Randomly controlled drivers using minimally invasive sampling: assessment of drug prevalence in Western Switzerland over two time periods.

BACKGROUND: According to the World Health Organization, road traffic injuries lead to 1.3 million deaths each year and represent the leading 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 pivotal to improving prevention and developing public awareness campaigns. METHODS: To assess the prevalence of psychoactive substances among drivers, roadside controls were performed in collaboration with local police, 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, minimally invasive sampling strategies (i.e., oral fluids during P1 and dried blood spots during P2) were performed on volunteer drivers after a road safety survey. A posteriori analyses and statistical interpretation were then performed. RESULTS: Among the 1605 drivers included in the study, 1048 volunteers provided an oral fluid sample, while 299 provided a dried blood spot sample. The percentage of drivers testing positive for at least one substance that can impact driving abilities was stable over time, with a rate of 10.5% positivity measured over both periods. Considering the different categories of substances, a slight variation was observed between both periods, with 7.6 and 6.3% of pharmaceuticals 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 while driving after drug consumption is low (3.3 on a 1-to-10 scale, N = 299). CONCLUSION: The 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 has decreased over time, 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.

Metabolomics reveals biomarkers in human urine and plasma to predict cytochrome P450 2D6 (CYP2D6) activity.

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. 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 precision medicine. This study focuses on the identification of markers of the CYP2D6 enzyme in human biofluids using an LC-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 7 days. CYP2D6 genotyping and phenotyping, using single oral dose of dextromethorphan 5 mg, were also performed in all participants. KEY RESULTS: We report 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 with 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). Mean relative intensities of these ions were significantly higher in the CYP2D6 normal-ultrarapid metabolizer group (n = 37) compared with the poor metabolizer group (n = 6). Solanidine intensity was more than 15 times higher in CYP2D6-deficient individuals compared with 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 personalized medicine.

Detecting early myocardial ischemia in rat heart by MALDI imaging mass spectrometry.

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.

High-Throughput Qualitative and Quantitative Drug Checking by MALDI HRMS.

Illicit drugs are a global health problem, since both their acute and chronic consumption have negative impacts on the drug user's health. Drug checking facilities are receiving growing interest as they allow drug users to chemically analyze their product prior to consumption to assess the presence of adulterants or other non-expected substances. Such harm reduction programs allow the reduction of the risks associated with drug consumption without encouraging it. In particular, the emergence of new psychoactive substances (NPS) emphasizes the risk for the population increasing the diversity and the lability of illicit drugs on the market. Analytical developments are required to catch up with this rapid evolution and reduce the potential harm caused by such consumption. In this study, we developed a matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) strategy for the high-throughput qualitative and quantitative analysis of drug checking samples. The use of online-based m/z cloud library for untargeted compound search improved the ability to identify unknown compounds. Sixty-seven drug checking samples were analyzed using this analytical strategy, allowing the detection of 10 designer drugs and several classical drugs of abuse (mainly cocaine and MDMA) as well as adulterants and contaminants. The results were then compared with routine analyses of the same samples using conventional approaches showing similar performance while removing the use of chromatographic separation thus resulting in a significant reduction of the time required for sample preparation and analysis. This study enlightens the potential of MALDI-HRMS as a high-throughput approach allowing to speed-up up to six times the identification and quantification of substances enabling to catch the fast changes on the drug of abuse market. This strategy could be an interesting alternative analytical approach, allowing better prevention and harm reduction for drug users.

Driving Under the Influence of Drugs: A Single Parallel Monitoring-Based Quantification Approach on Whole Blood.

Driving under the influence of psychoactive substances is a major cause of motor vehicle crashes. The identification and quantification of substances most frequently involved in impaired-driving cases in a single analytic procedure could be an important asset in forensic toxicology. In this study, a highly sensitive and selective liquid chromatography (LC) approach hyphenated with Orbitrap high-resolution mass spectrometry (HRMS) was developed for the quantification of the main drugs present in the context of driving under the influence of drugs (DUID) using 100 µL of whole blood. This procedure involves a simple sample preparation and benefit from the selectivity brought by parallel reaction monitoring (PRM) allowing to solve most DUID cases using a single multi-analyte injection. The method was fully validated for the quantification of the major classes of psychoactive substances associated with impaired-driving (cannabinoids, cocaine and its metabolites, amphetamines, opiates and opioids, and the major benzodiazepines and z-drugs). The validation guidelines set by the "Société Française des Sciences et des Techniques Pharmaceutiques" (SFSTP) were respected for 22 psychoactive substances using 15 internal standards. Trueness was measured to be between 95.3 and 107.6% for all the tested concentrations. Precision represented by repeatability and intermediate precision was lower than 12% while recovery (RE) and matrix effect (ME) ranged from 49 to 105% and from -51 to 3%, respectively. The validated procedure provides an efficient approach for the simultaneous and simple quantification of the major drugs associated with impaired driving benefiting from the selectivity of PRM.

Parallel Reaction Monitoring-Based Quantification of Cannabinoids in Whole Blood.

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.

Liquid chromatography-high resolution mass spectrometry for broad-spectrum drug screening of dried blood spot as microsampling procedure.

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.