Semiquantitative Activity-Based Detection of JWH-018, a Synthetic Cannabinoid Receptor Agonist, in Oral Fluid after Vaping.

The rapid proliferation of new synthetic cannabinoid receptor agonists (SCRAs) has initiated considerable interest in the development of so-called "untargeted" screening strategies. One of these new screening technologies involves the activity-based detection of SCRAs. In this study, we evaluated whether (synthetic) cannabinoid activity can be detected in oral fluid (OF) and, if so, whether it correlates with SCRA concentrations. OF was collected at several time points in a placebo-controlled JWH-018 administration study. The outcome of the cell-based cannabinoid reporter system, which monitored the cannabinoid receptor activation, was compared to the quantitative data for JWH-018, obtained via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. A total of 175 OF samples were collected and analyzed via both methods. The cannabinoid reporter assay correctly classified the vast majority of the samples as either negative (<0.25 ng/mL; 74/75 = 99%) or having low (0.25-1.5 ng/mL; 16/16 = 100% and 1.5-10 ng/mL; 37/41 = 90%), mid (10-100 ng/mL; 23/25 = 92%) or high (>100 ng/mL; 16/18 = 89%) JWH-018 concentrations. Passing-Bablok regression analysis yielded a good linear correlation, with no proportional difference between both methods (slope 0.97; 95% confidence interval 0.86-1.14) and only a small systematic difference. This is the first study to demonstrate the applicability of an untargeted, activity-based approach for SCRA detection in OF. Additionally, the outcome of the cannabinoid reporter assay was compared to the gold standard (LC-MS/MS), showing a good correlation between both methods, indicating that the cannabinoid reporter assay can be used for an estimation of drug concentrations.

Activity-based reporter assays for the screening of abused substances in biological matrices.

The (ab)use of designer drugs and steroid hormones has gained popularity due to the lower chance of getting caught, as routine drug or doping tests may miss these (novel) compounds. Current analytical approaches mostly make use of targeted, structure-based techniques, such as immunoassays or mass spectrometry (MS)-based methods. However, these approaches have limitations, including a lack of cross-reactivity and the need for prior knowledge of molecular identity. This has initiated considerable interest in the so-called "untargeted" screening strategies to detect these compounds. The use of "untargeted" MS-based screening methods (e.g. gas chromatography MS and especially high-resolution MS) has gained considerable interest to detect and identify novel compounds. However, due to their expensive and time-consuming character, very sophisticated analytical methods are not ideal as a first-line screening method and are not routinely implemented in most laboratories. Given the above, it is clear that there lies potential in novel "untargeted" screening approaches, which are less expensive, more high-throughput-amenable and more routinely applicable. Activity-based assays, capable of monitoring the biological activity of an abused substance in a biological matrix, have been proposed as an alternative. These biological assays do not require knowledge about a compound's structure and could be used as a first-line screening tool to identify potentially positive samples. In this review, we focus on activity-based reporter bioassays for the detection of steroids and drugs of abuse in biological matrices. As for drugs of abuse, only bioassays for detecting cannabinoid or opioid activity in biological matrices are available, only (synthetic) cannabinoid receptor agonists and opioids are discussed.

Activity-Based Detection of Cannabinoids in Serum and Plasma Samples.

BACKGROUND: Synthetic cannabinoids are the largest group of new psychoactive substances monitored by the European Monitoring Centre of Drugs and Drug Addiction. The rapid proliferation of novel analogs makes the detection of these new derivatives challenging and has initiated considerable interest in the development of so-called "untargeted" screening strategies to detect these compounds. METHODS: We developed new, stable bioassays in which cannabinoid receptor activation by cannabinoids led to recruitment of truncated ß-arrestin 2 (ßarr2) to the cannabinoid receptors, resulting in functional complementation of a split luciferase, allowing readout via bioluminescence. Aliquots (500 µL) of authentic serum (n = 45) and plasma (n = 73) samples were used for simple liquid-liquid extraction with hexane:ethyl acetate (99:1 v/v). Following evaporation and reconstitution in 100 µL of Opti-MEM® I/methanol (50/50 v/v), 10 µL of these extracts was analyzed in the bioassays. RESULTS: Truncation of ßarr2 significantly (for both cannabinoid receptors; P = 0.0034 and 0.0427) improved the analytical sensitivity over the previously published bioassays applied on urine samples. The new bioassays detected cannabinoid receptor activation by authentic serum or plasma extracts, in which synthetic cannabinoids were present at low- or sub-nanogram per milliliter concentration or in which Δ9-tetrahydrocannabinol was present at concentrations >12 ng/mL. For synthetic cannabinoid detection, analytical sensitivity was 82%, with an analytical specificity of 100%. CONCLUSIONS: The bioassays have the potential to serve as a first-line screening tool for (synthetic) cannabinoid activity in serum or plasma and may complement conventional analytical assays and/or precede analytical (mass spectrometry based) confirmation.

Activity-Based Concept to Screen Biological Matrices for Opiates and (Synthetic) Opioids.

BACKGROUND: Detection of new highly potent synthetic opioids is challenging as new compounds enter the market. Here we present a novel screening method for the detection of opiates and (synthetic) opioids based on their activity. METHODS: A cell-based system was set up in which activation of the µ-opioid receptor (MOR) led to recruitment of ß-arrestin 2, resulting in functional complementation of a split NanoLuc luciferase and allowing readout via bioluminescence. Assay performance was evaluated on 107 postmortem blood samples. Blood (500 µL) was extracted via solid-phase extraction. Following evaporation and reconstitution in 100 µL of Opti-MEM® I, 20 µL was analyzed in the bioassay. RESULTS: In 8 samples containing synthetic opioids, in which no positive signal was obtained in the bioassay, quadrupole time-of-flight mass spectrometry revealed the MOR antagonist naloxone, which can prevent receptor activation. Hence, further evaluation did not include these samples. For U-47700 (74.5-547 ng/mL) and furanyl fentanyl (<1-38.8 ng/mL), detection was 100% (8/8) for U-47700 and 95% (21/22) for furanyl fentanyl. An analytical specificity of 93% (55/59) was obtained for the opioid negatives. From an additional 10 samples found to contain other opioids, 5 were correctly scored positive. Nondetection in 5 cases could be explained by very low concentrations (<1 ng/mL alfentanil/sufentanil) or presence of inactive enantiomers. CONCLUSIONS: The MOR reporter assay allows rapid identification of opioid activity in blood. Although the cooccurrence of opioid antagonists is currently a limitation, the bioassay's high detection capability, specificity, and untargeted nature may render it a useful first-line screening tool to investigate potential opioid intoxications.

Determination of Antidepressants in Hair via UHPLC-MS/MS as a Complementary Informative Tool for Clinical and Forensic Toxicological Assessments.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 were <15% 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 deviation <15%) [except for demethylcitalopram, didemethylcitalopram, and trazodone (relative standard deviation <33%)]. 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. CONCLUSIONS: 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.

Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers.

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 µL) and in venous (V, 30 µL) 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.

Detection of Benzodiazepines and z-Drugs in Hair Using an UHPLC-MS/MS Validated Method: Application to Workplace Drug Testing.

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 <15% [except for clobazam (16%), loprazolam (20%), brotizolam (18%), and 7-aminoclonazepam (20%)]. The method was then successfully applied to the analysis of 40 hair samples from the workplace drug testing, containing alprazolam, estazolam, clonazepam, diazepam, zolpidem, and desalkylflurazepam (and metabolites). CONCLUSIONS: The method was completely validated and can be of interest to clinical and forensic laboratories.

Screening and confirmation methods for GHB determination in biological fluids.

The purpose of this review is to provide a comprehensive overview of reported methods for screening and confirmation of the low-molecular-weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polarity of the compound, its endogenous presence, its rapid metabolism after ingestion, and its instability during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone) are challenges for the analyst and for 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 liquid chromatography and capillary zone electrophoresis have also been used. Before injection, sample-preparation techniques include (a combination of) liquid-liquid, solid-phase, or headspace extraction, and chemical modification of the polar compound. Also simple "dilute-and-shoot" may be sufficient for urine or serum. Advantages, limitations, and trends are discussed.

Establishing effective interpretation criteria in hair analysis to distinguish between passive and active cocaine exposure: Insights from authentic hair samples collected from professional individuals exposed to cocaine.

Interpretation results of hair analysis, particularly for cocaine, can be challenging due to the need to differentiate between active use or passive contamination. Our study aimed to assess the impact of varying degrees of passive cocaine exposure hair analysis results and their interpretation. Hair samples (n = 25) were categorized based on the declared cocaine exposure of volunteers: (a) high, involving handling up to several kilograms of cocaine powder from dismantling illegal distribution sites; (b) medium, where staff dealt with cocaine blocks (up to kilograms); and (c) low, with staff in contact with up to grams of cocaine for laboratory analysis. Hair samples were decontaminated using dichloromethane, water, and methanol. The samples and final wash were analyzed for cocaine, benzoylecgonine, norcocaine, cocaethylene, m-OH-benzoylecgonine, and ecgonine methyl ester using a validated UPLC-MS/MS method. Cocaine hair concentrations ranges were as follows (pg/mg): high (n = 53 segments) < LLOQ(32)-7046; medium (n = 91) < LLOQ-939; and low (n = 54) < LLOQ-292. All hair samples had concentrations below the LLOQ for cocaethylene, ecgonine methyl ester, and m-OH-benzoylecgonine. Applying the SoHT cocaine cut-off in combination with a hair/wash ratio criterion identified 97% of the samples as contaminated. This study advocates for a comprehensive approach in evaluating cocaine hair concentrations. This involves integrating the 500 pg/mg decision limit for cocaine with a criterion comparing wash and hair concentration. Additionally, confirming the presence of specific metabolites is crucial. This multifaceted method effectively distinguishes between environmental contamination and active cocaine usage. The research contributes significantly to refining cocaine exposure assessment in professional contexts.

The complementarity of phosphatidylethanol in whole blood and ethyl glucuronide in hair as biomarkers for the monitoring of alcohol use.

Monitoring long-term alcohol use and/or abstinence is essential in clinical and medico-legal cases. Analysis of ethyl glucuronide (EtG) in hair provides information on alcohol consumption over several months. However, there is a lag time between ethanol consumption, incorporation of EtG in the hair bulb and hair growing out of the scalp. Phosphatidylethanol (PEth) 16:0/18:1 analysis in whole blood has a detection window of 2-4 weeks, allowing for the detection of recent alcohol consumption. In this study, 2340 paired samples (of hair and venous whole blood from 1170 individuals) were analysed for EtG in hair (hEtG) and PEth 16:0/18:1 in venous whole blood. PEth 16:0/18:1 and hEtG results were subdivided into three categories according to the consensus of SoHT (hEtG) and PEth-NET (PEth): abstinence/low, moderate or excessive alcohol consumption. For hEtG analysis, 446 individuals presented abstinence/low alcohol consumption, of which 2% were classified as excessive alcohol users through PEth 16:0/18:1 analysis. This suggests excessive alcohol consumption in the weeks before sample collection. Out of 483 individuals classified as heavy alcohol users based on hEtG analysis, 14% showed abstinence/low alcohol consumption for PEth 16:0/18:1 analysis, implying that these subjects stopped drinking 2-4 weeks before sample collection. Our results show that the analysis of the two different biomarkers can lead to a more accurate categorisation of individuals. Therefore, we emphasize that for the retrospective investigation of alcohol use, it is necessary to include two alcohol use biomarkers with different detection windows.

Driving under the influence of cannabis: pitfalls, validation, and quality control of a UPLC-MS/MS method for the quantification of tetrahydrocannabinol in oral fluid collected with StatSure, Quantisal, or Certus collector.

BACKGROUND: "Driving under the influence of drugs" (DUID) has a large impact on the worldwide mortality risk. Therefore, DUID legislations based on impairment or analytical limits are adopted. Drug detection in oral fluid is of interest due to the ease of sampling during roadside controls. The prevalence of Δ9-tetrahydrocannabinol (THC) in seriously injured drivers ranges from 0.5% to 7.6% in Europe. For these reasons, the quantification of THC in oral fluid collected with 3 alternative on-site collectors is presented and discussed in this publication. METHODS: An ultra-performance liquid chromatography-mass spectrometric quantification method for THC in oral fluid samples collected with the StatSure (Diagnostic Systems), Quantisal (Immunalysis), and Certus (Concateno) devices was validated according to the international guidelines. Small sample volumes of 100-200 µL were extracted using hexane. Special attention was paid to factors such as matrix effects, THC adsorption onto the collector, and stability in the collection fluid. RESULTS: A relatively high-throughput analysis was developed and validated according to ISO 17025 requirements. Although the effects of the matrix on the quantification could be minimized using a deuterated internal standard, and stability was acceptable according the validation data, adsorption of THC onto the collectors was a problem. For the StatSure device, THC was totally recovered from the collector pad after storage for 24 hours at room temperature or 7 days at 4°C. A loss of 15%-25% was observed for the Quantisal collector, whereas the recovery from the Certus device was irreproducible (relative standard deviation, 44%-85%) and low (29%-80%). During the roadside setting, a practical problem arose: small volumes of oral fluid (eg, 300 µL) were collected. However, THC was easily detected and concentrations ranged from 8 to 922 ng/mL in neat oral fluid. CONCLUSION: A relatively high-throughput analysis (40 samples in 4 hours) adapted for routine DUID analysis was developed and validated for THC quantification in oral fluid samples collected from drivers under the influence of cannabis.

Quantitative analysis of 26 opioids, cocaine, and their metabolites in human blood by ultra performance liquid chromatography-tandem mass spectrometry.

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 was <20%) determined with 2 internal and 2 external QC samples, and the bias was within ±15% (except for methylecgonine, which was <20%). 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.

Incorporation of doxylamine and N-doxylamine-oxide in human hair and the impact of a permanent oxidative hair dye.

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 and

Combined Use of Flubromazepam and Stimulants: Blood and Oral Fluid Concentrations and Impact on Driving Ability.

"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 (

Quantitative method validation for the analysis of 27 antidepressants and metabolites in plasma with ultraperformance liquid chromatography-tandem mass spectrometry.

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.

Evaluation of decontamination procedures for drug testing in undamaged versus damaged hair.

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.

Liquid Chromatography High-Resolution Mass Spectrometry in Forensic Toxicology: What are the Specifics of Method Development, Validation and Quality Assurance for Comprehensive Screening Approaches?

The use of high-resolution mass spectrometry (HRMS) has increased over the past decade in clinical and forensic toxicology, especially for comprehensive screening approaches. Despite this, few guidelines in this field have specifically addressed HRMS issues concerning compound identification, validation, measurement uncertainty and quality assurance. To fully implement this technique, certainly in an era in which the quality demands for laboratories are ever-increasing due to various norms (e.g. the International Organization for Standardization's ISO 17025), these specific issues need to be addressed. This manuscript reviews 26 HRMSbased methods for qualitative systematic toxicological analysis (STA) published between 2011 and 2021. Key analytical data such as samples matrices, analytical platforms, numbers of analytes and employed mass spectral reference databases/libraries as well as the studied validation parameters are summarized and discussed. The article further includes a critical review of targeted and untargeted data acquisition approaches, available HRMS reference databases and libraries as well as current guidelines for HRMS data interpretation with a particular focus on identification criteria. Moreover, it provides an overview on current recommendations for the validation and determination of measurement uncertainty of qualitative methods. Finally, the article aims to put forward suggestions for method development, compound identification, validation experiments to be performed, and adequate determination of measurement uncertainty for this type of wide-range qualitative HRMSbased methods.

The Future of Analytical and Interpretative Toxicology: Where are We Going and How Do We Get There?

(Forensic) toxicology has faced many challenges, both analytically and interpretatively, especially in relation to an increase in potential drugs of interest. Analytical toxicology and its application to medicine and forensic science have progressed rapidly within the past centuries. Technological innovations have enabled detection of more substances with increasing sensitivity in a variety of matrices. Our understanding of the effects (both intended and unintended) have also increased along with determination and degree of toxicity. However, it is clear there is even more to understand and consider. The analytical focus has been on typical matrices such as blood and urine but other matrices could further increase our understanding, especially in postmortem (PM) situations. Within this context, the role of PM changes and potential redistribution of drugs requires further research and identification of markers of its occurrence and extent. Whilst instrumentation has improved, in the future, nanotechnology may play a role in selective and sensitive analysis as well as bioassays. Toxicologists often only have an advisory impact on pre-analytical and pre-interpretative considerations. The collection of appropriate samples at the right time in an appropriate way as well as obtaining sufficient circumstance background is paramount in ensuring an effective analytical strategy to provide useful results that can be interpreted within context. Nevertheless, key interpretative considerations such as pharmacogenomics and drug-drug interactions as well as determination of tolerance remain and in the future, analytical confirmation of an individual's metabolic profile may support a personalized medicine and judicial approach. This should be supported by the compilation and appropriate application of drug data pursuant to the situation. Specifically, in PM circumstances, data pertaining to where a drug was not/may have been/was contributory will be beneficial with associated pathological considerations. This article describes the challenges faced within toxicology and discusses progress to a future where they are being addressed.

The Interest of a Systematic Toxicological Analysis Combined with Forensic Advice to Improve the Judicial Investigation and Final Judgment in Drug Facilitated Sexual Assault Cases.

The conviction rate in drug facilitated sexual assault (DFSA) cases is known to be very low. In addition, the potential impact of toxicological results on the case is often not well understood by the judicial authorities. The aims of this study were (1) to obtain more knowledge concerning the prevalence of incapacitating substances in DFSA cases, (2) to create a more efficient DFSA analysis strategy taking background information into account, and (3) to evaluate the potential impact of systematic toxicological analysis (STA) on the final judicial outcome. This small-scale epidemiological study (n = 79) demonstrates that 'commonly-used' illicit drugs, psychoactive medicines and ethanol are more prevalent in DFSA cases in contrast to the highly mediatized date rape drugs. Additionally, via case examples, the interest of performing STA-to prove incapacitation of the victim-in judicial procedures with mutual-consent discussions has been demonstrated as it led to increased convictions. However, more attention has to be paid to ensure a short sampling delay and to get more accurate information from the medical treatment of the alleged victim. This will improve the interpretation of the toxicological analysis and thus its applicability in a DFSA case. The future is multi-disciplinary and will certainly lead to an efficient and more cost-effective DFSA approach in which STA can impact the final judgment.