Gamma‐hydroxybutyric acid (GHB) is a sedative drug used in drug‐facilitated crimes. Its detection window is very short. GHB undergoes intensive phase I metabolism to organic acids (glycolic acid, succinic acid, dihydroxybutyric acids). These could be potential analytical targets to broaden the detection window. The aim of the present study was to enable the detection of endogenous levels of these metabolites in biological samples (blood and urine). A gas chromatographic–mass spectrometric method using liquid–liquid extraction and derivatization with N‐methyl‐N‐tri‐methylsilyltrifluoracetamide was developed for the quantification. Validation results were consistent with international guidelines, and the method was able to quantify endogenous levels of the substances in both urine and blood. Endogenous concentrations were shown to be <0.03–4.92 mg/L for glycolic acid, <0.03–1.28 mg/L for GHB, <0.28–18.1 mg/L for succinic acid, <0.12–1.38 mg/L for 2,4‐dihydroxybutyric acid, and <0.13–2.59 mg/L for 3,4‐dihydroxybutyric acid in serum samples of 101 volunteers. Urinary endogenous concentrations were shown to be 1.30–400 mg/L for glycolic acid, <0.03–1.94 mg/L for GHB, 1.17–2.73 mg/L for succinic acid, 0.72–26.2 mg/L for 2,4‐dihydroxybutyric acid, and 1.88–122 mg/L for 3,4‐dihydroxybutyric acid in urine samples of 132 volunteers. These endogenous concentrations represent a basis to which concentrations after the intake of GHB can be compared to in order to prove the intake of this substance.

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Phenyltetrahydroimidazothiazole (PTHIT, tetramisole) is the most frequently used adulterant of cocaine and exists in the two enantiomeric forms levamsiole (S) and dexamisole (R). Existing studies show diverse fractions of samples containing enantiopure levamsiole, levamisole-enriched mixtures, and racemic tetramisole as adulterant. However, blood samples have never been enantioselectively tested for PTHIT. Because enantiomers are usually metabolized stereoselectively, chiral analysis of blood samples can help estimate the time of drug use, provided that a racemic substance is ingested. Therefore, an enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed using a chiral column. Validation of the method was carried out for methanolic substance samples as well as serum samples and showed satisfactory selectivity, sensitivity, linearity (0.05-100 ng/mL), precision, and accuracy; 151 cocaine samples seized in Germany between 2018 and 2021 were analyzed. Most (94%, n = 48) of the 51 PTHIT-positive samples contained racemic tetramsiole, whereas there were two samples containing levamisole-enriched mixtures and one sample containing nearly enantiopure levamisole. Furthermore, 157 cocaine and/or benzoylecgonine-positive forensic serum samples were tested with cocaine-positive samples showing the highest frequency of PTHIT detection (43%). All positive samples contained either dexamisole alone or (R)/ (S)-concentration ratios >1 (1.05-70.6). Finally, a self-administration study was conducted with three subjects taking 10 mg of racemic tetramisole each. Although peak concentrations and corresponding times did not differ significantly between the enantiomers, dexamisole showed significantly longer apparent elimination half-lives (7.02-10.0 h) than levamisole (2.87-4.77 h). The resulting steadily increasing (R)/(S)-ratios can therefore be helpful in estimating the time of cocaine consumption.

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Over the last two decades, misuse of 4-fluoroamphetamine (4-FA) became an emerging issue in many European countries. Stimulating effects last for 4–6 hours and can impact psychomotor performance. The metabolism of amphetamine-type stimulants is stereoselective and quantification of (R)- and (S)-enantiomers has been suggested for assessing time of use. To date no data on enantioselective pharmacokinetics is available for 4-FA in serum samples. An enantioselective liquid chromatography−tandem mass spectrometry (LC–MS-MS) method was developed using a chiral Phenomenex® Lux 3 μm AMP column. Validation of the method showed satisfactory selectivity, sensitivity, linearity (0.5–250 ng/mL), precision and accuracy. Recreational stimulant users orally ingested two doses (100 mg, n=12, and 150 mg, n=5) of 4-FA. Blood samples were drawn prior to application and over a period of 12 hours after ingestion and analyzed for 4-FA enantiomers. Peak concentrations and corresponding times did not differ significantly between the enantiomers (mean (R)/(S)-ratio at tmax 1.05, 0.85–1.16). With mean 12.9 (8.3–16.1) hours, apparent elimination half-lives (t1/2) were significantly (p &lt; 0.01) longer for (R)-4-FA than for (S)-4-FA (6.0 hours; range 4.4–10.2 hours) and independent of the dose given. Over time, (R)/(S)-concentration-ratios were linearly increasing in all subjects to maximum ratios of 2.00 (1.08–2.77) in the last samples (after 12 hours). The slopes of the (R)/(S)-ratio exhibited marked inter-individual differences (0.023 to 0.157 h-1, mean 0.095 h-1). Ratios higher than 1.60 only appeared earliest after a minimum of 6 hours and therefore suggest the absence of acute drug effects. Different elimination half-lives of enantiomers lead to constantly increasing (R)/(S)-concentration-ratios. Consequently, ratios of 4-FA enantiomers in serum are a promising indicator for assessment of the time of drug consumption.