display a major role in the hair EtG incorporation.

In the wild, animals have developed survival strategies relying on their senses. The individual ability to identify threatening situations is crucial and leads to increase in the overall fitness of the species. Rodents, for example have developed in their nasal cavities specialized olfactory neurons implicated in the detection of volatile cues encoding for impending danger such as predator scents or alarm pheromones. In particular, the neurons of the Grueneberg ganglion (GG), an olfactory subsystem, are implicated in the detection of danger cues sharing a similar chemical signature, a heterocyclic sulfur- or nitrogen-containing motif. Here we used a “from the wild to the lab” approach to identify new molecules that are involuntarily emitted by predators and that initiate fear-related responses in the recipient animal, the putative prey. We collected urines from carnivores as sources of predator scents and first verified their impact on the blood pressure of the mice. With this approach, the urine of the mountain lion emerged as the most potent source of chemical stress. We then identified in this biological fluid, new volatile cues with characteristic GG-related fingerprints, in particular the methylated pyridine structures, 2,4-lutidine and its analogues. We finally verified their encoded danger quality and demonstrated their ability to mimic the effects of the predator urine on GG neurons, on mice blood pressure and in behavioral experiments. In summary, we were able to identify here, with the use of an integrative approach, new relevant molecules, the pyridine analogues, implicated in interspecies danger communication.

The antipsychotic drug clotiapine (Entumine) has been marketed for more than 35 years, however there is little published data on the therapeutic and toxic concentrations of this drug. To fill this gap, two rapid and sensitive methods were developed for the determination of clotiapine (2-chloro-11-(4-methyl-1-piperazinyl)dibenzo-[b,f][1,4]-thiazepine), in human plasma and post-mortem blood and tissue samples. After simple liquid-liquid extraction at pH 9.5 with n-hexane/dichloromethane (85/15, v/v), clotiapine was quantitated by HPLC-DAD and by GC-NPD. The calibration curve was linear between 10 and 1000 microg/L. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 2 and 6 microg/L for the GC-NPD method and 5 and 15 microg/L for the HPLC-method, respectively. These methods were applied to 12 plasma samples from patients treated with clotiapine, to seven autopsy cases and to one case of driving under the influence of drugs (DUID). Concentrations ranged for the clotiapine-treated patients between 6 and 155 microg/L (mean 46 microg/L), and for the autopsy cases between 22 and 341 microg/L (mean 123 microg/L).

Cigarettes may contain up to 10% by weight additives which are intended to make them more attractive. A fast and rugged method for a cigarette-screening for additives with medium volatility was developed using automatic headspace solid phase microextraction (HS-SPME) with a 65 microm carbowax-divinylbenzene fiber and gas chromatography-mass spectrometry (GC-MS) with standard electron impact ionisation. In three runs, each cigarette sample was extracted in closed headspace vials using basic, acidic and neutral medium containing 0.5 g NaCl or Na2SO4. Furthermore, the method was optimized for quantitative determination of 17 frequently occurring additives. The practical applicability of the method was demonstrated for cigarettes from 32 brands.

Analytical records concerning 440 living drivers suspected of driving under the influence of drug (DUID) were collected and examined during a 2 years period ranging from 2002 to 2003 in canton de Vaud, Valais, Jura and Fribourg (Switzerland). This study included 400 men (91%) and 40 women (9%). The average age of the drivers was 28+/-10 years (minimum 16 and maximum 81). One or more psychoactive drugs were found in 89% of blood samples. Half of cases (223 of 440, 50.7%) involved consumption of mixtures (from 2 to 6) of psychoactive drugs. The most commonly detected drugs in whole blood were cannabinoids (59%), ethanol (46%), benzodiazepines (13%), cocaine (13%), amphetamines (9%), opiates (9%) and methadone (7%). Among these 440 cases, 11-carboxy-THC (THCCOOH) was found in 59% (median 25 ng/ml (1-215 ng/ml)), Delta(9)-tetrahydrocannabinol (THC) in 53% (median 3 ng/ml (1-35 ng/ml)), ethanol in 46% (median 1.19 g/kg (0.14-2.95 g/kg)), benzoylecgonine in 13% (median 250 ng/ml (29-2430 ng/ml)), free morphine in 7% (median 10 ng/ml (1-111 ng/ml)), methadone in 7% (median 110 ng/ml (27-850 ng/ml)), 3,4-methylenedioxymethamphetamine (MDMA) in 6% (median 218 ng/ml (10-2480 ng/ml)), nordiazepam in 5% (median 305 ng/ml (30-1560 ng/ml)), free codeine in 5% (median 5 ng/ml (1-13 ng/ml)), midazolam in 5% (median 44 ng/ml (20-250 ng/ml)), cocaine in 5% (median 50 ng/ml (15-560 ng/ml)), amphetamine in 4% (median 54 ng/ml (10-183 ng/ml)), diazepam in 2% (median 200 ng/ml (80-630 ng/ml)) and oxazepam in 2% (median 230 ng/ml (165-3830 ng/ml)). Other drugs, such as lorazepam, zolpidem, mirtazapine, methaqualone, were found in less than 1% of the cases.

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

A sensitive and reproducible method for the quantitative determination of cathinone (CTN), norpseudoephedrine (NPE, cathine) and norephedrine (NE) from hair was developed. The compounds were extracted for 4 hours with phosphate buffer pH 2.0, followed by a standard solid phase extraction procedure on a mixed phase column, derivatization with heptafluorobutyric anhydride (HFBA) and GC–MS separation and quantification using D 3 -ephedrine (D 3 -E) and α-aminoacetophenone (AAP) as the internal standards. The diastereomers NPE and NE were satisfactorily separated. In the validation, the limits of detection and of quantification were determined at 0.03–0.08 ng/mg and 0.10–0.24 ng/mg, respectively and the interday standard deviation was between 10 and 15%. The method was applied to hair samples of 24 Yemenite khat chewers. All three compounds were detected in 23 of these cases. The concentrations ranged from 0.57 to 23.9 ng/mg for NPE, 0.19–25.0 ng/mg for NE and 0.11–22.7 ng/mg for CTN. A highly significant correlation was found between the self-reported data about the khat consumption habits of the volunteers (4–56 h chewing per week) and the concentrations of norephedrine and norpseudoephedrine in hair.

A new and in part automated headspace solid-phase microextraction method for quantitative determination of the highly toxic rodenticide fluoroacetic acid (FAA) in serum and other biological samples has been developed. FAA and deuterated acetic acid (internal standard) were extracted from acidified samples by a StableFlex divinylbenzene-Carboxen on polydimethylsiloxane fibre. The acids were derivatised on the fibre in-situ with 1-pyrenyldiazomethane and detected using gas chromatography-mass spectrometry with electron impact ionisation and selected ion monitoring. The calibration curve for FAA in serum was linear over the range from 0.02 to 5 microg/ml, with limits of detection and quantification of 0.02 and 0.07 microg/ml, respectively. The method was also tested with spiked whole blood, urine, stomach contents and kidney samples. It was sufficiently reliable, reproducible and sensitive for use in routine forensic toxicology applications.

Introduction The specificity of ethyl glucuronide (EtG) in hair as marker of alcohol consumption exceeds by far those of fatty acid ethyl esters. False positive cases are therefore very rare but not excluded as recent publications have shown. Especially, the use of plant extracts containing high percentages of ethanol can lead to EtG hair concentrations typically found in cases of chronic alcohol consumption. As proposed by Baumgartner et al., a nucleohilic substitution could most likely explain this phenomenon. Fresh and dried plants as well as commercial hair lotions based on plants extracts have been analysed for EtG presence or EtG formation. Methods Urtica dioica, Plantago lanceolata, Cortex Quercus, Sempervivum, Armoracia rusticana, Juniperus communis, Brassica alba, Thymian vulgaris, Salvia officinalis, Majorana hortensis, Aloe vera , birch gingko and green tea leafs, ginger, lemon grass were extracted in water, water/ethanol (50/50) and ethanol (100%). The extracts as well as diluted hair lotions were measured by immunological test (Microgenics DRI ® EtG assay) and by LC-MS/MS on Shimadzu Nexera UHPLC coupled with an AB Sciex 4500 QTrap. Results EtG could not be detected in water extracts of all tested plants. However, DRI ® EtG assay indicated the presence of EtG in 66% of the tested ethanolic plant extracts. That could only be confirmed by mass spectrometry in the cases of fresh thyme as well as in dried birch, oak and plantain extracts where EtG concentrations between of 0.25 and 2,09 mg/l were measured. In one hair lotion, the EtG concentration was 0,76 mg/l. Conclusion Ethanolic plant extracts represents a non-negligible risk for false positive EtG hair tests, especially when applied as lotion without following washing out. The use of hair care products must therefore be evaluated at every hair sampling. In case of doubt, the product should be analysed by mass spectrometric methods since the presence of EtG can’t be proven by use of the DRI ® EtG assay, only. Our results support Baumgartner's assumption of a nucleophilic substitution in presence of ethanol because EtG was only measured in the ethanolic extracts.

A simple method for analysis of methadone and its two main metabolites EDDP and EMDP in hair was developed using automatic headspace solid-phase microextraction (HS-SPME) at a multipurpose sampler and gas chromatography-mass spectrometry with electron impact ionization and selected ion monitoring (GC-MS-SIM). The washed hair pieces were digested in the closed headspace vial in 1 ml 1 M NaOH containing 0.5 g NaCl and each 10 ng of the internal standards D9-methadone and D3-EDDP at 110 degrees C for 20 min. Then the HS-SPME was performed with a 65 microm polydimethylsiloxan/ divinylbenzene fiber at the same temperature in the same vial for another 20 min followed by the desorption in the GC injection port. The calibration curves were linear between 0.1 and 3 ng/mg (methadone and EMDP) and 10 ng/mg (EDDP) respectively, at higher concentrations a negative deviation from linearity was found. The detection limits were 0.03 ng/mg (methadone) and 0.05 ng/mg (EDDP and EMDP), and the reproducibility was 9.2% for methadone and 11.2% for EDDP (n= 12). The method was applied to hair samples of 26 drug fatalities. 19 cases were positive with 0.36-11.8 ng/mg methadone and 0.19 -10.8 ng/mg EDDP. EMDP was found only in two cases with 0.18 and 0.84 ng/mg. The methadone concentration range was in agreement with previous data, but the EDDP/methadone concentration ratios (0.19-0.67) were definitely higher than those determined by other methods.