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Although substances incorporated by ingestion are strongly bound to hair, their loss may occur if aggressive decontamination procedures are applied, especially in highly damaged/porous hair. Evaluation of cleaning procedures using hair samples with different porosity obtained from ethanol or drug users (cocaine, heroin, methamphetamine, methadone, fentanyl, tramadol, diazepam, buprenorphine, dihydrocodeine, citalopram and trazodone). The effect of washing time and multiple wash steps with water and methanol were evaluated. Hair samples (n = 16) were selected and evaluated according to (a) the drug pattern consumption, (b) available amount, and (c) hair porosity (c1 'cosmetic treatment', c2: storage time). Six of them were soaked with an aqueous deuterated analogue solution. The samples were cut in 1-cm segments and homogenized. All hair samples were then decontaminated one or six times with 1.5 ml of water or methanol during 1, 5, 15, 30, 60 and/or 90 min (n = 1 to 3/sample, depending on the available amount of hair). Hair extracts were then cleaned up via a solid-phase extraction (SPE) or liquid-liquid extraction (LLE), while the washes were evaporated to dryness. All were thereafter reconstituted and analysed with routine ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods. Although concentrations of parent drugs and/or metabolites presented a negative trend along the washing time with methanol (up to 80%), the compounds were relatively well retained in hair even after a 90 min wash (with methanol or water) in most samples, and their retention would depend mostly on the hair nature rather than their physicochemical properties (whether incorporated by ingestion and/or from external contamination). Moreover, parent drugs and/or metabolites were detected in the washes in most samples, and the ratio between hair and washes decreased along the washing time. More than 50% of the deuterated analogues soaked into hair were still present after the different washing steps. Losses were observed more frequently for long-term stored hair samples, after decontamination with methanol for more than 30 min. Therefore, prolonged or repeated cleaning with methanol should be avoided in general procedures.

Cannabis sativa L. is undoubtedly the most used recreational drug worldwide because of its desired acute psychotropic effects, like relaxation, euphoria and altered perceptions. In addition, promising medical properties of Cannabis components have gained a lot of attention, resulting in a debate to permit recreational Cannabis use in several countries. In recent years, this controversial plant was increasingly studied and a large number of scientific papers were published. Herbal Cannabis consists of a variable and complex matrix, which makes it challenging to properly seize and prepare the sample for qualitative and quantitative analysis. Moreover, both the adoption of legal cut-off values in different countries for the Δ9-tetrahydrocannabinol (THC) content in seizures, and the emergence of cannabidiol (CBD) based products, containing generally small but variable amounts of THC, urged the need for sensitive and reliable analytical techniques to accurately identify and quantify the components of interest. This review presents detailed information on the procedure prior to analysis and covers chromatographic and spectroscopic methods developed for the analysis of cannabinoids in seizures for different forensic purposes, that is, identification/quantification, potency testing, drug- and fiber-type differentiation, age estimation, yield determination and Cannabis profiling. Advantages and drawbacks of existing methods, within a specific forensic context, are discussed. The application of chemometrics, which offers a powerful tool in interpreting complex data, is also explained.

Knowledge of the drug incorporation in hair and impact of cosmetic treatments remains essential to correctly interpret forensic cases. The study shows the analysis of doxylamine and doxylamine-N-oxide and the evaluation of the relationship between dose and hair concentration and the impact of hair treatment (oxidative dying). The study included (A) three subjects participated to the study: a regular user (Subject 1) and two single-dose users (Subject 2, 1 single dose; and Subject 3, 2 single doses spaced 5 months apart). Subject 3 applied a permanent oxidative hair dying monthly. (B) A permanent oxidative hair dying was applied twice to the hair collected from Subject 2. (A) The average concentrations in head hair for doxylamine and its N-doxylamine-oxide, respectively, were as follows: Subject 1, 1825 pg/mg and 16 pg/mg; Subject 2, 182 andlimit of quantification (LOQ); and Subject 3, Strand 1 (1 month after last single dose]; 1st cm: 367 pg/mg and 22 pg/mg; 2nd cm: 664 pg/mg and 37 pg/mg; 3rd cm 108 pg/mg and 13 pg/mg; 4th cm 13 pg/mg and 7 pg/mg); Strand 2 (5 months after last single dose:LOQ). (B) The oxidative hair dying increased the concentration of the metabolite from undetectedLOQ up to 79 pg/mg (n = 3, RSD 6%). The concentration of the parent drug decreased from 128 pg/mg to 40 pg/mg (n = 3, RSD 4%). This is the first time the incorporation of doxylamine and its metabolite was evaluated with adequate and controlled information concerning dose and timing of ingestion for the evaluation of their incorporation and the impact of a cosmetic treatment.

Spectroscopic techniques combined with chemometrics are a promising tool for analysis of seized drug powders. In this study, the performance of three spectroscopic techniques [Mid-InfraRed (MIR), Raman and Near-InfraRed (NIR)] was compared. In total, 364 seized powders were analyzed and consisted of 276 cocaine powders (with concentrations ranging from 4 to 99 w%) and 88 powders without cocaine. A classification model (using Support Vector Machines [SVM] discriminant analysis) and a quantification model (using SVM regression) were constructed with each spectral dataset in order to discriminate cocaine powders from other powders and quantify cocaine in powders classified as cocaine positive. The performances of the models were compared with gas chromatography coupled with mass spectrometry (GC–MS) and gas chromatography with flame-ionization detection (GC–FID). Different evaluation criteria were used: number of false negatives (FNs), number of false positives (FPs), accuracy, root mean square error of cross-validation (RMSECV) and determination coefficients (R2). Ten colored powders were excluded from the classification data set due to fluorescence background observed in Raman spectra. For the classification, the best accuracy (99.7%) was obtained with MIR spectra. With Raman and NIR spectra, the accuracy was 99.5% and 98.9%, respectively. For the quantification, the best results were obtained with NIR spectra. The cocaine content was determined with a RMSECV of 3.79% and a R2 of 0.97. The performance of MIR and Raman to predict cocaine concentrations was lower than NIR, with RMSECV of 6.76% and 6.79%, respectively and both with a R2 of 0.90. The three spectroscopic techniques can be applied for both classification and quantification of cocaine, but some differences in performance were detected. The best classification was obtained with MIR spectra. For quantification, however, the RMSECV of MIR and Raman was twice as high in comparison with NIR. Spectroscopic techniques combined with chemometrics can reduce the workload for confirmation analysis (e.g., chromatography based) and therefore save time and resources.

“Designer” benzodiazepines (DBZDs) are becoming increasingly available in Europe, with the European Monitoring Centre of Drugs and Drug Addiction currently monitoring ∼30 new benzodiazepines. The following driving under the influence of drug (DUID) case describes the oral fluid (OF) and blood concentrations, as well as the observed effects after the combined use of stimulants and flubromazepam. Both OF, collected via the Intercept i2 collector (Immunalysis, Pomona, CA, USA), and blood (collected in containers with various stabilizers) were screened using a liquid chromatographic (LC) time-of-flight (TOF) mass spectrometric (MS-MS) method. In addition, various LC–MS-MS methods in multi-reaction monitoring mode were applied for confirmation and quantification. The OF and blood samples were taken 2 h 25 min and 9 h 19 min after the accident, respectively. OF contained 789 ng/mL amphetamine, 5,173 ng/mL MDMA, 168 ng/mL benzoylecgonine, 492 ng/mL cocaine, 134 ng/mL 4-methylmethcathinone (4-MMC) and traces of flubromazepam (less than limit of quantification (LLOQ); 2 ng/mL). The sodium-fluoride blood samples contained 19 ng/mL amphetamine, 284 ng/mL MDMA, 20 ng/mL MDA, 38 ng/mL benzoylecgonine, 4 ng/mL methylecgonine, 161 ng/mL flubromazepam and traces of 4-MMC (

Increasing global production, trafficking and consumption of drugs of abuse is an emerging threat to people's health and safety. Electrochemical approaches have been proved to be useful for on-site analysis of drugs of abuse. However, less attention has been focused on the analysis of polydrug samples, even though these samples pose severe health concerns, especially when stimulants and depressants are combined, as is the case of speedball, a mixture of cocaine and heroin. In this work, we provide solutions for the selective detection of cocaine (stimulant) in polydrug samples adulterated with heroin and codeine (depressants). The presence of either one of these compounds in cocaine street samples leads to an overlap with the cocaine signal in square-wave voltammetry measurements at unmodified carbon screen-printed electrodes, leading to inconclusive screening results in the field. The provided solutions to this problem consist of two parallel approaches: (i) cathodic pretreatment of the carbon screen-printed electrode surface prior to measurement under both alkaline and neutral conditions and (ii) electropolymerization of orthophenylenediamine on graphene modified carbon screen-printed electrodes prior to measurement under neutral conditions. Both strategies allow simultaneous detection of cocaine and heroin in speedball samples as well as simultaneous detection of cocaine and codeine. Implementing these strategies in portable devices holds great potential for significantly improved accuracy of on-site cocaine screening in polydrug samples.

Electrochemical techniques have evidenced to be highly suitable for the development of portable, rapid and accurate screening methods for the detection of illicit drugs in seized samples. However, the redox inactivity of primary amines, one of the most common functional groups of illicit drugs, masks voltammetric detection in aqueous environment at carbon electrodes and, therefore, leads to false negative results if only these primary amines are present in the structures. This work explores the feasibility of a derivatisation approach that introduces formaldehyde in the measuring conditions in order to achieve methylation, via an Eschweiler-Clarke mechanism, of illicit drugs containing primary and secondary amines, using amphetamine (AMP) and methamphetamine (MET) as model molecules. As a result the electrochemical fingerprint is enriched and thereby the detectability enhanced. A combination of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOFMS) and square-wave voltammetric (SWV) measurements is employed to identify reaction products and link them to the observed redox peaks. Although an alkaline environment (pH 12.0) proved to increase the reaction yield, a richer electrochemical fingerprint (EF) is obtained in neutral conditions (pH 7.0). Similarly, the addition of formate improved the reaction conversion but reduced the EF by eliminating a redox peak that is attributed to side products formed in the absence of formate. To illustrate the applicability, the derivatisation strategy is applied to several prominent illicit drugs containing primary and secondary amines to demonstrate its EF enriching capabilities. Finally, real street samples from forensic seizures are analysed. Overall, this strategy unlocks the detectability of the hitherto undetectable AMP and other drugs only containing primary amines, while strongly facilitating the identification of MET and analogues. These findings are not limited to illicit drugs, the insights can ultimately be applied to other target molecules containing similar functional groups. (C) 2020 Published by Elsevier Ltd.

Using the Belgian Drugs and Driving procedure, 36% of the cocaine-positive oral fluid (OF) screening results were not confirmed in plasma. This study investigates the impact of the choice of screening devices and confirmation matrix on the detection of cocaine use. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method quantifying cocaine, benzoylecgonine (BZE), and other basic drugs in OF was developed and validated. This method monitored OF samples obtained either from a roadside (n = 12) or a double-blind controlled study with cocaine users (n = 10) who were given either a capsule containing 300 mg of cocaine-HCl or a placebo. The OF data were compared to plasma concentrations to obtain concentration-time profiles. In addition, the sensitivity and accuracy of the Drugwipe5S® was assessed. A significant difference between the OF volume collected at baseline/placebo (median 0.93 mL [range 0.43-1.92 mL]) or after cocaine-HCL intake (0.79 mL [0.30-1.21 mL]) was observed. The median OF/Plasma at the 3 collection time points were 10.7, 13.8, 6.7 for cocaine and 0.8, 1.7, 0.8 for BZE, respectively. The Drugwipe5S® detected cocaine use until at least 4 hours after intake. When applying the Belgian legal confirmation decision limit of 10 ng/mL in OF, an accuracy of 75%-98% was observed, depending on the study setting. Cocaine concentrations in OF were much higher and were detected longer as compared to plasma, when applying the same decision limit. From a toxicological viewpoint, the longer detection window with the higher sensitivity of Cocaine and BZE is beneficial to detect drivers in the crash/fatigue phase.

Summary New psychoactive substances (NPS) are a challenge for the worldwide forensic community. More than 450 NPS are currently being monitored by the UNODC and EMCDDA. NPS are a major challenge for laboratories, not only because of the large numbers of NPS, but also due to the continuous chemical development of the substances and thus the rapid turnover of the drug market. The aims of our publication are to give an overview of practical considerations when applying oral fluid (OF) analysis in the context of NPS detection, to highlight the current limitations and to address options to improve current research and legislative actions. OF sample collection and protocols will be discussed as differences will have an impact on the final obtained concentrations via dilution, drug (in)stability and recovery issues. In addition, the current state and the future possibilities for NPS detection in OF using direct assays or chromatographic techniques are described. Finally, judicial challenges and applications are discussed.

Summary Action against driving under the influence of drugs (DUID) often starts with an on-site immunological screening which is not yet developed for new psychoactive substances (NPS). Our aim was to determine the prevalence of NPS in drivers screened positive for a classical illicit drug. Blood samples (n = 556) were obtained between January–August 2015 in Belgium. The on-site Drugwipe 5S (Securetec) results and the subjects’ signs of recent drug use were available. Classical illicit drugs were confirmed in blood via LC-MS/MS methods. NPS screening was performed using 2 methods: LC-HRMS and LC-MS/MS. Of the 256 samples yet analysed, NPS were detected in 13 samples (5%) including following substances (n): ketamine (5), methoxetamine (2), diphenidine (2), 5-MeO-DALT (1), 4-AcO-DiPT (1), methiopropamine (1), methedrone (1), α-PVP (1), a mix of 5-MAPB/5-EAPB (1), and AB FUBINACA (1). This preliminary study demonstrates a prevalence of 5%. However, this result should be confirmed and reassessed at the end of our study.

Seizures of synthetic opioids have increased since 2012, with a 45 % increase in synthetic opioid related deaths between 2016 and 2017 in US. Recently, concerns have arisen around these substances and their illicit use also in several European countries. Our aim was to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of 16 synthetic opioids in segmented hair, including fentanyl, norfentanyl, acetylfentanyl, U-47700, AH-7921, acrylfentanyl, crotonylfentanyl, butyrylfentanyl, methoxacetylfentanyl, U-49900, valeryfentanyl, 4-fluoro-iso-butyrylfentanyl, ocfentanyl, furanylfentanyl, tetrahydrofuranylfentanyl, and alfetanyl. Sample preparation involved washing the hair in dichloromethane, water and methanol, and extraction in methanol, followed by solid phase extraction clean-up. This method was validated for linearity, limit of quantification (LLOQ), precision and bias, selectivity, stability, matrix effects, extraction efficiency of the clean up procedure, and carryover. LLOQs ranged from 0.15-1pg/mg, and the calibration ranged from the LLOQ up to 500pg/mg. Intra and inter-day precision were evaluated at low and high concentrations, with spiked QCs, during 8 days and the results were satisfactory with RSD ULOQ (500) pg/mg, norfentanyl (n=14) 1-38pg/mg, acetylfentanyl (n=7) 0.6->ULOQ (250) pg/mg, furanylfentanyl (n=5) 2-123pg/mg, tetrahydrofuranylfentanyl (n=1) 0.5-63pg/mg and valerylfentanyl (n=1) 2.1->ULOQ (50) pg/mg, along the hair strands. To our knowledge, this is the first time where concentrations of tetrahydrofuranylfentanyl, and valerylfentanyl in hair are reported. The same samples were also analysed for the determination of other drugs of abuse using our routine method (also in 1cm/segment for head hair when available). The results demonstrated poly-drug use in these fentanyl-analogue users population (mean drugs: n=5): amphetamine and/or methamphetamine (n=10), buprenorphine (n=5), cocaine (n=8), methadone (n=8), 6-MAM (n=17), meperidine (n=1), oxycodone (n=11), tramadol (n=3). Evaluation of the concentrations of these drugs, together with the fentanyl analogues is discussed in the present paper. Two authentic samples from two Belgian post-mortem cases, were also analysed showing fentanyl use and in one case polydrug use. The results demonstrated multi-analyte quantitative methods, including fentanyl analogues, are becoming useful in forensic laboratories involved in hair analysis, and in particular when polydrug use is suspected.

Monitoring of alcohol consumption by living persons takes place in various contexts, amongst which workplace drug testing, driving under the influence of alcohol, driving licence regranting programs, alcohol withdrawal treatment, diagnosis of acute intoxication or fetal alcohol ingestion. The matrices that are mostly used today include blood, breath and urine. The aim of this review is to present alternative sampling strategies that allow monitoring of the alcohol consumption in living subjects. Ethanol itself, indirect (carbohydrate deficient transferrin, CDT%) as well as direct biomarkers (ethyl glucuronide, EtG; ethyl sulphate, EtS; fatty acid ethyl esters, FAEEs and phosphatidylethanol species, PEths) of ethanol consumption will be considered. This review covers dried blood spots (CDT%, EtG/EtS, PEths), dried urine spots (EtG/EtS), sweat and skin surface lipids (ethanol, EtG, FAEEs), oral fluid (ethanol, EtG), exhaled breath (PEths), hair (EtG, FAEEs), nail (EtG), meconium (EtG/EtS, FAEEs), umbilical cord and placenta (EtG/EtS and PEth 16:0/18:1). Main results, issues and considerations specific to each matrix are reported. Details about sample preparation and analytical methods are not within the scope of this review.

Previous studies have postulated that four structural compartments may be differentiated in hair: surface protein domain, water-accessible protein domain, water-inaccessible protein domain, and melanin. Drugs contained in blood, sweat, sebum, and environment would be deposited in the first two domains, with primarily drugs in blood being incorporated in the latter two domains during hair synthesis. Drugs in the first two domains would be removed by washing procedures. Use of enzymatic extraction procedures and evaluation of hair for damage from harsh cosmetic treatments might help to separately identify and quantify the drugs incorporated in the second two domains.a) Development of an UPLC-MS/MS method for the simultaneous quantification of the following 19 antipsychotic drugs and metabolites in hair: amisulpride, aripiprazole, chlorpromazine, clotiapine, clozapine, desmethylclozapine, desmethylolanzapine, haloperidol, norchlorpromazine, 7-OH-quetiapine, 9-OH-risperidone, olanzapine, pimozine, pimpamperone, quetiapine, risperidone, sertindole, sulpride, and tiapride; b) evaluation of measurement of patient adherence to prescribed medication use, c) determination of the influence of biochemical individuality effects on hair drug content, d) evaluation of relative binding of antipsychotic drugs to protein and to melanin hair structures.Approximately 10 mg of intact hair were decontaminated with isopropanol and phosphate buffer, and then enzymatically digested overnight with dithiothreitol. After centrifugation, the supernatant digest (protein fraction) was separated from the remaining melanin hair pellet (melanin fraction). Melanin fraction was washed with water, and the drugs were extracted with dimethyl sulfoxide with ball-mill pulverization. Both fractions were purified with solid-phase cation exchange cartridges and injected in the UHPLC-MS/MS system.Validation of the method was carried out on the protein fraction following international guidelines. The limits of quantification ranged from 1.6-40 pg/mg. The method was applied to 59 head hair samples from prisoners from an antipsychotic compliance study in the criminal justice system in US. The patients were under chlorpromazine, haloperidol, risperidone, olanzapine, or quetiapine multiple antipsychotic treatment, during incarceration. The first head hair centimeter, closest to the scalp, was analyzed. The results were evaluated in relation to the type of hair, colour, hair damage, drug melanin affinity, and prescribed dose. In general, no good correlation between the prescribed dose/concentration in hair was obtained. A wide range of antipsychotic concentrations were observed 'dose mg/day (d); pg/mg protein fraction (A)': chlorpromazine (d:50-400;A:LOQ-1600) and its metabolite norchlorpromazine (A:LOQ-1600), haloperidol (d:4-20;A:LOQ-2902), olanzapine (d:5-20;A:LOQ-223) and its metabolite desmethylolanzapine (A:LOQ -136), quetiapine (d:4-400;A:LOQ -2754) and its metabolite 7-OH-quetiapine (A:LOQ -1448), risperidone (d:2-20;A:LOQ-1600) and its metabolite 9-OH-quetiapine (A:LOQ-296;B). Melanin was found to have a 2- to 85-fold higher drug-binding affinity relative to the protein fraction CONCLUSION: This report describes a different viewpoint method of quantifying the antipsychotics in the melanin and water-inaccessible protein fraction of hair, separately. No correlation between the prescribed dose and the concentrations found in the protein and melanin domains were observed. The results show that hair melanin would have a much higher affinity for the antipsychotic medications than do hair proteins, being influenced mainly by biochemical individuality effects and less by hair colour. Future studies would be of interest using the proposed extraction method applied to hair of different colours.

Since gamma-hydroxybutyric acid (GHB) is present in hair of the general population under physiological concentrations, special attention has to be given to the hair analysis of GHB and its interpretation. Normal levels of endogenous GHB can vary in each individual. As a result, strands of hair from a subject have to be cut in small segments (0.3–0.5 cm long) with analysis of each segment. As such, each subject can be used as its own control with a continuous endogenous signal. If one segment has a GHB concentration 10 times higher than the others, this suggests possible administration of exogenous GHB according to the UNODC guideline for Drug Facilitated Assault Cases. Aim As cosmetic treatments were found to decrease drug concentrations in hair, the aim of the study was to develop an UPLC®–MS/MS method for the analysis of GHB in hair. An in vitro study was then carried out in order to evaluate the impact of a hair straightener or a bleaching treatment on endogenous GHB concentrations. Method Hair samples (10 mg) were washed with dichloromethane and water. After drying overnight in an oven at 35 °C the samples were pulverized in disposable plastic tubes. Methanol/acetonitrile/ammonium formate buffer 1 mM (25:25:50, v/v/v) was used to extract the drug from the hair matrix in a water bath for 1.5 h at 37 °C. Thereafter, the samples were filtered and evaporated to dryness. The dried extracts were then reconstituted in mobile phase and injected in a UPLC®–MS/MS (Waters, Winslow, UK) with a BEH C18 column. Results The method was validated using untreated hair samples from three healthy volunteers. The calibration curve ranged from 0.06 to 25 ng/mg and the repeatability and intra-batch precision was lower than 20% evaluated in 8 different batches. Processed samples were stable for 3 days in the auto-sampler. To demonstrate the method applicability, 54 hair samples from healthy volunteers were analysed for endogenous GHB resulting in a concentration range from 0.2 to 6 ng/mg. Three different hair treatments experiments were carried out, in which a hair straightener and/or a bleaching treatment were applied. These experiments demonstrated that hair treatments decreased up to 80% of the GHB endogenous concentrations. Conclusion This in vitro study showed that hair bleaching or a heat source treatment influences GHB concentrations in hair. For a correct interpretation of GHB results in hair, cosmetic treatments should be considered, certainly in cases where only a part of the hair is treated.

Phosphatidylethanol species (PEths) are promising biomarkers of alcohol consumption. Here, we report on the set-up, validation, and application of a novel UHPLC-ESI-MS/MS method for the quantification of PEth 16:0/18:1, PEth 18:1/18:1, and PEth 16:0/16:0 in whole blood (30 mu L) and in venous (V, 30 mu L) or capillary (C, 3 punches (3 mm)) dried blood spots (DBS). The methods were linear from 10 (LLOQ) to 2000 ng/mL for PEth 16:0/18:1, from 10 (LLOQ) to 1940 ng/mL for PEth 18:1/18:1, and from 19 (LLOQ) to 3872 ng/mL for PEth 16:0/16:0. Extraction efficiencies were higher than 55 % (RSD < 18 %) and matrix effects compensated for by IS were between 77 and 125 % (RSD < 10 %). Accuracy, repeatability, and intermediate precision fulfilled acceptance criteria (bias and RSD below 13 %). Validity of the procedure for determination of PEth 16:0/18:1 in blood was demonstrated by the successful participation in a proficiency test. The quantification of PEths in C-DBS was not significantly influenced by the hematocrit, punch localization, or spot volume. The stability of PEths in V-DBS stored at room temperature was demonstrated up to 6 months. The method was applied to authentic samples (whole blood, V-DBS, and C-DBS) from 50 inpatients in alcohol withdrawal and 50 control volunteers. Applying a cut-off value to detect inpatients at 221 ng/mL for PEth 16:0/18:1 provided no false positive results and a good sensitivity (86 %). Comparison of quantitative results (Bland-Altman plot, Passing-Bablok regression, and Wilcoxon signed rank test) revealed that V-DBS and C-DBS were valid alternatives to venous blood for the detection of alcohol consumption.

Driving under the influence of drugs (DUID) is a worldwide problem. Several countries have adopted DUID legislations which prove their deterrent effect and impact on road safety. However, the use of new psychoactive substances (NPS) and prescription drugs is not known, as the applied roadside screening tests have not yet been adapted for these compounds. In this study, 558 blood samples obtained during roadside controls in Belgium (January to August 2015) after a positive Drugwipe 5S® test and 199 oral fluid (OF) samples obtained from negatively screened test pads were analyzed. The NPS positivity rate was 7% in blood, while it reached 11% in OF. NPS detected were: diphenidine, ketamine, 4-fluoroamphetamine, 2-amino-indane, methoxetamine, α-PVP, methiopropamine, a mix of 5-MAPB/5-EAPB, TH-PVP, mephedrone, methedrone, 4-methylethylcathinone, 5-MeO-DALT, 4-Acetoxy-DiPT, AB Fubinaca, FUB-JWH018, JWH020, trifluoromethylphenylpiperazine, and ethylphenidate. Moreover, 17% of blood samples (and 5% of OF) contained an analgesic drug, 10% (0.5%) a benzodiazepine/hypnotic, 5% (2%) an antidepressant, 2% (3%) an antipsychotic, 2% an antiepileptic drug, and 1% methylphenidate. The presence of NPS in the young (and predominately male) DUID population is proven. Furthermore, a high level of poly-drug use including combinations of NPS, licit, and drugs of abuse was observed. Further research concerning the development of on-site NPS detection techniques should be established. Meanwhile, the effects of combined drug use on driving ability and the physical/psychological signs after NPS use should be performed to improve the on-site DUID detection of NPS by police officers, so they can engage in blood sampling for a general unknown screening.

Forensic testing for drugs of abuse in hair has become a useful diagnostic tool in determining chronic drug use as well as examining long-term drug history thorough segmental analysis. However, sensitive and specific analytical methods are needed. A simple, rapid and highly sensitive and specific method for the extraction and quantification of 33 opioids, opiates, cocaine, and amphetamines is presented. The method was fully validated according to international guidelines. Twenty milligrams of hair sample was pulverized and then incubated in the same disposable tube with methanol (under sonication at 45°C) during 4h. After centrifugation the supernatant was evaporated up to about 100 μL and a solid phase extraction (SPE) followed by separation and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UHLC-MS/MS) were carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. Good intra-assay and inter-assay precision (relative standard deviations (RSDs) were observed (15%) for most of the compounds. The lower limit of quantification was fixed at the lowest calibrator in the linearity experiments and it ranged from 0.006 to 0.063 ng/mg. No instability was observed in processed samples. Extraction efficiency varied from 37 to 107% (except for EDDP with a recovery of 5%) and matrix effects ranged from 52 to 160%, and for most of the compounds it was compensated by the internal standard (IS). The method was subsequently applied to authentic hair samples obtained from forensic and toxicology cases and to proficiency test (obtaining z-scores lower than 1 for most of the compounds). The validation and actual sample analysis results show that this method is rugged, precise, accurate, and well-suited for routine hair analysis.

Oral fluid (OF) is potentially useful to detect driving under the influence of drugs because of its ease of sampling. While cannabis is the most prevalent drug in Europe, sensitivity issues for Δ(9) -tetrahydrocannabinol (THC) screening and problems during OF collection are observed. The ability of a recently improved OF screening device - the DrugWipe5S(®) , to detect recent THC use in chronic cannabis smokers, was studied. Ten subjects participated in a double-blind placebo-controlled study. The subjects smoked two subsequent doses of THC; 300 µg/kg and 150 µg/kg with a pause of 75 min using a Volcano vapourizer. DrugWipe5S(®) screening and OF collection using the Quantisal™ device were performed at baseline, 5 min after each administration and 80 min after the last inhalation. Blood samples were drawn simultaneously. The screening devices (n = 80) were evaluated visually after 8 min, while the corresponding OF and serum samples were analyzed respectively with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) or gas chromatography-mass spectrometry (GC-MS). Neat OF THC concentrations ranged from 12 361 ng/g 5 min after smoking down to 34 ng/g 80 min later. Under placebo conditions, a median THC concentration of 8 ng/g OF (0-746 ng/g) and

The aim of this review is to provide a comprehensive overview of screening and confirmation methods reported to determine the low-molecular weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polar nature, the endogenous presence, rapid metabolization following ingestion and stability issues during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone), impose challenges for the analyst and for the interpretation of a positive result. First, possible screening procedures for GHB are discussed, including colorimetric, enzymatic and chromatography-based procedures. Confirmation methods for clinical and forensic cases mostly involve gas chromatography (coupled to mass spectrometry), although also liquid chromatography and capillary zone electrophoresis have been applied. Prior to injection, sample preparation techniques include (a combination of) liquid-liquid, solid-phase or headspace extraction, as well as chemical modification of the polar compound. Also simple dilute-and-shoot may be sufficient in the case of urine or serum samples. Advantages, limitations and trends are discussed.

Background When head hair is not suitable or not available, body hair, such as leg or beard hair might be the most suitable sample for drug hair analysis. Drugs may be detected in beard, from the first day up to more than one week. Specific information about the time course detection of drugs, after a single intake, in body hair has not been yet well documented for the different body hair samples. Aim The aim of this study was to determine and compare the detection window of dihydrocodeine in frequently shaved legs and beard after a single intake. Unshaved hair from head hair, chest hair, leg hair, and/or arm hair were also analysed. Method Before a single intake of 12 mg dihydrocodeine by test subject 1 (female), the leg hair was shaved in the morning. The second test subject 2 (male) shaved his beard in the morning and 30 min later he had a dose of 10 mg of dihydrocodeine. The samples were washed with water and shampoo, dried and collected as follows: subject 1: every 3-days shaved leg hair (n = 10) and 1-month-later head hair (n = 1). Subject 2: daily shaved beard hair (n = 15), 2 months later head hair (n = 50, different regions), and every 20 days unshaved arm, leg and chest hair (from different regions) (n = 4/area). The samples were analysed for dihydrocodeine using a validated liquid chromatography tandem mass spectrometry method with a limit of quantification (LOQ) of 16 pg/mg for dihydrocodeine. About 20 mg of hair samples were weighted, washed with dichloromethane, centrifuged, dried, and pulverized in the same disposable tubes. Then the samples were incubated with methanol (under sonication at 45 °C) during 4 h. After centrifugation, the supernatant was evaporated and a cation exchange solid phase extraction followed by separation and quantification using ultra performance liquid chromatography tandem mass spectrometry (UHLC-MS/MS) was carried out. Chromatographic separation was achieved using a BEH phenyl column eluted with 0.1% formic acid: methanol (0.1% formic acid). The UPLC-MS/MS method was validated and used in routine for drug hair analysis for already several years [1] . Results and discussion In the present study leg hair was collected every 3 days, as an average of frequent shaved hair in western woman population. Leg hair was very limited and only one hair sample from both woman legs was available per analysis. Beard was collected daily and in a higher amount. Dihydrocodeine was detected in leg hair from the first sample (3 days after the intake). Maximum concentration at 68 pg/mg for the single intake was obtained after 15 days (± 2 days), decreasing later to the LOQ from the 21st day. Beard hair was already positive from the first day sample, and the maximum concentration was observed at 66 pg/mg, 6 days after the intake, decreasing later to the LOQ from day 10. This may be explained by the ‘Richards–Meharg body and head hair growth table, where it indicates that legs have only 20% of growing hairs in anagen phase, while beard goes from 50 to 70%. In other body hair samples, dihydrocodeine was negative or detected from 1 month after the intake. Conclusion Body hair presents different time course window detection due to the different growth rates. Frequently shaved leg and beard hair may be suitable samples for recent single dihydrocodeine dose detection from the first days up to 21 or 13 days after the intake, respectively, when an LOQ of 16 pg/mg is applied.

Drug facilitated crimes (DFCs) can be defined as criminal acts carried out by means of administering a substance to a person with the intention of impairing behaviour, perceptions, or decision-making capacity. It also extends to taking advantage of an impaired person after voluntary intake of an incapacitating substance. Crimes include robbery, money extortion, and maltreatment of the elderly, children, or mentally ill patients. Rape or other types of sexual assault are referred to as drug facilitated sexual assault (DFSA). In DFSA cases, the victim is subjected to a sexual act without legal consent as a result of the pharmacological effects of alcohol and/or drug(s). A large number of psychoactive substances have the potential to alter the victim’s state of mind, alcohol being the number one candidate because of its widespread use. Illicit drugs, psychoactive prescription drugs and even over-the-counter medicines are also likely candidates, either consumed alone or in combination with alcohol. The resulting pharmacological effects may include relaxation, euphoria, and lack of inhibition on the one hand and drowsiness, loss of motor function, unconsciousness, and amnesia on the other hand. Information on the frequency of DFC cases is scarce but there has been a significant increase in reports worldwide. However, several factors complicate the registration of the actual number of DFSA cases. Governmental statistics show a general underreporting of sexual assault crimes. The impact that central nervous system depressant drugs have on memory and consciousness might lead to a situation where the victim is not able to remember what has actually happened and chooses not to report the incident. If the incident is reported, police officers often assume that the victim was simply drunk rather than drugged. If an investigation is initiated, the delay between the collection of biological evidence and the alleged assault can be between several hours and more than a day. This case report describes a DFSA case in a medical setting: a gynaecologist was suspected of assaulting a young patient after drug administration. In the presented case, the time delay between the alleged assault and the sampling was 30 h. Evidence collection via a standardized sampling kit and the choice of sample in such cases is discussed. The analytical strategy is described with regard to the choice of methods, uncertainty measurement, and limitations of the methods which are important during interpretation of the analytical results. Finally, interpretation of this specific case, but also interpretation issues of DFSA cases in general, is discussed.

A sensitive and selective ultra performance liquid chromatographic-tandem mass spectrometric method was developed and fully validated for the simultaneous determination of (in order of chromatographic elution) methylecgonine, pholcodine, morphine, hydromorphone, oxymorphone, norcodeine, codeine, dihydrocodeine, oxycodone, 6-Monoacetylmorphine (6-MAM), hydrocodone, ethylmorphine, norfentanyl, benzoylecgonine, tramadol, normeperidine, meperidine, cocaine, pentazocine, cocaethylene, fentanyl, norbuprenorphine, 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, propoxyphene, and methadone in blood. The matrixes analyzed during the validation experiments were as follows: citrated blank plasma for calibrators, fluoride blank plasma for internal quality control (QC), lyophilized serum for external QC, fluoride plasma and whole blood for authentic samples, and lyophilized serum and whole blood for proficiency testing schemes. Samples were extracted with cation exchange solid-phase extraction cartridges. The target drugs were separated and quantified in a chromatographic run of 8.1 minutes using 0.1% formic acid in water and methanol (with 0.1% formic acid) as mobile phase. The limit of quantification ranged from 0.5 to 2.5 ng/mL depending on the compound and the therapeutic concentration. The intra- and interassay precision was less than 15% for all the compounds (except for pentazocine and EDDP, which was20%) determined with 2 internal and 2 external QC samples, and the bias was within ±15% (except for methylecgonine, which was20%). Extraction efficiency was greater than 70% for all the compounds except for EDDP. Matrix effects were evaluated with authentic blood samples (n = 10), and they ranged from 47 to 95%, but they were compensated for most analytes using deuterated analogs as internal standards. Prepared samples were stable for 62 hours in the autosampler. This method was successfully applied to authentic samples (n = 120), involving the use of heroin, cocaine, tramadol, and methadone, and to proficiency testing schemes.

Hair analysis is a complementary approach for the detection of antidepressants (ADs) in clinical and forensic schemes because it yields a picture of long-term exposure over a time window depending on the length of the hair.A fast and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method using a BEH C18 column with a mobile phase consisting of ammonium acetate/acetonitrile was developed and validated according to international guidelines for the simultaneous analysis of 24 ADs in hair. Methanol/acetonitrile/ammonium formate buffer 1 mmol/L (25:25:50, vol/vol/vol) was used to extract the drugs from the hair matrix before a solid-phase extraction using cation exchange cartridges was applied. Hair samples (n = 18) obtained from a US workplace drug testing center were analyzed to demonstrate the method applicability.The limit of quantification values ranged from 0.006 to 0.05 ng/mg hair, and the calibration curves ranged from the LOQ up to 10 ng/mg hair. The bias and imprecision were15% for all the compounds except maprotiline (17%). This was evaluated with 2 "in-house" QCs and 1 authentic hair sample from an amitriptyline user. No significant matrix effects for most of the compounds were observed, and the extraction efficiency of the sample cleanup procedure ranged from 40% to 80% (relative standard deviation15%) [except for demethylcitalopram, didemethylcitalopram, and trazodone (relative standard deviation33%)]. The method was then successfully applied to the analysis of hair samples from workplace drug testing. The samples were analyzed in 1-cm segments to determine the medication history of the patient. When a sample was reported positive, information concerning the prescription was obtained anonymously for several samples. Concentrations of (minimum-maximum value in ng/mg) citalopram (0.01-132: extrapolated), trazodone (0.01-5.3), sertraline (0.05-0.1), paroxetine (0.02-1.0), bupropion (0.05-0.6), fluoxetine (0.5-8), and amitriptyline (0.2-4.8), including metabolites, are reported.This study may be of interest to clinical and forensic laboratories for interpretation because it demonstrates the AD concentration windows in hair and the link to the prescribed drugs.

Traditionally, fast screening for the presence of cocaine in unknown powders is performed by means of colour tests. The major drawbacks of these tests are subjective colour evaluation depending on the operator ('50 shades of blue') and a lack of selectivity. An alternative fast screening technique is Fourier Transform InfraRed (FTIR) spectrometry. This technique provides spectra that are difficult to interpret without specialized expertise and shows a lack of sensitivity for the detection of cocaine in mixtures. To overcome these limitations, a portable FTIR spectrometer using Attenuated Total Reflectance (ATR) sampling was combined with a multivariate technique, called Support Vector Machines (SVM). Representative street drug powders (n = 482), seized during the period January 2013 to July 2015, and reference powders (n = 33) were used to build and validate a classification model (n = 515) and a quantification model (n = 378). Both models were compared with the conventional chromatographic techniques. The SVM classification model showed a high sensitivity, specificity, and efficiency (99%). The SVM quantification model determined cocaine content with a root mean squared error of prediction (RMSEP) of 6% calculated over a wide working range from 4 to 99 w%. In conclusion, the developed models resulted in a clear output (cocaine detected or cocaine not detected) and a reliable estimation of the cocaine content in a wide variety of mixtures. The ATR-FTIR technique combined with SVM is a straightforward, user-friendly, and fast approach for routine classification and quantification of cocaine in seized powders. Copyright © 2016 John WileySons, Ltd.

Background In Belgium, the analysis of indirect biomarkers such as carbohydrate deficient transferrin (CDT%), gamma-glutamyltransferase (GGT), aspartate aminotransferase/alanine aminotransferase (AST/ALT) and mean corpuscular volume (MCV), is currently used to monitor the alcohol consumption in cases of fitness to drive assessment. We evaluated the use of direct ethanol markers for this purpose, exclusively determined in matrices obtained via non- or minimally invasive sampling. Methods Three validated quantitative methods (ethylglucuronide (EtG) in hair and urine, ethylsulfate (EtS) in urine, and phosphatidylethanol species (PEth 16:0/18:1, PEth 18:1/18:1 and PEth 16:0/16:0) in capillary dried blood spots (C-DBS)) were used. Fifty volunteers, for whom fitness to drive had to be assessed and for whom a blood analysis for indirect biomarkers was requested, were included in the study. The sampling and analysis of hair, urine and C-DBS were added to the process currently used. Results Hair EtG (24/50) and C-DBS PEths (29/50) are more sensitive than the currently used indirect biomarkers (13/50 for CDT%) to detect excessive and chronic alcohol consumption and allow to disprove an abstinence period. Urinary EtG and EtS are useful parameters to determine recent alcohol consumption. Conclusion The combined use of the three strategies allows better inference about the evolution of the alcohol consumption prior to the sampling. Moreover, the exclusive use of non- or minimally invasive sampling (hair, urine and C-DBS) allows this to be performed directly during the fitness to drive assessment by regular staff members. This approach offers the potential to improve the Belgian driver’s licence regranting process.

A fast and selective ultraperformance liquid chromatographic-tandem mass spectrometric method was developed and validated for the simultaneous quantification of amitriptyline, citalopram, clomipramine, desipramine, desmethylcitalopram, desmethylclomipramine, desmethyldosulepin, desmethyldoxepin, desmethylfluoxetine, desmethylvenlafaxine, didesmethylcitalopram, dosulepin, doxepin, duloxetine, fluoxetine, fluvoxamine, imipramine, maprotiline, mianserin, mirtazapine, moclobemide, nortriptyline, paroxetine, reboxetine, sertraline, trazodone, and venlafaxine in 100 μL of plasma. After liquid-liquid extraction with 1-chlorobutane, analytes were separated on a BEH (Ethylene Bridged Hybrid) C18 analytical column with gradient elution. The compounds were ionized and detected over 7-minute analysis time by electrospray ionization tandem mass spectrometry with multiple reaction monitoring. Limits of quantification and limits of detection ranged from 2.5 to 10 ng/mL and 0.2 to 10 ng/mL, respectively. Intra- and interassay imprecision were lower than 15% for all the compounds except for mirtazapine, moclobemide, and desmethylclomipramine [relative standard deviation (RSD) < 20%], and the bias of the assay was lower than 15% for all the compounds except for fluvoxamine (bias < 20.5%), evaluated with 5 commercial quality control and 3 "in-house" quality control. The extraction was found to be reproducible (RSD < 16%) (except for duloxetine RSD 21.9%) and with recoveries varying from 59% to 86%. Furthermore, the stability studies demonstrated that the processed samples were stable in the autosampler for 24 hours for all the compounds. The method was successfully applied to the analysis of authentic samples from forensic toxicology cases and external quality control assays from the Society of Toxicology and Forensic Chemistry (GTFCh). The method was completely validated and can be of interest to clinical and forensic laboratories.

A fast and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of amphetamines (amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, ephedrine, and p-methoxyamphetamine) in plasma has been developed and validated. Sample preparation was performed by liquid-liquid extraction using ethyl acetate. For optimized chromatographic performance with repeatable retention times, narrow and symmetrical peaks, and focusing all analytes at the column inlet, a gradient start, with acid mobile phase consisting of 0.1% formic acid and methanol was chosen. Positive electrospray ionization MS-MS detection was performed with two multiple reaction monitoring transitions for each analyte. Deuteriumlabeled internal standards were used for five of the analytes. The limit of detection was in the range 0.25-1.25 ng/mL, and the limit of quantification was fixed at the lowest calibrator of 2.5 ng/mL for all of the compounds. The RSD values of the intra- and interassay precision and accuracy were lower than 11% at four concentration levels, including two external quality controls. No or only minor matrix effects were observed, and the extraction method presented recoveries higher than 93% for all the compounds. Total run time, including equilibration, was 12 min. The method is routinely used at the National Institute of Criminalistics and Criminology for quantitative determination of the main amphetamines in plasma from forensic and driving under the influence cases.

This paper describes a sensitive UPLC-MS/MS method for quantification of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in single empty puparial case of Lucilia sericata. Larvae were reared on substrates spiked with different concentrations of methadone (0-4 μg/g). Methadone was quantified in puparia reared on high concentrated substrates (0.8-4 μg/g). The major metabolite of methadone (EDDP) was not detected, confirming rapid elimination of metabolites by the larvae before pupation. The effects of methadone on the development of L. sericata were also investigated. No effect on sex ratio was detected. A significant difference was calculated for emerged adults but no trends could be observed. Concerning the developmental curve, a significant difference was observed between control and high methadone concentrations using the Kolmogorov-Smirnov test.

Forensic entomotoxicology studies the usefulness of insects as alternative toxicological samples. Use of insects as alternative matrix for drug detection is well documented and recommended when conventional matrices such as blood, urine or internal organs are no longer available. However, several limitations of entomotoxicology have been highlighted, especially concerning interpretation of the drug concentrations in insects on human forensic cases. In addition, the lack of knowledge in pharmacokinetic of drugs in insects, large variability of experimental set-up and toxicological analysis compromise the utility of this science. This review focuses on the current knowledge of factors influencing drug detection in insects. Reasons for the current limitations, but also recommendations for future research are discussed and proposed in this paper.

Reporting reliable analytical data is the backbone of forensic and clinical bioanalytical research and applications. Therefore, international agreement concerning validation and quality control requirements is needed. Several international guidelines provide a standard for fundamental validation parameters such as selectivity, matrix effects, method limits, calibration, accuracy, precision and stability. However, it is not always easy for the analyst to ‘translate’ these guidelines into practice. International guidelines remain nonbinding protocols that need to be updated according to the type of application and the analyst’s method requirements and depends on the evolution of analytical techniques. In this publication, suggestions for experimental set-up, statistical approaches and straightforward acceptance criteria for validation of forensic bioanalytical applications are suggested. Furthermore, permanent quality control, to ensure state-of-the-art quantitative analytical performances, as well as measurement uncertainty influencing interpretation is discussed.

Objectif En Belgique comme en France, la procedure reprimant la conduite automobile sous influence de produits stupefiants (Drugs Of Abuse, DOA) consiste en un depistage salivaire suivi d’une confirmation sanguine (ou salivaire en France) par chromatographie couplee a la spectrometrie de masse. Ce travail presente une evaluation de la prevalence de l’usage de nouveaux produits de synthese (NPS) dans les fluides oraux de populations de conducteurs controles en 2016 et 2017, reposant sur l’analyse de DSS (Dried Saliva Spots) recuperes dans les dispositifs usages des tests immunochimiques salivaires (DrugWipe©-5S tests, DW, Securetec) utilises pour le depistage des DOA. Methode Une recherche de produits stupefiants, de principes actifs de medicaments et d’autres toxiques incluant les NPS a ete realisee dans les DSS : (i) screening non cible par LC-HRMS (Xevo G2®-XS QToF, Waters), (ii) screenings « stupefiants », « medicaments » et « NPS » par LC-MS/MS en MRM (Xevo® T-QS, Waters) [1] , [2] . Resultats Les principaux resultats des analyses des echantillons salivaires collectes en 2016 et 2017 (6 premier mois de l’annee) en France (region de Lille) et en Belgique (regions de Mons et de Malines) sont presentes dans le Tableau 1 . Dans ces DSS, des produits stupefiants (y compris dans les echantillons depistes negatifs) et des medicaments ont ete egalement identifies. Ces pourcentages d’usage de NPS sont comparables aux donnees sanguines disponibles dans des populations similaires [3] . Conclusion Ces resultats (i) confirment l’interet de l’analyse des DSS des tests de depistage salivaire, et (ii), meme si ces resultats ne concernent que des conducteurs qui ont ete soumis a un controle en France et en Belgique, temoignent de la realite d’une conduite automobile apres avoir fait usage de NPS, dans ces deux pays.

Objectives About 620 new psychoactive drugs (NPS) have appeared on the worldwide drug market the last decade. Several European countries have recently published a generic legislation, making a broad range of NPS illegal. The major challenge is the continuous chemical development of NPS and the rapid turnover of the drug market, making it difficult to obtain up-to-date monitoring techniques. The aim of this presentation is to discuss the challenges of the NPS detection workflows for toxicological analysis of biological matrices. Methods A literature overview of the current methodology for NPS detection will be achieved via a Pubmed search. In addition, the experience of the researchers’ group concerning the detection of NPS will be discussed using either (a) high-resolution mass spectrometry via the (LC-HRMS) Xevo-G2-QTOF-XS (Waters, Manchester, UK) with UNIFI software (Wille et al., https://doi.org/10.1002/dta.2232 ) or (b) a newly developed cell-based G-protein coupled receptor activation assay using the NanoLuc® Binary technology (Promega), detecting in vitro cannabinoid (CB1 and 2) or μ-opioid receptor (MOR) activation, via the recruitment of β-arrestin 2 and resulting in a luminescent signal (Cannaert et al., https://doi.org/10.1373/clinchem.2017.285361 ). Results Amongst the major challenges for NPS detection continuous adaptation of the methodology to ensure up-to-date screening of correct target compounds and their metabolites with adequate sensitivity, limited research concerning stability, the lack of reference materials or their access, and the development of up-to-date libraries. Immunoassays focusing on classical drugs have insufficient NPS cross-reactivity, while immunoassays focusing on specific NPS can be quickly outdated as they target specific chemical structures. The bioassay developed by Cannaert et al. detects cannabinoid or opioid receptor activity. The detection of synthetic cannabinoids resulted in an overall sensitivity of 75% and a specificity of 91% in urine and a sensitivity of 82% and a specificity of 100% in serum. Several semi-synthetic opioids (hydromorphone and desomorphine) and synthetic opioids (fentanyl and derivatives) and other unrelated opioids (U-49900 and U-47700) demonstrated different levels of MOR activation in the opioid bioassay. A sensitivity and specificity of 100% was obtained for authentic blood samples from morphine users and non-users. In the NPS detection workflow of a toxicological laboratory, this technique may be of interest as an initial screening technique. However, chromatographic detection will still be necessary to obtain identification and quantification of NPS. Advanced techniques involving high-resolution mass spectrometry combine the detection using developed standard libraries with possible elucidation of unknown chemical structures via accurate mass. In addition, NPS metabolites can be in vitro produced, identified and subsequently added in mass libraries to improve NPS detection. One of the challenges of accurate mass instruments is the development of an adequate procedure to generate a time-efficient interpretation of the obtained analytical data. A practical and time-efficient workflow for detection of NPS in biological matrices using time-of-flight is described and applied to blood (n = 558) as well as oral fluid (n = 199) samples obtained during roadside testing by Wille et al. An NPS positivity rate of 7% in blood and 11% in oral fluid was observed for the cohort that was examined. Conclusion A feasible and straightforward workflow for the detection of NPS in biological matrices for toxicological screening consisting of first-line screening tools using receptor activity in combination with a practical workflow for accurate mass detection is demonstrated.

Objectif Presenter une evaluation de la prevalence de l’usage de nouveaux produits de synthese (NPS) par l’analyse des fluides oraux de populations de conducteurs soumis a un controle au bord de la route en 2016 et 2017, en France (region de Lille) et en Belgique (regions de Mons et de Malines). Methodes Elle repose sur l’analyse de residus de salive recuperes dans les dispositifs usages des tests immunochimiques salivaires DW (DrugWipe©-5S tests, Securetec) utilises pour le depistage des produits stupefiants, DOA (cannabis, cocaine, opiaces, amphetamines et derives). Une recherche de produits stupefiants, de principes actifs de medicaments et d’autres toxiques incluant les NPS a ete realisee dans les residus de salive des conducteurs, dried saliva spots (DSS) : (i) screening non cible par LC-HRMS (Xevo G2®-XS QToF, Waters), (ii) screenings « stupefiants », « medicaments » et « NPS » par LC-MS/MS en MRM (Xevo® T-QS, Waters) [1] . Resultats Les principaux resultats des analyses des echantillons salivaires collectes en 2016 et 2017 en France et en Belgique sont presentes dans le Tableau 1 . Les NPS deceles sont : A ethylone (1), methylone (1), et MDPV(1) ; B JWH020 (2), HU210 (1), MDMB-Chmica (1), 5F-AKB48 (4), AM-694 (1), FUB-JWH018 (1), NEB (1), 3 ou 4 MMC (10), MDPV (2), 4-MEC (4), 3-FPM (1), 4-FA (1), EPH (2), 5-MeO-DALT (1), mCPP (1), ketamine (4) et MXE (1) ; C 4-methyl-NEP (1), methamnetamine (1), LSD (1) et EPH (1) ; D THJ-2201 (1), N-ethylhexedrone (1), alpha-PVP (4), 4-FMA (1), 3-FMP (1), EPH (2) et isopropylphenidate (2). Dans ces DSS, des produits stupefiants (y compris dans les echantillons depistes negatifs) et des medicaments ont ete egalement deceles. Ces pourcentages d’usage de NPS (3 % en France, 10 % et 3 % en Belgique, en 2016 et 2017) sont a rapprocher des donnees sanguines disponibles en Belgique en 2015 (7 %) [2] et des resultats observes dans la salive de conducteurs aux abords d’un festival de musique en France a l’ete 2017 (7,4 %) [3] . Conclusion Ces resultats (i) confirment l’interet de l’analyse des DSS des tests de depistage salivaire, (ii) temoignent de la realite d’une conduite automobile apres avoir fait usage de NPS en France et en Belgique meme si ces resultats ne concernent que des conducteurs qui ont ete soumis a un controle dans ces deux pays (iii) tout en montrant une evolution des NPS consommes fournissant ainsi des donnees longitudinales dans ces populations de conducteurs.

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid-liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C18 column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (10.7%) at each measured concentration of two external quality controls (QC) and three "in house" QC. No matrix effects were observed and good recoveries (70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.

An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 microL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation11.2% and bias13%) for two external quality control samples (QC) for both matrices and three and two 'in house' QCs for blood and urine, respectively. Responses were linear over the investigated range (r(2)0.99, 2.5-400 microg/L for blood and 25-1000 microg/L for urine). Limits of quantification were determined to be 2.5 and 25 microg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 microg/L for blood and 0.25 to 2.5 microg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 microg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.

Cannabis is considered to be the most widely abused illicit drug in Europe. Consequently, sensitive and specific analytical methods are needed for forensic purposes and for cannabinoid pharmacokinetic and pharmacodynamic studies. A simple, rapid and highly sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy- Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy- Delta(9)-tetrahydrocannabinol (THC-COOH) in blood is presented. The method was fully validated according to international guidelines and comprises simultaneous liquid-liquid extraction (LLE) of the three analytes with hexane:ethyl acetate (90:10, v/v) into a single eluant followed by separation and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved using a XBridge C(18) column eluted isocratically with methanol:0.1% formic acid (80:20, v/v). Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions. The use of the LLE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Validation of the method was performed using 250 microL of blood. The method was linear over the range investigated (0.5-40 microg/L for THC, 1-40 microg/L for 11-OH-THC, and 2-160 microg/L for THC-COOH) with excellent intra-assay and inter-assay precision; relative standard deviations (RSDs) were

A validated method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and several metabolites is presented. The procedure comprises a sample clean-up step, using mixed-mode solid-phase extraction followed by liquid chromatography (LC)-tandem mass spectrometry analysis. Chromatographic separation was achieved using a Sunfire C(8) column eluted with a mixture of formate buffer, methanol, and acetonitrile. The applied LC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated analogues. Validation of the method was performed using 500 microL of urine. The limits of quantification (LOQ) for LSD and 2-oxo-3-hydroxy-LSD were 0.05 and 1 ng/mL, respectively, and ranged, for the other hallucinogens, from 0.5 to 10 ng/mL. Linear and quadratic regression was observed from the LOQ of each compound to 12.5 ng/mL for LSD, 50 ng/mL for 2-oxo-3-hydroxy-LSD and 500 ng/mL for the others (r(2)0.99). Precision for the QC samples, spiked at a minimum of two concentrations, was calculated [%CV and %bias20% for most of the compounds, except for bufotenine and cathinone (%bias24%), and ibogaine (%bias30%)]. Extraction was found to be both reproducible and efficient with recoveries87% for all the analytes. Furthermore, the processed samples were demonstrated to be stable in the autosampler for at least 24 h. Finally, the validated method was applied to the determination of chlorpheniramine, ketamine, LSD, and psilocin in authentic urine samples.

Sedative agents are used to facilitate sexual assault due to their ability to render the victim passive, submissive and unable to resist. The primary pharmacological effect of the benzodiazepine tetrazepam is muscle relaxation, whereas the benzodiazepine diazepam acts on the central nervous system (CNS) exerting mainly sedation effects. Therefore, contrary to tetrazepam, diazepam is an often-abused drug, which can potentially be used as a date-rape drug. In this study, we describe the detection of low amounts of diazepam in Myolastan® (Sanofi–Synthelabo S.A., Brussels, Belgium) and Epsipam® (Will-Pharma, Wavre, Belgium) 50mg tablet preparations by LC-MS-MS, GC-FID and HPLC-DAD. Considering the important forensic implication of this finding, a study was conducted with volunteers receiving a single or repeated dosage of Myolastan®. Urine, hair and preserved oral fluid samples were analysed using a previously described sensitive and specific LC-MS-MS detection method allowing for the simultaneous quantification of tetrazepam, diazepam, nordiazepam, oxazepam and temazepam. This study demonstrates that diazepam can be observed in urine samples even after a single dose of Myolastan®. In addition, maintaining therapy for 1 week results in the detection of both diazepam and nordiazepam in urine samples and of diazepam in the first hair segment. Importantly, comparing urine and hair samples after a single intake of diazepam versus the single and 1 week administration of Myolastan® shows that the possible metabolic conversion of tetrazepam to diazepam is a more plausible explanation for the detection of diazepam in biological samples after the intake of Myolastan®. As such, these results reveal that the presence of diazepam and/or nordiazepam in biological samples from alleged drug-facilitated assault cases should be interpreted with care.

Recent advances in analytical techniques have enabled the detection of drugs and drug metabolites in oral fluid specimens. Although GC-MS is still commonly used in practice, many laboratories have developed and successfully validated methods for LC-MS(-MS) that can detect a large number of compounds in the limited sample volume available. In addition, several enzyme immunoassays have been commercialized for the detection of drugs of abuse in oral fluid samples, enabling the fast screening and selection of presumably positive samples. A number of concerns are discussed, such as the variability in the volume of sample collected and its implications in terms of quantitative measurements, and the drug recoveries of the many different specimen collection systems on the market. Additional considerations that also receive attention are the importance of providing complete validation data with respect to analyte stability, matrix effect, and the choice of collection method.

Background: Enantioselective analysis of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) helps interpret toxicological results. Methods have been described for various matrices, but so far not for oral fluid, a matrix of increasing importance in testing for drugs of abuse, especially in the context of driving under the influence of drugs (DUID). Methods: After dilution with 200 μL carbonate buffer (pH 9), oral fluid samples (10–50 μL) were derivatized with S-heptafluorobutyrylprolyl chloride. The resulting diastereomers were extracted into 100 μL of cyclohexane, separated by gas chromatography (HP-5MS column), and detected by mass spectrometry in the negative-ion chemical ionization mode (GC-NICI-MS). The method was validated and applied to samples from a controlled study with MDMA and from authentic DUID cases. Results: The derivatized AM, MA, MDA, MDMA, and MDEA enantiomers were well separated from each other. The method was linear from 5–250 μg/L per enantiomer of MDA and from 25–1250 μg/L per enantiomer of AM, MA, MDMA, and MDEA. With the exception of MDEA, analytical recoveries, repeatability, and intermediate precision were within required limits. The analyte concentrations and enantiomer ratios in the application samples correlated only weakly with corresponding published plasma data. Conclusions: This sensitive, reliable, and fast GC-NICI-MS assay enantioselectively measures AM, MA, MDA, and MDMA in oral fluid samples. Prediction of plasma concentrations and enantiomer ratios from respective oral fluid data is not possible.

Introduction Previous studies on the acute effects of MDMA on psychomotor performance and impulsivity showed that MDMA acts as a stimulant. These studies assessed performance during daytime, whereas in real life, dance-attendees leaving a party use the drug during the night. Objectives The present study aimed to assess the effects of nocturnal doses of MDMA on psychomotor performance and impulsivity during the night and after a night of sleep deprivation. Materials and methods Fourteen healthy subjects participated in a double-blind, placebo-controlled, two-way within-subject study. The treatment was MDMA (75 and 50 mg) divided over the evening or double placebo. Psychomotor and impulsivity tasks were conducted four times throughout the evening and night. A vigilance test was conducted once, at 5 A.M., and a sleepiness scale was presented to the subjects ten times throughout the evening and night. Results MDMA impaired tracking performance in a simple tracking task. Divided attention task performance was also impaired as indicated by a decrease in secondary task performance under the influence of MDMA compared with placebo. MDMA did not affect impulsivity measures. Vigilance performance decreased as a function of time on task, but this decrement was less during MDMA treatment compared to placebo. After the administration of MDMA, the sleepiness scale scores were lower during the night when compared with placebo. This difference between MDMA and placebo disappeared in the morning. Conclusion It is concluded that nocturnal doses of MDMA may produce impairments of tracking performance and divided attention throughout the night that are additive to performance impairment produced by sleep loss.