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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.

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

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

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