Analysis of tetramisole metabolites - Is "Aminorex" found in forensic samples of cocaine users actually 4-phenyl-2-imidazolidinone?

Phenyltetrahydroimidazothiazole (PTHIT, tetramisole) is a common adulterant in cocaine samples. Little is known about its human metabolism. p-hydroxy-PTHIT has long been the only proven phase-I-metabolite. Another putative metabolite is the stimulant aminorex. However, data on its analytical proof is rare and contradictory. Even less known is its constitutional isomer 4-phenyl-2-imidazolidinone which has only been proven in animal samples so far. The aim of the study was to get insight into the metabolism of PTHIT after controlled nasal uptake of PTHIT and in real forensic cocaine/benzoylecgonine-positive samples. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated for quantification of 4-phenyl-2-imidazolidinone and p-hydroxy-PTHIT (LOQ 0.05 ng/ml each). Selectivity was ensured for 4-phenyl-2-imidazolidinone and aminorex (LOD 0.05 ng/ml). After controlled nasal uptake of tetramisole (10 mg, n = 3) a shorter half-life for p-hydroxy-PTHIT (3.4-5.8 h) was determined than for 4-phenyl-2-imidazolidinone (14.0-15.9 h). p-hydroxy-PTHIT (33%) and 4-phenyl-2-imidazolidinone (51%) were also detected in serum samples from cocaine users tested previously positive for PTHIT (n = 73). Aminorex was never detected. The potential of misinterpreting 4-phenyl-2-imidazolidinone as aminorex was tested using a gas chromatography-mass spectrometry (GC-MS) method used in the literature and an in-house liquid chromatography-time-of-flight mass spectrometry (LC-QTOF) screening-method. Using GC-MS the analysed bis-trimethylsilyl-derivatives cannot be differentiated due to co-elution. Both substances were chromatographically separated using the LC-QTOF method, but library comparison workflows misinterpreted 4-phenyl-2-imidazolidinone as aminorex. It seems likely that aminorex, which was allegedly identified as a metabolite of PTHIT in samples of cocaine users in previous studies, is in fact 4-phenyl-2-imidazolidinone.

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

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.