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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 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.

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.

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.

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.

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.

Background: The enantiomers of the designer drugs 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) differ in their pharmacologic and toxicologic potency. The aim of this study was to develop an assay for measuring these enantiomers in small plasma volumes and to analyze samples from a controlled study with MDMA. Methods: The analytes were extracted from ≤0.2 mL of plasma by mixed-mode solid-phase extraction. After derivatization with S-(−)-heptafluorobutyrylprolyl chloride, the resulting diastereomers were separated by gas chromatography (HP-5MS) within 17 min and detected by mass spectrometry in the negative-ion chemical ionization mode. The method was fully validated and applied to samples from a controlled study in which a single dose of racemic MDMA (75 mg) was administered. Results: The derivatized enantiomers were well separated and detected with good sensitivity. The assay was linear (per enantiomer) at 1–50 μg/L for MDA and 5–250 μg/L for MDMA and MDEA. Analytical recovery, accuracy, repeatability, and intermediate precision data were within required limits. Extraction yields were 82.1%–95.3%. In the study samples, concentrations of R-(−)-MDMA significantly exceeded those of S-(+)-MDMA. Their ratios (R vs S) were always >1.0 and increased over time. Concentrations of S-(+)-MDA exceeded those of R-(−)-MDA, their ratios (R vs S) also increasing over time but remaining Conclusions: This assay enables sensitive, reliable, and fast enantioselective measurement of MDA, MDMA, and MDEA in small volumes of plasma. The controlled study data confirm previous findings of MDMA and MDA enantiomer ratios (R vs S) increasing over time after ingestion of racemic MDMA.

The use of amphetamine and 'ecstasy' (MDMA) has increased exponentially in many European countries since the late nineties, leading to a rapid growth in the number of clinical and forensic analyses. Therefore, a rapid screening procedure for these substances in biological specimens has become an important part of routine toxicological analysis in forensic laboratories. The objective of this study was to evaluate the Cozart(R) amphetamine enzyme-linked immunosorbent assay (ELISA) for the screening of plasma samples and oral fluid samples (collected with the Intercept(R) device). Authentic plasma samples from drivers (n = 360) were screened, using an 1:5-fold dilution. True positive, true negative, false positive and false negative results were determined relative to the in-house routine GC-MS analysis. Samples consisted of 144 amphetamine-only positives, 141 MDMA/MDA-only positives, and 74 negatives when using the limit of quantitation as the cutoff level for confirmation (10 ng/mL). Using these results, receiver operating characteristic (ROC) curves were generated and optimal cut-off values for the screening assay were calculated. Analysis showed that the ELISA is able to predict the presence of either amphetamine or *MDMA/MDA (*MDMA as its metabolite MDA) in plasma samples with 98.3% sensitivity and 100% specificity at a cut-off value of 66.5 ng/mL D-amphetamine equivalents. A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind study. Subjects received placebo or a high (100 mg) or low (75 mg) dose of MDMA. Oral fluid samples were collected at 1.5 and 5.5 h after administration. Combined results of the analysis of the high and low dose oral fluid samples indicated a screening cut-off of 51 ng/mL D-amphetamine equivalents with both a sensitivity and specificity of 98.6% (using a LC-MS/MS confirmation cut-off of 10 ng/mL). In conclusion, these data indicate that the Cozart(R) AMP EIA plates constitute a fast and accurate screening technique for the identification of amphetamine and MDMA/MDA positive plasma samples and oral fluid specimens (collected with Intercept(R)). It should be emphasized that method validation should be performed for each type of biological matrix.

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC-MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC 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. Oral fluid was collected with the Intercept (R), a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC-MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries > 76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2-200 mu g/L) with an excellent intra-assay and inter-assay precision (coefficients of variation

Target analysis of amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. At present, most laboratories analyze such samples by gas chromatography-mass spectrometry. However, this procedure is labor-intensive and time-consuming, particularly as a preliminary extraction and derivatization are usually unavoidable. Here we describe the development of an alternative method. Amphetamines were isolated from human plasma and oral fluid using a simple methanol precipitation step and subsequently analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. Quantitation of the drugs was performed using multiple reaction monitoring. The developed method, which requires only 50 microL of biological sample, has a total analysis time of less than 20 min (including sample preparation) and enables the simultaneous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, methamphetamine, and ephedrine in a single chromatographic run. Limits of detection of 2 microg/L or better were obtained. The method has been validated and subsequently applied to the analysis of plasma and oral fluid samples collected from current drug users.

BACKGROUND A sensitive and reproducible Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry method has been developed for the simultaneous quantification of the 29 commonly prescribed benzodiazepines and z-drugs in hair. The method was validated according to international guidelines. METHODS After decontamination (with dichloromethane and water), compounds were extracted from 20 mg of pulverized hair samples using methanol at 45°C and sonication for 2 hours. The drugs were recovered by liquid-liquid extraction using 1-chlorobutane, evaporated to dryness, and reconstituted with 100 μL of methanol before injection in the UPLC-MS/MS. RESULTS The applied gradient ensured the elution of all the compounds within 7 minutes using 0.1% formic acid in water and methanol as mobile phase. The lower limit of quantification values ranged from 0.5 to 5 pg/mg of hair. Calibration curves were linear for almost all the compounds and ranged from the limit of quantification to 620 pg/mg hair. The bias and relative standard deviation of the intraday and interday imprecision were lower than 15% in 3 fortified "in-house" quality control samples, 1 external quality control sample, and 1 authentic hair sample (from a diazepam user). No significant matrix effects were observed for most of the compounds, and the extraction efficiency of the sample cleanup procedure ranged from 19% to 82% with a relative standard deviation

Although administered as a short-acting hypnotic for sleeping disorders, flunitrazepam, often in combination with alcohol or other drugs, was one of the most frequently abused benzodiazepines over the last 10 years. It has been reported in cases of driving under the influence, and its use is associated with marked psychomotor impairment. Studies over the last five years have investigated the use of oral fluid as an alternative matrix to blood and urine, especially when non-intrusive and quick sampling procedures are important (e.g., screening for drugs of abuse at the roadside and screening and confirmatory workplace drug testing). In this study, Rohypnol (flunitrazepam) was administered to four healthy volunteers, and oral fluid samples were collected by spitting into a polypropylene tube at fixed times between 0 and 6 h after the intake of a tablet of 1 mg. A specific and very sensitive method was developed, both for flunitrazepam and for its main metabolite 7-aminoflunitrazepam, based on solid-phase extraction of the oral fluid samples, stored at +4 degrees C, and gas chromatographic-mass spectrometric analyses using negative chemical ionization with methane as the ionization gas. The heptadeuterated parent compound and metabolite were used as internal standards. The respective limits of detection and quantitation were 0.05 microg/L and 0.1 microg/L for flunitrazepam, and 0.1 and 0.15 microg/L for 7-aminoflunitrazepam. The parent drug could only be detected when the analyses were performed within 12-24 h after collection of the oral fluid samples or when 2% of NaF was added to the collection tubes. The stability of flunitrazepam in oral fluid was poor, even at +4 degrees C, when no NaF was added to the sample. In any case, concentrations remained below 1 microg/L. The metabolite was detected in slightly higher concentrations, with or without the presence of NaF, reaching a maximum of 1-3 microg/L within 2-4 h after administration. In all cases the drug was detectable, but at extremely low concentrations, for 6 h after intake of a normal dose of Rohypnol and it will be an analytical challenge to come up with a sufficiently sensitive onsite test for low-dose benzodiazepines in oral fluid.

The Society of Hair Testing (SoHT) has provided cutoffs for the quantification of ethyl glucuronide (EtG) in hair to indicate occasional or chronic/excessive alcohol consumption. Although several sensitive methods have been reported, past proficiency test results show a lack of reproducibility. An ultra-performance liquid chromatographic mass spectrometric method (LLOQ of 10 pg EtG/mg hair) has been validated according to the international guidelines, including the successful participation in five proficiency tests. This method was subsequently used to evaluate the impact of different grinding conditions (cut, weakly or extensively pulverized hair samples) on the final measured EtG concentration. Hair from alcohol consumers (n = 2) and commercially available quality control samples (QCs) (n = 2) was used. For the QCs, extensive pulverization led to a significantly higher amount of measured EtG. In the hair samples obtained from volunteers, cut or weakly pulverized hair resulted in EtG concentrations below the LLOQ, while the mean concentrations of 14 and 40 pg EtG/mg hair were determined after extensive pulverization. Differences in sample preparation could partially explain inter-laboratory variability. As the differences in results can lead to a different interpretation even when applying the SoHT cutoffs, it is of interest to standardize sample preparation techniques in the field of EtG hair testing.

The aim of this work was to automate a sample preparation procedure extracting morphine, hydromorphone, oxymorphone, norcodeine, codeine, dihydrocodeine, oxycodone, 6-monoacetyl-morphine, hydrocodone, ethylmorphine, benzoylecgonine, cocaine, cocaethylene, tramadol, meperidine, pentazocine, fentanyl, norfentanyl, buprenorphine, norbuprenorphine, propoxyphene, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine from urine samples. Samples were extracted by solid-phase extraction (SPE) with cation exchange cartridges using a TECAN Freedom Evo 100 base robotic system, including a hydrolysis step previous extraction when required. Block modules were carefully selected in order to use the same consumable material as in manual procedures to reduce cost and/or manual sample transfers. Moreover, the present configuration included pressure monitoring pipetting increasing pipetting accuracy and detecting sampling errors. The compounds were then separated in a chromatographic run of 9 min using a BEH Phenyl analytical column on a ultra-performance liquid chromatography – tandem mass spectrometry system. Optimization of the SPE was performed with different wash conditions and elution solvents. Intra- and inter-day relative standard deviations (RSDs) were within +15% and bias was within + +15% for most of the compounds. Recovery was >69% (RSD < 11%) and matrix effects ranged from 1 to 26% when compensated with the internal standard. The limits of quantification ranged from 3 to 25 ng/mL depending on the compound. No crosscontamination in the automated SPE system was observed. The extracted samples were stable for 72 h in the autosampler (488C). This method was applied to authentic samples (from forensic and toxicology cases) and to proficiency testing schemes containing cocaine, heroin, buprenorphine and methadone, offering fast and reliable results. Automation resulted in improved precision and accuracy, and a minimum operator intervention, leading to safer sample handling and less time-consuming procedures.

A method has been developed for the qualitative and quantitative determination of enzymatic lipolysis products in the presence of high triglyceride concentrations, using aminopropyl bonded silica cartridges and capillary gas chromatography. Two reactions are catalyzed in the presence of the Rhizopus javanicus lipase: hydrolysis of triglycerides to diglycerides, and diglycerides to monoglycerides. Lipids were extracted from the hydrolyzed olive oil emulsion and separated into fractions with aminopropyl bonded silica cartridges by means of sequential elution. The elution procedure was well characterized and optimized to obtain a maximum recovery and purity for each lipid fraction. Further analysis was done with capillary gas chromatography. The method is suitable to study the properties of lipases in vitro in the presence of high triglyceride concentrations and different amounts of bile salts. The expected inhibition of the fungal lipase by higher concentrations of bile salts, under physiological conditions, was limited.

A method for the quantification of ethyl glucuronide (EtG) and ethyl sulphate (EtS) in human urine is developed and fully validated according to international guidelines. Protein precipitation is used as sample preparation. During the development of the method on an UPLC-ESI-MS/MS system using a CSH C18 column, special attention was paid to reduce matrix effects to improve assay sensitivity and to improve detection of the second transition for EtS for specificity purposes. The method was linear from 0.1 to 10μg/mL for both analytes. Ion suppression less than 24% (RSD15%) was observed for EtG and no significant matrix effect was measured for EtS. The recovery was around 80% (RSD14%) for both compounds. This method provides good precision (RSDr and RSDt10%) and bias (15%) for internal and external quality control samples. The reproducibility of the method was demonstrated by the successful participation to proficiency tests (z-score0.86). This method was finally used to analyze urine samples obtained from twenty-seven volunteers whose alcohol consumption during the 5 days before sampling was monitored. Concentrations between 0.5 and 101.9μg/mL (mean 10.9, median 1.4) for EtG and between 0.1 and 37.9μg/mL (mean 3.6, median 0.3) for EtS were detected in urine samples of volunteers who declared having consumed alcohol the day before the sampling. EtG and EtS concentrations in urine were highly correlated (r=0.996, p0.001). A moderate correlation between the number of drinks the day before sampling and the concentration of EtG (r=0.448, p0.02) or EtS (r=0.406, p0.04) was observed. Using a cut-off value at 0.1μg/mL for EtG and EtS, this method is able to detect social alcohol consumption approximately 24h after the intake, without showing any false positive result.

Entomotoxicology studies the application of toxicological analysis on necrophageous insects present on human remains. This paper describes the development and validation of a sensitive liquid chromatography tandem mass spectrometry method for quantification of methadone and its main metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in developmental stages of Lucilia sericata. One single larva was pulverized in a disposable vial and then extracted with 1-chlorobutane. After evaporation of the organic layer, samples were reconstituted in the mobile phase. Chromatographic separation was achieved on a NUCLEODUR Sphinx RP column with a liquid chromatographic gradient (0.1% formic acid and methanol), ensuring the elution of methadone and EDDP within 15 min. The method was fully validated according to international guidelines. The use of liquid liquid extraction was demonstrated to be effective (matrix effect27% and recovery66%). The method was linear over the dynamic range (10-400 pg/mg larva) with excellent within- and between-run precision and bias (CV%5%). The lower limit of quantification was fixed at 10 pg/mg larva. No instability of the extracted samples was observed in the autosampler after three freeze/thaw cycles and after two months at -20 degrees C. The validated method was applied to third instar larvae of Lucilia sericata reared on beef heart spiked with 4 microg/g methadone and on a postmortem methadone overdose case. The validation and actual sample analysis showed that the method is sensitive, rugged, precise, accurate, and well-suited for routine analysis of methadone and EDDP in a single larva obtained from forensic cases.

In recent years, the interest in the use of oral fluid as a biological matrix has increased significantly, particularly for detecting driving under the influence of drugs (DUID). In this study, the relationship between the oral fluid and the blood concentrations of drugs of abuse in drivers suspected of DUID is discussed. Blood and oral fluid samples were collected from drivers suspected of DUID or stopped during random controls by the police in Belgium, Germany, Finland, and Norway for the ROSITA-2 project. The blood samples were analyzed by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), sometimes preceded by immunoassay screening of blood or urine samples. The oral fluid samples were analyzed by GC-MS or LC-MS(/MS). Scatter plots and trend lines of the blood and oral fluid concentrations and the median, mean, range, and SD of the oral fluid to blood (OF:B) ratios were calculated for amphetamines, benzodiazepines, cocaine, opiates, and Delta(9)-2 tetrahydrocannabinol. The ratios found in this study were comparable with those that were published previously, but the range was wider. The OF:B ratios of basic drugs such as amphetamines, cocaine, and opiates were >1 [amphetamine: median (range) 13 (0.5-182), methylenedioxyamphetamine: 4 (1-15), methylenedioxymethamphetamine: 6 (0.9-88), methamphetamine: 5 (2-23), cocaine: 22 (4-119), benzoylecgonine: 1 (0.2-11), morphine: 2 (0.8-6), and codeine: 10 (0.8-39)]. The ratios for benzodiazepines were very low, as could be expected as they are highly protein bound and weakly acidic, leading to low oral fluid concentrations [diazepam: 0.02 (0.01-0.15), nordiazepam: 0.04 (0.01-0.23), oxazepam: 0.05 (0.03-0.14), and temazepam: 0.1 (0.06-0.54)]. For tetrahydrocannabinol, an OF:B ratio of 15 was found (range 0.01-569). In this study, the time of last administration, the dose, and the route of administration were unknown. Nevertheless, the data reflect the variability of the OF:B ratios in drivers thought to be under the influence of drugs. The wide range of the ratios, however, does not allow reliable calculation of the blood concentrations from oral fluid concentrations.

The aim of the study was to determine the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA) and other amphetamine-type stimulants (ATS) in segmented hair specimens of self-declared ecstasy abusers, who took part in a double-blind placebo-controlled six-way crossover study during approximately 7 weeks, during which they received a 75 and a 100 mg dose of racemic MDMA twice. Hair specimens were washed and cut into pieces of 2 cm length. After digestion and solid phase extraction, the enantiomers were derivatized with a chiral agent (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, developed at the authors laboratory and quantified by gas chromatography coupled to mass spectrometry operating in the negative chemical ionization mode. Most of the hair specimens that were tested positive for MDMA showed a predominance of the (R)-enantiomer. The R/S ratios of MDMA varied between 1.02 and 2.75 and total concentrations ranged from 0.1 to 20.1 ng/mg. The enantiomers of its metabolite 3,4-methylenedioxyamphetamine (MDA) were also quantified in most hair segments. The R/S ratios of MDA varied between 0.60 and 1.60, while the concentrations of the enantiomers ranged from 10 to 160 pg/mg hair. When segmental analysis was performed on single hair specimens, no inversion of the R versus S ratios of MDMA and MDA was observed. The predominance of (R)-MDMA in hair was in accordance with those already published for other matrices. Furthermore, both enantiomers of amphetamine (AM) were also detected in hair segments of four volunteers and the R/S ratios ranged from 1.00 to 1.47.

We have developed a rapid method that enables the simultaneous analysis of gamma-hydroxybutyrate (GHB) and its precursors, i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in urine. The method comprised a simple dilution of the urine sample, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation was achieved using an Atlantis dC18 column, eluted with a mixture of formic acid and methanol. The method was linear from 1-80 mg/L for GHB and 1,4-BD and from 1-50 mg/L for GBL. The limit of quantification was 1 mg/L for all analytes. The procedure, which has a total analysis time (including sample preparation) of less than 12 min, was fully validated and applied to the analysis of 182 authentic urine samples; the results were correlated with a previously published GC-MS procedure and revealed a low prevalence of GHB-positive samples. Since no commercial immunoassay is available for the routine screening of GHB, this simple and rapid method should prove useful to meet the current increased demand for the measurement of GHB and its precursors. © 2004 Elsevier B.V. All rights reserved.

Larvae of the Calliphora vicina (Diptera: Calliphoridae) were reared on artificial food spiked with different concentrations of nordiazepam. The dynamics of the accumulation and conversion of nordiazepam to its metabolite oxazepam in post-feeding larvae and empty puparia were studied. Analysis was performed using a previously developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. This method enabled the detection and quantitation of nordiazepam and oxazepam in single larvae and puparia. Both drugs could be detected in post-feeding larvae and empty puparia. In addition, the influence of nordiazepam on the development and growth of post-feeding larvae was studied. However, no major differences were observed for these parameters between the larvae fed on food containing nordiazepam and the control group. To our knowledge, this is the first report describing the presence of nordiazepam and its metabolite, oxazepam, in single Calliphora vicina larvae and puparia.

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is emerging as the tool of choice for rapid analysis and the detection of biologically active compounds in complex mixtures. We describe the development of a sensitive method for the simultaneous quantitation of 10 benzodiazepines in Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of larvae for toxicological analyses offers some technical advantages over putrefied tissue. Four sample pretreatment methods for isolating the benzodiazepines out of larvae were evaluated. A simple homogenization, followed by acetonitrile precipitation yielded the highest recoveries. Puparia were pulverized and extracted by ultrasonification in methanol. All extracts were subsequently analyzed using reversed-phase LC-MS-MS. Larvae and puparia calibrators containing benzodiazepines at concentrations ranging from 25 to 750 pg/mg and 50 to 500 pg/mg, respectively, were prepared and analyzed. The method was demonstrated to be linear over the ranges investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and from 6.28 to 19.03 pg/mg puparium. The developed method was applied to the determination of nordiazepam and its metabolite oxazepam in larvae and puparia of the Calliphora vicina fly that had been reared on artificial foodstuff (beef heart) spiked with 1 microg/g nordiazepam. The larvae were harvested at day 5 for analysis of drug content. The method was sufficiently sensitive to allow the detection of nordiazepam and oxazepam in a single larva or puparium.

1 We report on a patient who was resuscitated after a suicide attempt with the veterinary euthanasia pro duct T-61 and treated with N-acetylcysteine (NAC) to prevent hepatotoxicity from N,N-dimethylformamide (DMF), the solvent of T-61. 2 Serum concentrations of DMF were high as compared with values published on occupational exposure. 3 The patient showed only a transient increase in liver enzymes with eventually a full recovery. 4 The hepatoprotective effect of NAC was studied in a rat model using the rise in serum sorbitol dehydrogenase (SDH) as a marker for DMF-induced hepatotoxicity. 5 Four series of randomized, controlled and double- blind experiments were carried out and consistently showed a lower increase in SDH in NAC-treated animals in each series. The difference was statistically significant only when the data of the 4 series were pooled. This is probably due to the large interindivi dual variations in the effect of DMF. 6 We hypothesize that in the rat NAC may have a protective effect. Whether NAC is also protective in patients, in which it is administered after exposure to DMF, cannot be concluded from the present experi ments.