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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] .

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.

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: 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 ( Case 5. A 30-year old man was involved in a car accident. Blood (+4h after the accident): cocaine (29 μg/L), benzoylecgonine ( 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.