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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.

Cannabis is the most consumed drug of abuse, making it the primary target for identification and quantification in human whole blood regarding forensic and clinical toxicology analyses. Among biological matrices, blood is the reference for toxicological interpretation. A highly sensitive and selective liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS) was developed for the quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxytetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and cannabidiol (CBD). Those cannabinoids were extracted from 1 mL of whole blood by a simple liquid–liquid extraction (LLE) in acidic conditions. HRMS was performed on an Orbitrap-based instrument using its trapping capabilities and increased selectivity for parallel reaction monitoring (PRM) quantification in positive polarity with a negative polarity switching for THC-OH and THC-COOH. Although selected reaction monitoring (SRM) and PRM-targeted methods have similar performance in terms of linearity, dynamic range, precision and repeatability, Orbitrap-based PRM provides a higher specificity due to the use of high-resolution mode separating background ions from the targeted molecules. The method was fully validated according to guidelines set forth by the “Société Française des Sciences et des Techniques Pharmaceutiques” (SFSTP). Trueness was measured below 107% for all tested concentrations. Repeatability and intermediate precision were found to be lower than 12% while the assay was found to be linear in the concentration range of 0.4–20 ng/mL for THC, THC-OH and CBD and of 2–100 ng/mL for THC-COOH. Recovery (RE) and matrix effect (ME) ranged from 70.6 to 102.5% and from −40 to 6.6%, respectively. The validated method provides an efficient procedure for the simultaneous and rapid quantification of cannabinoids in PRM mode providing an alternative over classical SRM.

Background and purpose Individualized assessment of cytochrome P450 2D6 (CYP2D6) activity is usually performed through phenotyping following administration of a probe drug to measure the enzyme's activity. In order to avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-burdensome tools for real-time phenotyping of CYP2D6 could significantly contribute to the expansion of precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using a liquid chromatography-high-resolution mass spectrometry-based metabolomic approach. Experimental approach Plasma and urine samples from healthy volunteers were analysed before and after intake of a daily dose of paroxetine 20 mg over seven days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. Key results In this study, we reported four metabolites of solanidine and two unknown compounds as possible novel CYP2D6 markers. Mean relative intensities of these features were significantly reduced during the inhibition session compared to the control session (n = 37). Semi-quantitative analysis showed that the largest decrease (-85%) was observed for the ion m/z 432.3108 normalized to solanidine (m/z 398.3417). Furthermore, mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared to the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared to other volunteers. Conclusion and implications The applied untargeted metabolomic strategy identified potential novel markers capable of semi-quantitatively predicting CYP2D6 activity, a promising discovery for personalised medicine.

Addiction is a complex brain disease, whose molecular and cellular underpinnings remain largely unknown. Repeated drug use induces long-term plasticity in various brain areas that is considered to potentially trigger further compulsive use. Converging evidence has revealed that memory and addiction share both neural circuitry and molecular mechanisms. While many studies investigated the mesocorticolimbic system, the hippocampus has become a region of interest. Matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) presents a unique ability to map the toxicodynamic changes associated to drugs of abuse. In this study, the acute effect of cocaine in rat brain was investigated with or without co-administration of alcohol. Brain tissue sections for each condition were analyzed using a MALDI-MSI in negative polarity after 9-aminoacridine (9-AA) deposition on the tissue surface. Multivariate analysis including principal component analysis (PCA) and spatial shrunken centroids have been performed to differentiate the metabolic profiles in hippocampus according to the type of administration. This study offers promising results showing the potential of MSI for investigating the metabolomic changes in the brain associated with drug consumption. It may bring new insights into the study of toxicodynamic responses of cocaine consumption in reward-learning related regions.

Diagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6–12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely corresponding to S-NADHX—a damage product of nicotinamide adenine dinucleotide (NADH)). Based on the previously reported role of NAD+/NADH ratio in regulating localization of the sodium channel (Nav1.5) at the plasma membrane, Nav1.5 was evaluated by immunofluorescence. As expected, a fainter signal was observed at the plasma membrane in the predicted ischemic region starting 30 min of ischemia and the change became the most pronounced by 4 h. Metabolomic changes occur early during ischemia, can assist in identifying markers for post-mortem diagnostics and improve understanding of molecular mechanisms.

Background and Purpose: Individualized assessment of the activity of cytochrome P450 2D6 (CYP2D6), a highly variable drug-metabolizing enzyme, is performed through phenotyping during which a probe drug is administered to measure the enzyme’s activity. In order to avoid any iatrogenic harm (allergic drug reaction, dosing error) related to the probe drug, the development of non-invasive tools for real-time phenotyping of CYP2D6 could significantly contribute to the expansion of precision medicine in clinical practice. This study focuses on the identification of endogenous markers of the CYP2D6 enzyme in human biofluids using a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS)-based metabolomics approach. Experimental Approach: Data from a control session were compared to data from an inhibition session. Before the latter, healthy volunteers (extensive and ultrarapid metabolizers) received a daily dose of paroxetine 20 mg over seven days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. Key Results: In CYP2D6 extensive and ultrarapid metabolizers (n = 37), mean relative intensities of five features were significantly reduced during the inhibition session compared to the control session (fold changes ≤ 0.67, FDR-adjusted P < 0.0001). Furthermore, mean relative intensities of these candidates were significantly higher in the CYP2D6 extensive-ultrarapid metabolizer group (n = 37) compared to the poor metabolizer group (n = 6) (fold changes ≤ 0.67, P < 0.0001). Conclusion and Implications: The applied untargeted metabolomics strategy was able to identify five CYP2D6 endogenous metabolites, a promising discovery for non-invasive phenotyping and personalised medicine.

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.

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.