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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.

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

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.

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.

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

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.

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.

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.

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.

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.