Atypical postmortem redistribution in chronic methadone consumers.

Available literature demonstrates that methadone is prone to moderate postmortem redistribution, but subject to high interindividual variability in the central to peripheral blood concentration ratios (C/P). In this case series, 10 cases of chronic methadone users displaying C/P < 1 (range 0.26-0.82) are described. Femoral, cardiac and ante-mortem blood concentrations of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) are reported for all cases, as well as sex, age, case history, results of the pathological investigation, other toxicological findings and cause and manner of death. EDDP blood concentrations, similar in both central and peripheral blood, as well as antemortem blood concentration results in Case 4, demonstrate that this atypical C/P < 1 finding is attributable to postmortem changes and not analytical or pre-analytical artifacts. Case 4 is a particularly instructive example, with femoral blood concentration (966 ng/mL) approximately twice as high as cardiac blood (499 ng/mL) and ante-mortem blood (418 ng/mL, collected 38 min prior to death)-clearly demonstrating that cardiac blood methadone concentration is more representative of the antemortem blood concentration in this case. In Case 4 and four others, toxicological interpretation based on femoral blood concentration alone would have been misleading. Based on these results and evidence from the literature, it is hypothesized that methadone bioaccumulates in the tissues of chronic users and redistributes from thigh tissues into femoral blood, increasing the concentration postmortem. This case series highlights how femoral blood is not always preserved from postmortem changes and that the analysis of multiple blood sources is necessary to avoid a misleading toxicological interpretation-particularly for cases of chronic methadone users.

Postmortem redistribution of cannabinoids: Statistical analysis of a novel dataset and meta-analysis.

The assessment of human postmortem concentrations of Δ9-THC (THC) and its metabolites, 11-nor-9-carboxy-THC (THCCOOH) and 11-hydroxy-THC (11-OH-THC), is routinely performed in forensic toxicology laboratories. However, the literature on cannabinoids postmortem redistribution (PMR) is scarce and highlights their complex postmortem changes. This study aims to investigate the postmortem behavior of THC and its metabolites in order to provide practitioners with potential indicators of PMR. To do so, antemortem and postmortem cases positive for cannabinoids were compiled in a database. Its analysis shows significantly higher THC concentrations in postmortem blood than in antemortem blood. Antemortem and postmortem blood also present significantly different profiles for their THC to THCCOOH ratios. Whereas antemortem blood generally shows THCCOOH concentrations higher or equal to THC, several postmortem cases show the opposite, with THC concentrations higher than THCCOOH. While occurrence of postmortem redistribution (PMR) is difficult to measure directly, an evaluation was performed using the central to peripheral (C/P) blood concentrations ratio as a proxy. With a C/P significantly lower than 1.0 for THC and significantly higher than 1.0 for THCCOOH, the PMR hypothesis is supported for both compounds, with redistribution towards peripheral blood for THC and towards central blood for THCCOOH. On the other hand, 11-OH-THC does not show a C/P significantly different than 1.0, suggesting the absence of PMR. Influence of body mass index, conservation state and postmortem interval on C/P was statistically analyzed and no significant impact was observed. To compare and contrast C/P observed in the database with those published in the literature, a meta-analysis was performed using a median of median (MM) model. THC PMR towards peripheral blood is supported by a global estimate of 0.81 (CI95%: 0.51 to 1.2). Redistribution towards femoral blood appears to be stronger than towards iliac blood; indeed, the median estimate of C/P decreases to 0.64 (CI95%: 0.40 to 1.1) when studies with iliac blood were removed from the meta-analysis. THCCOOH PMR towards central blood is supported by a C/P median estimate of 1.3 (CI95%: 0.97 to 1.6). THC PMR can be suspected when these indicators are observed (i) high THC blood concentration (>50 ng/mL), (ii) THC C/P lower than 1.0 (iii) blood THC/THCCOOH concentration ratios greater than 1.0 and (iv) non-detectability of THCCOOH in urine. In postmortem samples, many factors may contribute to the overestimation of THC concentration, therefore a careful interpretation is required, relying on both central and peripheral blood samples.

On-site drug detection coasters: An inadequate tool to screen for GHB and ketamine in beverages.

With drug facilitated sexual assault (DFSA) being alleged in 15-20 % of sexual assault cases, drink spiking is a serious concern for several people, casting doubts over the expected safety at events in public spaces. On-site drug testing material is often touted as a solution, allowing attendees to test their drinks for the presence of certain so-called "date-rape drugs". In this manuscript, we aim to evaluate the efficiency of such a coaster device, manufactured by Drink Safe Technologies (Tallahassee, Florida, United States) and sold by Alco Prevention Canada (Laval, Québec, Canada), in detecting drink spiking by GHB and ketamine. From the onset, several generic arguments call into question the practicality of the test: limitations set by the manufacturer on drinks that can be tested, cost, waiting time, interpretation in suboptimal lighting and elevated limits of detection (LODs) compared to a standard recreational or impairing dose. More importantly, the test simply isn't effective at detecting the targeted drugs. The GHB test reagent was identified as bromocresol green using surface-enhanced Raman spectroscopy (SERS). Therefore, it does not detect GHB, but any matrix with a pH higher than 5.5. The ketamine test reagent was identified as cobalt thiocyanate, a non-specific chemical commonly used in colorimetric drug testing. Performance tests were carried with more than 22 drug-free and drug-spiked (≥125 % of the LOD) matrices, including solvent solutions (water, methanol), fixed pH solutions, and an array of popular drinks (including wine, beer, cocktails and spirits). While specificity in drug-free drinks was 100 % for both GHB and ketamine, provided that the manufacturer's limitations on drinks were respected, sensitivity in drug spiked drinks (at 150 % of the LOD) was 0 % for ketamine and between 31 % and 69 % for GHB, depending on whether one classifies inconclusive results as negatives or positives. We conclude that these coasters are an inadequate tool to screen for GHB and ketamine in beverages.

Squaring Things Up with R2: What It Is and What It Can (and Cannot) Tell You.

The coefficient of correlation (r) and the coefficient of determination (R2 or r2) have long been used in analytical chemistry, bioanalysis and forensic toxicology as figures demonstrating linearity of the calibration data in method validation. We clarify here what these two figures are and why they should not be used for this purpose in the context of model fitting for prediction. R2 evaluates whether the data are better explained by the regression model used than by no model at all (i.e., a flat line of slope = 0 and intercept $\bar y$), and to what degree. Hopefully, in the context of calibration curves, the fact that a linear regression better explains the data than no model at all should not be a point of contention. Upon closer examination, a series of restrictions appear in the interpretation of these coefficients. They cannot indicate whether the dataset at hand is linear or not, because they assume that the regression model used is an adequate model for the data. For the same reason, they cannot disprove the existence of another functional relationship in the data. By definition, they are influenced by the variability of the data. The slope of the calibration curve will also change their value. Finally, when heteroscedastic data are analyzed, the coefficients will be influenced by calibration levels spacing within the dynamic range, unless a weighted version of the equations is used. With these considerations in mind, we suggest to stop using r and R2 as figures of merit to demonstrate linearity of calibration curves in method validations. Of course, this does not preclude their use in other contexts. Alternative paths for evaluation of linearity and calibration model validity are summarily presented.

Drugs and driving prior to cannabis legalization: A 5-year review from DECP (DRE) cases in the province of Quebec, Canada.

OBJECTIVES: To examine alleged drug-impaired driving in the province of Quebec (Canada), including drug use profile amongst suspected impaired drivers, prior to recreational cannabis legalization and major modifications to the legislation regarding driving under the influence of drugs (DUID). METHODS: The Laboratoire de sciences judiciaires et de médecine légale (LSJML) performs toxicological analyses on all biological samples collected during a Drug Evaluation and Classification Program (DECP) evaluation by a Drug Recognition Expert (DRE). A targeted analysis of 137 drugs and metabolites by liquid chromatography - tandem mass spectrometry (LC-MS/MS) is systematically carried out, enhanced by gas chromatography - mass spectrometry (GC-MS) general unknown screening if deemed necessary. Data from all DECP cases analyzed from January 2014 to December 2018 was compiled and summarized. RESULTS: In the 5-year period studied, a total of 2 982 DECP cases underwent toxicological analysis. Age of the intercepted drivers varied between 14-82 years old (average 33 years old), with 79 % men and 21 % women. At least one substance with impairing potential was detected in 98 % of cases. In 89 % of cases (n = 2 640), at least one substance detected matched a category of drug suspected of causing impairment. At the other end of the spectrum, there were 270 cases (9%) where the findings did not match any of the categorie(s) suspected by the DRE, 66 negative cases (2%) and 6 incomplete evaluations. Substances most commonly detected belonged to the central nervous system (CNS) stimulants (72 %), CNS depressants (61 %) and cannabis (48 %) categories. Most prevalent substances were methamphetamine (54%), cannabis (11-nor-9-carboxy-tetrahydrocannabinol or THC-COOH, 48 %), cocaine (29 %) and gamma-hydroxybutyrate (GHB, 24 %). Polydrug consumption was common, with two or more substances with impairing potential detected in 79 % of cases. There were 113 occurrences of new psychoactive substances (NPS), the most prevalent being rolicyclidine (PCPy, 47 %), methylenedioxypyrovalerone (MDPV, 17 %), methylbenzylpiperazine (MBZP, 13 %) and flubromazolam (10 %). Drug prevalence patterns varied geographically, as well as with age and gender. Indeed, methamphetamine and GHB were more popular amongst women, whereas cannabis and cocaine were more prevalent amongst men. Cannabis and CNS stimulants were more common amongst younger drivers (14-34 years old); CNS depressants, dissociative anesthetics and non-psychoactive drugs prevalence increased with age. CONCLUSIONS: An up-to-date database of DUID cases is a powerful tool in identifying trends and threats, focusing resources and orienting research and development activities. Such a database, combined with the data presented in the current study, will be key in evaluating the impact of new regulations, i.e., recreational cannabis legalization and modifications to the DUID legislation.

A comprehensive analytical process, from NPS threat identification to systematic screening: Method validation and one-year prevalence study.

Several New Psychoactive Substances (NPS) enter the illicit drug market each year. This constant evolution of compounds to screen is challenging to law enforcement and drug chemists, and even more so to forensic toxicologists, who need to detect such compounds which might be at low concentrations in complex biological matrices. While some technological solutions are better suited than others to address such a challenge (e.g., high resolution mass spectrometry), laboratories with limited instrumental and financial resources are faced with a complex task: systematically screening for a rapidly evolving NPS panel using an accredited method run on standard equipment (e.g., liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). This work presents a solution to this challenge: a complete workflow from the detection of a regional NPS threat to its implementation in a method accredited under the ISO 17025:2017 norm. Initial LC-MS/MS method included 55 NPS and metabolites (31 Novel Synthetic Opioids (NSO), 22 NSO metabolites and 2 designer benzodiazepines). Following their identification as relevant territorial threats, flualprazolam, then isotonitazene, were added to the contingent. By relying on development aiming for maximal integration to the current analysis workflow, systematic NPS screening using this method was easily implemented. Between March 2019 and March 2020, the 5 079 forensic cases analyzed in the province of Québec (Canada) revealed a NPS positivity rate of 3.4%. While 94% involved designer benzodiazepines, 5% involved NSO. This process, combining high efficiency, simple detection technology, ISO accreditation and rapid response to new threats resulted in a four-fold increase in NPS detection.

A threshold LC-MS/MS method for 92 analytes in oral fluid collected with the Quantisal® device.

A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal® device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was developed and validated. This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g., α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal® device has polyglycol in its stabilizing buffer, but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency testing was performed, where six OF samples were tested and analytes were classified as "negative", "likely positive" or "positive" with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high throughput analysis.

Qualitative threshold method validation and uncertainty evaluation: A theoretical framework and application to a 40 analytes liquid chromatography-tandem mass spectrometry method.

Qualitative methods hold an important place in drug testing, filling central needs in screening and analyses, among others, linked to per se legislation. Nevertheless, the bioanalytical method validation guidelines do not discuss this type of method or describe method validation procedures ill-adapted to qualitative methods. The output of qualitative methods are typically categorical, binary results, such as presence/absence or above cut-off/below cut-off. As the goal of any method validation is to demonstrate fitness for use under production conditions, qualitative validation guidelines should evaluate performance based on discrete, binary results instead of the continuous measurements obtained from the instrument (e.g. area). A tentative validation guideline for threshold qualitative methods was developed by in silico modelling of measurements and derived binary results. This preliminary guideline was applied to a liquid chromatography-tandem mass spectrometry method for 40 analytes, each with a defined threshold concentration. Validation parameters calculated from the analysis of 30 samples spiked above and below the threshold concentration (false negative rate, false positive rate, selectivity rate, sensitivity rate and reliability rate) showed a surprisingly high failure rate. Overall, 13 out of the 40 analytes were not considered validated. A subsequent examination found that this was attributable to an appreciable shift in the standard deviation of the area ratio on a day-to-day basis, a previously undescribed and unaccounted-for behaviour in the qualitative threshold method validation literature. Consequently, the developed guideline was modified and used to validate a qualitative threshold method, based on the binary results for performance evaluation and incorporating measurement uncertainty.

Challenges Related to Three Cases of Fatal Intoxication to Multiple Novel Synthetic Opioids.

In the last two decades, a large increase in opioid overdose death rates has been recorded in North America. This phenomenon, related to the misuse of prescription opioids, has been dubbed an "opioids crisis". Recent years have seen the entrance of novel synthetic opioids (NSO) on the market, compounding the fatal intoxications issue. This brings several challenges for forensic toxicology laboratories: an increased number of cases, a large number of novel structurally similar compounds to include in screening analytical methods, the low concentration of drugs in biological fluids, and the challenging interpretation in the absence of sufficient literature. Three cases of fatal intoxication highlighting those challenges are presented, complete with post-mortem concentrations in cardiac blood, femoral blood and urine. Toxicological screening and quantitative analyses were performed on the biological specimens. In the first and second cases, furanylfentanyl, U-47700 and 4-anilino-N-phenethylpiperidine (4-ANPP) were detected at similar concentrations in cardiac blood. In the third case, a total of seventeen different NSO were detected. All intoxications showed a combination of NSO and other drugs. These three cases appear to be the harbinger of an increased NSO prevalence in the province of Québec, Canada.

A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC-MS-MS and GC-MS.

Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample's concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC-MS-MS), allowing the rectification of BHB biases of up to 30 µg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations.

Toxic Myocarditis Caused by Acetaminophen in a Multidrug Overdose.

We report the case of an 18-year-old woman with personality disorders who was hospitalized a few hours after suicidal ingestion of acetaminophen, quetiapine, acetylsalicylic acid, and ethanol. Twelve hours after admission, severe liver damage was evident, but the patient was stable and awaiting hepatic transplantation. Electrolytes were successfully controlled. The condition of the liver stabilized. Cardiac biomarkers then deteriorated unexpectedly. Localized ST-segment elevations were noted on electrocardiogram, but angiography ruled out myocardial infarction. A computed tomographic scan ruled out cerebral edema. The patient died of irreversible cardiac arrest 40 hours after admission. Heart failure remained unexplained, and the body underwent forensic autopsy.At autopsy, histologic findings were indicative of acute toxic myocarditis and were concluded to be caused by acetaminophen intoxication. Acetaminophen overdose is common and typically leads to liver failure requiring supportive treatment and emergency liver transplantation. Toxic myocarditis is an extremely rare complication of acetaminophen overdose. It has only been reported 4 times in the literature despite the widespread use and misuse of acetaminophen. Toxic myocarditis remains a possibility in many cases of overdose but can be overlooked in a clinical picture dominated by hepatorenal failure and encephalopathy. Clinicians and forensic pathologists should be aware of this rare potential complication.

A case of fatal idiosyncratic reaction to the designer drug 3,4-methylenedioxypyrovalerone (MDPV) and review of the literature.

The stimulant designer drug 3,4-methylenedioxypyrovalerone (MDPV) was first synthesized by Boehringer Ingelheim in 1969 and introduced on the black market in 2006. Only a small number of fatal intoxication cases have been reported in the literature, all with significant blood MDPV concentrations. In this report, we describe one fatality attributed to an idiosyncratic reaction to MDPV. The victim displayed agitation, violent behavior and delirium followed by cardiac arrest. Hyperthermia was observed at the hospital. The MDPV cardiac and femoral blood concentrations were 6 ng/mL. The presence of excited delirium syndrome and MDPV, a drug with a pharmacology similar to cocaine, leads to the conclusion that the victim suffered a fatal adverse reaction to MDPV. This is the first published case of idiosyncratic reaction to MDPV.

Procedure for the Selection and Validation of a Calibration Model II-Theoretical Basis.

In the first part of this paper (I-Description and application), an automated, stepwise and analyst-independent process for the selection and validation of calibration models was put forward and applied to two model analytes. This second part presents the mathematical reasoning and experimental work underlying the selection of the different components of this procedure. Different replicate analysis designs (intra/inter-day and intra/inter-extraction) were tested and their impact on test results was evaluated. For most methods, the use of intra-day/intra-extraction measurement replicates is recommended due to its decreased variability. This process should be repeated three times during the validation process in order to assess the time stability of the underlying model. Strategies for identification of heteroscedasticity and their potential weaknesses were examined and a unilateral F-test using the lower limit of quantification and upper limit of quantification replicates was chosen. Three different options for model selection were examined and tested: ANOVA lack-of-fit (LOF), partial F-test and significance of the second-order term. Examination of mathematical assumptions for each test and LC-MS-MS experimental results lead to selection of the partial F-test as being the most suitable. The advantages and drawbacks of ANOVA-LOF, examination of the standardized residuals graph and residuals normality testing (Kolmogorov-Smirnov or Cramer-Von Mises) for validation of the calibration model were examined with the last option proving the best in light of its robustness and accuracy. Choosing the correct calibration model improves QC accuracy, and simulations have shown that this automated scheme has a much better performance than a more traditional method of fitting with increasingly complex models until QC accuracies pass below a threshold.

Procedure for the Selection and Validation of a Calibration Model I-Description and Application.

Calibration model selection is required for all quantitative methods in toxicology and more broadly in bioanalysis. This typically involves selecting the equation order (quadratic or linear) and weighting factor correctly modelizing the data. A mis-selection of the calibration model will generate lower quality control (QC) accuracy, with an error up to 154%. Unfortunately, simple tools to perform this selection and tests to validate the resulting model are lacking. We present a stepwise, analyst-independent scheme for selection and validation of calibration models. The success rate of this scheme is on average 40% higher than a traditional "fit and check the QCs accuracy" method of selecting the calibration model. Moreover, the process was completely automated through a script (available in Supplemental Data 3) running in RStudio (free, open-source software). The need for weighting was assessed through an F-test using the variances of the upper limit of quantification and lower limit of quantification replicate measurements. When weighting was required, the choice between 1/x and 1/x2 was determined by calculating which option generated the smallest spread of weighted normalized variances. Finally, model order was selected through a partial F-test. The chosen calibration model was validated through Cramer-von Mises or Kolmogorov-Smirnov normality testing of the standardized residuals. Performance of the different tests was assessed using 50 simulated data sets per possible calibration model (e.g., linear-no weight, quadratic-no weight, linear-1/x, etc.). This first of two papers describes the tests, procedures and outcomes of the developed procedure using real LC-MS-MS results for the quantification of cocaine and naltrexone.

Determination of confidence intervals in non-normal data: application of the bootstrap to cocaine concentration in femoral blood.

Calculating the confidence interval is a common procedure in data analysis and is readily obtained from normally distributed populations with the familiar [Formula: see text] formula. However, when working with non-normally distributed data, determining the confidence interval is not as obvious. For this type of data, there are fewer references in the literature, and they are much less accessible. We describe, in simple language, the percentile and bias-corrected and accelerated variations of the bootstrap method to calculate confidence intervals. This method can be applied to a wide variety of parameters (mean, median, slope of a calibration curve, etc.) and is appropriate for normal and non-normal data sets. As a worked example, the confidence interval around the median concentration of cocaine in femoral blood is calculated using bootstrap techniques. The median of the non-toxic concentrations was 46.7 ng/mL with a 95% confidence interval of 23.9-85.8 ng/mL in the non-normally distributed set of 45 postmortem cases. This method should be used to lead to more statistically sound and accurate confidence intervals for non-normally distributed populations, such as reference values of therapeutic and toxic drug concentration, as well as situations of truncated concentration values near the limit of quantification or cutoff of a method.

Cyanide quantification in post-mortem biological matrices by headspace GC-MS.

Cyanide is a powerful chemical asphyxiant found in some forensic cases following voluntary (suicide) or involuntary ingestion (fire, accidental exposure). A quantification method for cyanide that is specifically suited to post-mortem forensic purposes was developed. Determination was performed by headspace gas chromatography coupled to mass spectrometry using a GS-GASPRO column on an HP-6890 gas chromatograph with an HP-5973N mass detector. The biological sample was treated with an internal standard, frozen, glacial acetic acid was added and the sample was then incubated at 60°C for 15 min. The headspace was sampled with a disposable syringe, and analyzed to quantify hydrogen cyanide. Isotopically labeled cyanide ((13)C(15)N) was used as the internal standard to minimize matrix effect and sampling error. The method produced an extended linear dynamic range (0.07-50 µg/mL), and a method detection limit of 0.02 µg/mL. Identical calibration curves were obtained when blood, gastric contents and aqueous solutions were used as the calibration standard matrix. This method was also successful in quantitating cyanide in gastric contents, one of the most variable biological fluids. The method has been validated and is being used for current forensic cases such as fire victims and suicides.

Enzymatic assay for GHB determination in forensic matrices.

Current procedures for the determination of gamma-hydroxybutyric acid (GHB) require time-consuming extraction and derivatization steps before chromatographic detection, making a high-throughput alternative desirable. Bühlmann Laboratories offers an enzymatic assay for the quantitative determination of GHB in urine and serum. We report the adaptation of this photometric assay to the Thermo Scientific MGC-240 analyzer and its use in the determination of GHB in forensic matrices including urine, whole blood and vitreous humour. Most matrices require only a brief centrifugation before analysis, while blood requires an additional protein precipitation step. A variety of cases (sexual assaults, impaired drivers and death investigations) have been analyzed alongside the gas chromatography-mass spectrometry (GC-MS) reference method. Correlation with the GC-MS has been found to be acceptable, with no false negatives and few false positives, although postmortem samples appear more prone to testing false positive than do antemortem samples. Simple sample preparation and high throughput allow for a significant reduction in analysis time relative to chromatographic methods. This assay is used as a screening method in our laboratory, with a quantitative GC-MS method serving for the confirmation of positive results. To our knowledge, this represents the first evaluation of an enzymatic assay for GHB in a forensic context.