Chiral pharmacokinetics of tetramisole stereoisomers—Enantioselective quantification of levamisole and dexamisole in serum samples from users of adulterated cocaine

Abstract

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.

Analysis of tetramisole metabolites – Is “Aminorex” found in forensic samples of cocaine users actually 4‐phenyl‐2‐imidazolidinone?

“…In our previous study, the chiral pharmacokinetics of dexamisole and levamisole were examined. 6 Therefore, an adulterated cocaine consumption was simulated, and a 10 mg dose of racemic tetramisole hydrochloride was administered nasally using a straw by three male healthy volunteers (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38) were re-analysed for metabolites approx. 15 months after the initial analysis.…”

“…Moreover, serum samples of cocaine users (cocaine ≥ 1 ng/ml and/or benzoylecgonine ≥ 25 ng/ml), were analysed for the presence of dexamisole and levamisole in our previous study. 6 The samples were…”

“…During method development, it was noted that 4-phenyl-2-imidazolidinone showed a weak and unreproducible response after the solid phase extraction (SPE) sample preparation technique used in our previous study. 6 Therefore, different SPE techniques routinely used in our laboratory, including C18-phases as well as mixed-mode-phases consisting of a non-polar C8 sorbent and a strong cation exchanger, were tested for their extraction efficiency for the analytes p-hydroxy-PTHIT and 4-phenyl-2-imidazolidinone. Moreover, a simple protein precipitation omitting an SPE step and the effect of evaporation under a steam of nitrogen were evaluated.…”

“…4 Regarding the enantiomeric composition of PTHIT-tainted cocaine, PTHIT is currently predominantly added in its racemic form as tetramisole, while levamisole or non-racemic mixtures with an excess of levamisole are less frequently found. 5,6 In general, little is known about the metabolism of PTHIT in humans. Although there is evidence suggesting an excessive hepatic metabolism, it has been studied only rudimentarily using levamisole.…”

“…This entails the potential of mass-spectrometric misinterpretation and may be a reason for the contradictory findings in the detection of aminorex in forensic sample material. 10 After having quantified dexamisole and levamisole in serum samples of cocaine users and having elucidated the chiral pharmacokinetics of tetramisole enantiomers after a controlled nasal application, 6 the aim of this study was to detect PTHIT metabolites in human blood…”