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BackgroundAccording to the World Health Organization, road traffic injuries lead to 1.3 million deaths each year and represent the first cause of death for young adults under 30 years old. The use of psychoactive substances, including alcohol, drugs and pharmaceuticals is a well-known risk factor for road traffic injuries. Our study aims to assess the prevalence of substances consumed by drivers in Western Switzerland. Such studies are capital to improve prevention and develop public awareness campaigns.MethodsTo assess the prevalence of psychoactive substances among drivers, roadside controls were performed in collaboration with local polices, using their classical sampling procedures to detect drivers under the influence of drugs or alcohol, over two time periods (P1: 2006-2008, P2: 2017-2020). When impaired-driving was not suspected by the police, minimal-invasive sampling strategies were performed on volunteer drivers, after a road safety survey. A posteriori analyses and statistical interpretation were then performed.ResultsAmongst the 1’605 drivers included in the study, 1’048 volunteers provided an oral fluid sample, whilst 299 provided a dried blood spot sample. The percentage of drivers testing positive to at least one substance which may impair driving ability was stable over time, with a rate of 10.5% of positivity measured over both periods. The proportion of medicinal drugs and drugs of abused showed a slight variation between both periods, with 7.6 % and 6.3 % of prescribed drugs and 3.6 % and 4.9 % of illicit drugs for P1 and P2 respectively. Regarding the consumption of illicit drugs, the highest percentage of positivity was measured in biological fluids of drivers under the age of 35, during nights and week-ends, periods which are considered particularly prone to fatal accidents for this age group. Disturbingly, the road safety survey highlighted that drivers’ perception of the risk of getting positively controlled whilst driving after drug consumption is low (3.3 on a 1-to-10 scale, N=299).ConclusionThe number of positive cases measured in voluntary drivers who passed the preliminary police check demonstrates the importance of systematic biofluid sampling strategies regarding driving under the influence of psychoactive substances. Although the number of fatal road accidents globally decreases overtime, the results of this study reveal the need for both better prevention and deterrent processes that could potentially reduce the risk of fatal road accidents associated with drug consumption.

Tramadol is a synthetic opioid drug used in the treatment of chronic and acute pain. An abnormal prevalence of its misuse in elite sport to overcome pain resulting from prolonged physical effort was recently reported. However, besides its antinociceptive effects, tramadol consumption is associated with negative effects such as numbness, confusion, and reduced alertness. This fact prompted the Union Cycliste Internationale to ban the use of tramadol in cycling competitions. Herein, we present the development of a dried blood spot (DBS) sample collection and preparation method followed by a liquid‐chromatography mass spectrometry (LC‐MS) analysis to rapidly determine the presence of tramadol and its two main metabolites in blood samples. The detection window of each analyte was evaluated and the analysis of performance on various MS platforms (HRMS and MS/MS) was assessed. Tramadol and its two main metabolites were detected up to 12 h after the intake of a single dose of 50 mg of tramadol in positive controls. In professional cycling competitions, 711 DBS samples collected from 361 different riders were analysed using the developed methodology, but all returned negative results (absence of parent and both metabolite compounds). In the context of professional cycling, we illustrate a valid method bringing together the easiness of collection and minimal sample preparation required by DBS, yet affording the performance standards of MS determination. The proposed method to detect tramadol and its metabolites was successfully implemented in cycling races with a probable strong deterrent effect.

Cytochrome P450 (CYP) activity can be assessed using a 'cocktail' phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low-dose probes with a low-invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre-treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. To further validate this cocktail, in this study, we have verified whether probe drugs contained in the latter cause mutual drug-drug interactions. In a randomized, four-way, Latin-square crossover study, 30 healthy volunteers received low-dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug-drug interactions); fexofenadine alone; bupropion alone; or all seven drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self-sampling. The 90% confidence intervals for the geometric mean ratios of AUC metabolite/AUC probe for CYP probes administered alone or within Geneva cocktail fell within the 0.8-1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices, that is metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post-cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping.

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.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients., Scientific Reports, 8, ISSN:2045-2322

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] .

It is well known that the standard doses of a given drug may not have equivalent effects in all patients. To date, the management of depression remains mainly empirical and often poorly evaluated. The development of a personalized medicine in psychiatry may reduce treatment failure, intolerance or resistance, and hence the burden and costs of mood depressive disorders. The Geneva Cocktail Phenotypic approach presents several advantages including the “in vivo” measure of different cytochromes and transporter P-gp activities, their simultaneous determination in a single test, avoiding the influence of variability over time on phenotyping results, the administration of low dose substrates, a limited sampling strategy with an analytical method developed on DBS analysis. The goal of this project is to explore the relationship between the activity of drug-metabolizing enzymes (DME), assessed by a phenotypic approach, and the concentrations of Venlafaxine (VLX) + O-demethyl-venlafaxine (ODV), the efficacy and tolerance of VLX. This study is a multicentre prospective non-randomized open trial. Eligible patients present a major depressive episode, MADRS over or equal to 20, treatment with VLX regardless of the dose during at least 4 weeks. The Phenotype Visit includes VLX and ODV concentration measurement. Following the oral absorption of low doses of omeprazole, midazolam, dextromethorphan, and fexofenadine, drug metabolizing enzymes activity is assessed by specific metabolite/probe concentration ratios from a sample taken 2 h after cocktail administration for CYP2C19, CYP3A4, CYP2D6; and by the determination of the limited area under the curve from the capillary blood samples taken 2–3 and 6 h after cocktail administration for CYP2C19 and P-gp. Two follow-up visits will take place between 25 and 40 days and 50–70 days after inclusion. They include assessment of efficacy, tolerance and observance. Eleven french centres are involved in recruitment, expected to be completed within approximately 2 years with 205 patients. Metabolic ratios are determined in Geneva, Switzerland. By showing an association between drug metabolism and VLX concentrations, efficacy and tolerance, there is a hope that testing drug metabolism pathways with a phenotypical approach would help physicians in selecting and dosing antidepressants. The MARVEL study will provide an important contribution to increasing the knowledge of VLX variability and in optimizing the use of methods of personalized therapy in psychiatric settings. ClinicalTrials.gov NCT02590185 (10/27/2015). This study is currently recruiting participants.

Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurological disorders. Current approaches using microdialysis catheters are however drastically limited in term of temporal resolution. Here we show a functional in vivo validation of a droplet collection system included at the tip of a neural probe. The system comprises an advanced droplet formation mechanism which enables the collection of neurochemicals present in the brain ECF at high-temporal resolution. The probe was implanted in a rat brain and could successfully collect fluid samples organized in a train of droplets. A microfabricated target plate compatible with most of the surface-based detection methods was specifically developed for sample analysis. The time-resolved brain-fluid samples are analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results provide a time evolution picture of the cerebral tissues neurochemical composition for selected elements known for their involvement in neurodegenerative diseases., Current approaches studying the collection of extracellular fluid to monitor neurological disorders have temporal resolution limitations. Here the authors show functional in vivo validation of a droplet collection system included at the tip of a neural probe.

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.Clinical Pharmacology & Therapeutics (2014); advance online publication 7 May 2014. doi:10.1038/clpt.2014.83.

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.

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

Busulfan (Bu) is an important component of the myeloablative conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) especially in children. Intravenously administered Bu exhibits a therapeutic window phenomenon requiring therapeutic drug monitoring. Analytical methods developed for Bu routine monitoring were aimed at using low volumes of biological fluids and development of simple procedures to facilitate the dosage adjustment. In this report, we describe a simple, rapid method for Bu measurement using dried blood spots (DBS) from only 5 μL of whole blood.

Because of the emergence of dried blood spots (DBS) as an attractive alternative to conventional venous plasma sampling in many pharmaceutical companies and clinical laboratories, different analytical approaches have been developed to enable automated handling of DBS samples without any pretreatment. Associated with selective and sensitive MS-MS detection, these procedures give good results in the rapid identification and quantification of drugs (generally less than 3min total run time), which is desirable because of the high throughput requirements of analytical laboratories. The objective of this review is to describe the analytical concepts of current direct DBS techniques and to present their advantages and disadvantages, with particular focus on automation capacity and commercial availability. Finally, an overview of the different biomedical applications in which these concepts could be of major interest will be presented. Figure Direct analysis of dried blood spots

This work presents a strategy for the evaluation of differences in plasma phospholipid content between atherosclerotic and healthy mice from micro volumes (2 microL) spotted on filter paper. Dried plasma spots (DPS) were directly desorbed into a triple quadrupole linear ion trap mass spectrometer using a homemade prototype, ensuring high-throughput analysis of dried spots without any sample pretreatment. Multiple positive and negative neutral loss and precursor ion scans were simultaneously acquired in a single loop, allowing oriented fingerprinting until 2700 potential species including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM) classes. The phospholipidic variations between 15 healthy and 15 atherosclerotic mice were evaluated using t tests, matrix reduction and merging, and principal component analysis (PCA) as a chemometric statistical approach. The discriminating ions in PCA analysis were qualitatively identified in an information dependent acquisition (IDA) manner using enhanced resolution and enhanced product ion scans. PCA demonstrates a clear clustering between healthy and diseased mice. Regarding the most relevant variables identified, this procedure has confirmed the role of SM and PS classes in atherosclerosis and has established potential biomarkers shown to be significantly up- or down-regulated with the disease. The results presented in this work demonstrate the sample processing and analysis potential of the developed strategy to evaluate new biomarkers and the state of a disease.

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro-whole blood samples (i.e. 5 microL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.

The objective of this work was to combine the advantages of the dried blood spot (DBS) sampling process with the highly sensitive and selective negative-ion chemical ionization tandem mass spectrometry (NICI-MS-MS) to analyze for recent antidepressants including fluoxetine, norfluoxetine, reboxetine, and paroxetine from micro whole blood samples (i.e., 10 microL). Before analysis, DBS samples were punched out, and antidepressants were simultaneously extracted and derivatized in a single step by use of pentafluoropropionic acid anhydride and 0.02% triethylamine in butyl chloride for 30 min at 60 degrees C under ultrasonication. Derivatives were then separated on a gas chromatograph coupled with a triple-quadrupole mass spectrometer operating in negative selected reaction monitoring mode for a total run time of 5 min. To establish the validity of the method, trueness, precision, and selectivity were determined on the basis of the guidelines of the "Société Française des Sciences et des Techniques Pharmaceutiques" (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500 ng mL(-1) for fluoxetine and norfluoxetine and 20 to 500 ng mL(-1) for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20 pg mL(-1) for all the analytes. The stability of DBS was also evaluated at -20 degrees C, 4 degrees C, 25 degrees C, and 40 degrees C for up to 30 days. Furthermore, the method was successfully applied to a pharmacokinetic investigation performed on a healthy volunteer after oral administration of a single 40-mg dose of fluoxetine. Thus, this validated DBS method combines an extractive-derivative single step with a fast and sensitive GC-NICI-MS-MS technique. Using microliter blood samples, this procedure offers a patient-friendly tool in many biomedical fields such as checking treatment adherence, therapeutic drug monitoring, toxicological analyses, or pharmacokinetic studies.

Objectifs : Cet article presente un nouveau concept d’extraction en ligne de composes d’interet pharmaceutique a partir de spots de sang seche (DBS) et avant analyse par LC-MS/MS. Methodes : Bien qu’il existe un nombre important de publications dediees a l’analyse de composes d’interet pharmaceutique, le couplage en ligne DBS-LC-MS/MS reste encore un defi serieux pour l’analyste toxicologue. Ce travail, a la lumiere de donnees de la litterature et de la propre experience des auteurs fait le point sur l’etat de la question en 2010. Conclusion : Le couplage en ligne DBS-LC-MS/MS va sans aucun doute modifier l’approche analytique en toxicologie clinique et forensique.

The aim of this work is to present a new concept, called on-line desorption of dried blood spots (on-line DBS), allowing the direct analysis of a dried blood spot coupled to liquid chromatography mass spectrometry device (LC/MS). The system is based on an inox cell which can receive a blood sample (10 microL) previously spotted on a filter paper. The cell is then integrated into LC/MS system where the analytes are desorbed out of the paper towards a column switching system ensuring the purification and separation of the compounds before their detection on a single quadrupole MS coupled to atmospheric pressure chemical ionisation (APCI) source. The described procedure implies that no pretreatment is necessary in spite the analysis is based on whole blood sample. To ensure the applicability of the concept, saquinavir, imipramine, and verapamil were chosen. Despite the use of a small sampling volume and a single quadrupole detector, on-line DBS allowed the analyses of these three compounds over their therapeutic concentrations from 50 to 500 ng/mL for imipramine and verapamil and from 100 to 1000 ng/mL for saquinavir. Moreover, the method showed good repeatability with relative standard deviation (RSD) lower than 15% based on two levels of concentration (low and high). Function responses were found to be linear over the therapeutic concentration for each compound and were used to determine the concentrations of real patient samples for saquinavir. Comparison of the founded values with those of a validated method used routinely in a reference laboratory showed a good correlation between the two methods. Moreover, good selectivity was observed ensuring that no endogenous or chemical components interfered with the quantitation of the analytes. This work demonstrates the feasibility and applicability of the on-line DBS procedure for bioanalysis.

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.

Microsampling, mainly as DBS, has been significantly expanded in the biomedical and pharmaceutical communities in the last 10 years. In parallel, technology and methodology have evolved to overcome some of the issues associated with this sampling procedure. Despite the continuous developments and interest, only a few validated and routinely implemented clinical applications have arisen beyond the initial inborn screening. Based on the latest developments in this field, this perspective aims to discuss some of the missing steps (i.e., the habits to change, the Health Authorities acceptance and the shift for dried plasma generation), which may turn the current use of microsampling into an established and standard procedure in clinical and pharmaceutical analysis.

Background: Direct-infusion ESI–MS/MS is a powerful approach for the identification of substances in complex mixtures. The aim of this work was to investigate its applicability to the toxicological screening of blood samples. A simple protein precipitation was used, followed by a 15 min infusion of the extract in the mass spectrometer. Results: The application of the procedure to commercial quality controls and authentic post-mortem blood samples demonstrated that the direct-infusion ESI–MS/MS approach enables the simultaneous identification of substances that require different chromatographic conditions. However, poor sensitivity was observed for benzodiazepine, amphetamines and opiate compounds. Conclusion: Considering the facile implementation and positive performance of direct-infusion ESI–MS/MS, this approach may to be a valuable complementary technique for systematic toxicological analysis procedures.

Background: An LC–MS/MS method has been developed for the simultaneous quantification of P-glycoprotein (P-gp) and cytochrome P450 (CYP) probe substrates and their Phase I metabolites in DBS and plasma. P-gp (fexofenadine) and CYP-specific substrates (caffeine for CYP1A2, bupropion for CYP2B6, flurbiprofen for CYP2C9, omeprazole for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4) and their metabolites were extracted from DBS (10 µl) using methanol. Analytes were separated on a reversed-phase LC column followed by SRM detection within a 6 min run time. Results: The method was fully validated over the expected clinical concentration range for all substances tested, in both DBS and plasma. The method has been successfully applied to a PK study where healthy male volunteers received a low dose cocktail of the here described P-gp and CYP probes. Good correlation was observed between capillary DBS and venous plasma drug concentrations. Conclusion: Due to its low-invasiveness, simple sample collection and minimal sample preparation, DBS represents a suitable method to simultaneously monitor in vivo activities of P-gp and CYP.

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid,5 ng/ml; pericardial fluid, 14 ng/ml; brain,5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

This article describes a rapid LC-MS/MS target screening method based on an automated extraction of 5 mu L dried blood spots (DBS), two 5 min chromatographic runs on orthogonal phase columns (RP and Hilic) and a data dependent acquisition (DDA) of product ions spectra for the reliable identification of the detected compounds. The extraction step was performed in 2 min by using the LC autosampler itself in 96-well plates. This procedure was evaluated using 22 model compounds frequently encountered in forensic investigations, i.e., cocaine, benzodiazepines, amphetamines, opioids, antidepressants and antipsychotics. These investigations showed that even if the extraction step was reduced to a minimum, the extraction recoveries were satisfactory (median value of 40 %) and allowed for the detection of the model compounds in their therapeutic ranges, with the exception of morphine. Moreover, the use of two different chromatographic columns broadened the number of screening targets to those that behaved poorly under RP conditions, such as amphetamines or glucuronides, while keeping chromatographic gradients very short. This procedure was applied to 34 authentic post-mortem cases. It allowed the detection of 89 % of the compounds that were quantified in the routine procedures and the formal identification of 77 % of the compounds using their product ions spectra. These results were considered more than satisfactory compared to routine screening alone (GC-MS and LC-DAD, 55 % compound identification). The method described in this article is therefore a powerful approach for a fast, reliable and efficient target screening of drugs in forensic and clinical investigations.

We investigated whether a single blood measurement using the minimally invasive technique of a finger prick to draw a blood sample of 5 µl (to yield a dried blood spot (DBS)) is suitable for the assessment of flurbiprofen (FLB) metabolic ratio (MR). Ten healthy volunteers who had been genotyped for CYP2C9 were recruited as subjects. They received FLB alone in session 1 and FLB with fluconazole in session 2. In session 3, the subjects were pretreated for 4 days with rifampicin and received FLB with the last dose of rifampicin on day 5. Plasma and DBS samples were obtained between 0 and 8 h after FLB administration, and urine was collected during the 8 h after administration. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. FLB's apparent clearance values decreased by 35% in plasma and DBS during session 2 and increased by 75% in plasma and by 30% in DBS during session 3. Good correlations were observed between MRs calculated from urine, plasma, and DBS samples.

Background: Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC–MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. Results: The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. Conclusion: The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.

Busulfan (Bu) is an important component of the myeloablative conditioning regimen prior to hematopoietic stem cell transplantation (HSCT) especially in children. Intravenously administered Bu exhibits a therapeutic window phenomenon requiring therapeutic drug monitoring. Analytical methods developed for Bu routine monitoring were aimed at using low volumes of biological fluids and development of simple procedures to facilitate the dosage adjustment. In this report, we describe a simple, rapid method for Bu measurement using dried blood spots (DBS) from only 5 μL of whole blood.Bu extracted from DBS with methanol was measured by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry in multiple reaction monitoring mode using D8-Bu as an internal standard. The method was in-house validated evaluating trueness, repeatability, within-laboratory reproducibility, specificity and the lower limit of quantification (LLOQ).The method was linear in the calibration range of 100-2000 ng mL(-1) (r(2)0.99) encompassing the therapeutic concentrations of Bu. A good trueness (14%), precision (10%), and recovery (100%) were observed during validation of the method with quality controls of 300, 600 and 1400 ng mL(-1). The LLOQ was determined as 50 ng mL(-1) and no matrix or carryover effects were observed. The validated method was applied to measure Bu levels in four children receiving infusion of Bu prior to HSCT. A good correlation was observed between the Bu levels measured by DBS and dried plasma spot (DPS) (r(2) =0.96) and between DPS and the GC/MS method (r(2) =0.92). Bu was found to be stable in DBS up to 6 h at room temperature and for 24 h at 4 °C.The new DBS method facilitates earlier dosage adjustment during Bu therapy by its specific and simple procedure using 5 μL of whole blood.

SummaryThe identification and quantification of proteins and lipids is of major importance for the diagnosis, prognosis and understanding of the molecular mechanisms involved in disease development. Owing to its selectivity and sensitivity, mass spectrometry has become a key technique in analytical platforms for proteomic and lipidomic investigations. Using this technique, many strategies have been developed based on unbiased or targeted approaches to highlight or monitor molecules of interest from biomatrices. Although these approaches have largely been employed in cancer research, this type of investigation has been met by a growing interest in the field of cardiovascular disorders, potentially leading to the discovery of novel biomarkers and the development of new therapies. In this paper, we will review the different mass spectrometry- based proteomic and lipidomic strategies applied in cardiovascular diseases, especially atherosclerosis. Particular attention will be given to recent developments and the role of bioinformatics in data treatment. This review will be of broad interest to the medical community by providing a tutorial of how mass spectrometric strategies can support clinical trials.

This paper illustrates the development of an automated system for the on-line bioanalysis of dried blood spots (on-line DBS). In this way, a prototype was designed for integration into a conventional LC/MS/MS, allowing the successive extraction of 30 DBS toward the analytical system without any sample pretreatment. The developed method was assessed for the DBS analysis of flurbiprofen (FLB) and its metabolite 4-hydroxyflurbiprofen (OH-FLB) in human whole blood (i.e. 5 μL). The automated procedure was fully validated based on international criteria and showed good precision, trueness, and linearity over the expected concentration range (from 10 to 1000 ng/mL and 100 to 10,000 ng/mL for OH-FLB and FLB respectively). Furthermore, the prototype showed good results in terms of recovery and carry-over. Stability of both analytes on filter paper was also investigated and the results suggested that DBS could be stored at ambient temperature for over 1 month. The on-line DBS automated system was then successfully applied to a pharmacokinetic study performed on healthy male volunteers after oral administration of a single 50-mg dose of FLB. Additionally, a comparison between finger capillary DBS and classic venous plasma concentrations was investigated. A good correlation was observed, demonstrating the complementarity of both sampling forms. The automated system described in this article represents an efficient tool for the LC/MS/MS analysis of DBS samples in many bioanalytical applications.

InnovAtIve APProAch to Blood sAmPlIng usIng drIed Blood sPots. APPlIcAtIon to PhArmAcokInetIcs And cytochrome P450 PhenotyPIng M. Bosilkovska; J. Deglon; A. Thomas; C. Samer; J. Desmeules; and Y. Daali Geneva University Hospitals, Geneva, Switzerland; and University Center of Legal Medicine, Lausanne-Geneva, Switzerland Background: The use of dried blood spots (DBS) has gained in popularity in the last few years over conventional whole blood or plasma sampling for PK or drug monitoring. In order to overcome the impact that haematocrit has on the spreading of the applied drop of blood, precise knowledge of the collected volume is crucial for the determination of drug/metabolites concentrations. Material and Methods: Although the collection of an accurate capillary volume using a volumetric micropipette is simpler than venous blood collection, it still needs to be conducted by trained technicians using dedicated instruments. To simplify this process a new capillary blood collection device has been developed. The prototype integrates a patented microfluidic plate (WO/2013/144743) allowing for accurate volume control and a conventional filter paper card for blood storage. The concentrations and pharmacokinetic profiles of a P-glycoprotein (P-gp) and six cytochrome P450 (CYP) probes and their metabolites obtained with the new sampling device have been compared with a conventional volumetric micropipetting method in a clinical trial including 30 volunteers who have received the Geneva cocktail for CYP and P-gp phenotyping. The quantification was done using a previously validated LC/MS-MS method. Results: Concentrations obtained with the new microfluidic sampling device showed excellent correlation with conventional micropipetting concentrations with slopes values close to 1 (0.91 – 1.03) and determination coefficients R> 0.90 for all of the 13 analysed substances. Sampling could be successfully performed by the volunteers themselves with almost no previous training. Conclusion: DBS technique combined with an innovative sampling device and a sensitive analytical method can be used as a self-test for CYP and P-gp phenotyping The use of this technique can be further enlarged to the quantification of other substances for PK studies and therapeutic drug monitoring.

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.