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.

Determination of the enantiomeric composition of amphetamine, methamphetamine and 3,4-methylendioxy-N-methylamphetamine (MDMA) in seized street drug samples from southern Germany.

Amphetamine (speed), methamphetamine (crystal meth), and 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) represent the most frequently abused amphetamine-type stimulants (ATS). Differences in pharmacological potency and metabolism have been shown for the enantiomers of all three stimulants. Legal consequences in cases of drug possession may also differ according to the German law depending on the enantiomeric composition of the seized drug. Therefore, enantioselective monitoring of seized specimens is crucial for legal and forensic casework. Various kinds of samples of amphetamine (n = 143), MDMA (n = 94), and methamphetamine (n = 528) that were seized in southern Germany in 2019 and 2020 were analyzed for their chiral composition using different chromatographic methods. Whereas all samples of amphetamine and MDMA were racemic mixtures, the chiral composition of the methamphetamine specimens was diverse. Although the vast majority (n = 502) was present as (S)-methamphetamine, also specimens containing pure (R)-methamphetamine (n = 7) were found. Furthermore, few samples (n = 8) were of racemic nature or contained non-racemic mixtures of both enantiomers (n = 10). Because methamphetamine appears in varying enantiomeric compositions, any seizure should be analyzed using an enantioselective method. Amphetamine and MDMA, on the other hand, currently appear to be synthesized exclusively via racemic pathways and are not chirally purified. Nevertheless, regular monitoring of the chiral composition should be ensured.

Enantioselective Quantification of Amphetamine and Metabolites in Serum Samples: Forensic Evaluation and Estimation of Consumption Time.

In forensic toxicology, amphetamine intoxications represent one of the most common case groups and present difficult questions for toxicologists. Estimating the time of consumption and the current influence of the stimulant is particularly difficult when only total amphetamine concentrations are considered. Stereoselective analysis and the consideration of metabolites can provide valuable information to facilitate interpretation. An enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of amphetamine, norephedrine and 4-hydroxyamphetamine was developed. Validation showed satisfactory selectivity, sensitivity, linearity (0.5-250 ng/mL), precision and accuracy for all enantiomers. The method was applied to a collective of 425 forensic serum samples and 30 serum samples from psychiatric inpatients stating their last time of amphetamine consumption. Norephedrine and 4-hydroxyamphetamine were detected more frequently at higher amphetamine concentrations and at lower amphetamine (R)/(S) concentration ratios, possibly indicating recent consumption. Mean (R)/(S) ratio of amphetamine was 1.14, whereas higher ratios (mean 1.36) were found for amphetamine concentrations below 100 ng/mL. The (R)/(S) ratios of psychiatric inpatients significantly correlated with the reported time intervals to last consumption. The use of amphetamine (R)/(S) ratios and the simultaneous detection of metabolites are promising factors that can facilitate estimation of consumption time and current impairment.

Enantioselective determination of plasma protein binding of common amphetamine-type stimulants.

Amphetamine-type stimulants (ATS) like amphetamine ('speed'), methamphetamine ('crystal meth') and 3,4-methylenedioxy-N-methylamphetamine (MDMA, 'ecstasy') represent some of the most frequently abused drugs worldwide. Another less frequently abused ATS is 4-fluoroamphetamine (4-FA). The enantiomers of these four compounds exhibit different pharmacokinetic and pharmacodynamic properties. According to the free drug theory, the pharmacological properties of a substance are dependent on its plasma protein binding (PPB). However, data on PPB of stimulant enantiomers in humans are rare or non-existent. Human plasma samples were spiked with racemic mixtures of the stimulants and subjected to ultrafiltration to extract the unbound fraction. Enantioselective liquid chromatography - tandem mass spectrometry (LC-MS/MS) methods were applied using a chiral Phenomenex® Lux3 µm AMP column. Method validation showed satisfactory selectivity, linearity (0.5 250 ng/mL), accuracy and precision. Enantiomers were quantified before and after ultracentrifugation to determine PPB. For all analytes, low to medium plasma protein binding was found. For (R)-amphetamine a slightly but significantly higher PPB was found compared to the (S)-enantiomer (31.7 % vs 29.0 %). (R)-MDMA also showed only slightly but significantly significantly higher PPB than (S)-MDMA, although the mean difference was negligible (21.6 % vs 21.3 %). For the enantiomers of methamphetamine and 4-FA, no significant differences in PPB were found. In summary, there were no or only minor differences in PBB for the enantiomers of all investigated compounds. The different pharmacological properties of the stimulant enantiomers can therefore not be explained by differences in PPB.

Detectability of cannabinoids in the serum samples of cannabis users: Indicators of recent cannabis use? A follow-up study.

Forensic toxicologists are frequently required to predict the time of last cannabis consumption. Several studies suggested the utility of minor cannabinoids as indicators of recent cannabis use. Because several factors influence blood cannabinoid concentrations, the interpretation of serum cannabinoid concentrations remains challenging. To assess the informative value of serum cannabinoid levels in cannabis users (in total N = 117 patients, including 56 patients who stated an exact time of last cannabis use within 24 h before blood sampling), the detectability of cannabinoids, namely, delta-9-tetrahydrocannabinol (delta-9-THC), 11-hydroxy-delta-9-THC, 11-nor-9-carboxy-delta-9-THC, cannabichromene (CBC), cannabidiol (CBD), cannabinol (CBN), cannabidivarin, tetrahydrocannabivarin, cannabigerol (CBG), cannabicyclol, delta-8-THC, tetrahydrocannabinolic acid A, cannabichromenic acid, cannabidiolic acid (CBDA), cannabigerolic acid, cannabicyclolic acid (CBLA), 11-nor-9-carboxy-THCV (THCVCOOH), and 11-nor-CBN-9-COOH, was investigated. Excluding CBDA and CBLA, all investigated cannabinoids were detected in at least one analyzed sample. The interval between cannabis consumption and sample collection (reported by the patients) was not correlated with cannabinoid concentrations. Minor cannabinoids tended to be more easily detected in samples obtained shortly after consumption. However, some samples tested positive for minor cannabinoids despite an interval of several hours or even days between consumption and sampling (according to patients' statements). For instance, CBC, CBG, THCVCOOH, CBD, and CBN in certain cases could be detected more than 24 h after the last consumption of cannabis. Thus, findings of minor cannabinoids should always be interpreted with caution.

Chiral Serum Pharmacokinetics of 4-Fluoroamphetamine after Controlled Oral Administration: Can (R)/(S)-Concentration Ratios Help in Interpreting Forensic Cases?

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; 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 < 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 interindividual differences (0.023-0.157 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.

Chromatographic separation of R-(-)/S-(+)-enantiomers of amphetamine and methamphetamine: differentiation between single methamphetamine consumption and co-consumption with amphetamine using enantioselective quantitative LC-MS/MS analysis.

The differentiation between single methamphetamine consumption and co-consumption with amphetamine is difficult, however possible by enantioselective analysis due to different preferred synthesis pathways of both substances. We quantified (R)-(-) and (S)-(+)-enantiomers of methamphetamine and amphetamine by a fast liquid chromatographic tandem-mass spectrometric method using a Lux® 3-µm AMP 150 × 3.0 mm analytical column after simple protein precipitation with methanol. Method validation for quantitative detection showed limits of quantification < 5 ng/mL, linearity in a range between 5 and 300 ng/mL and bias and imprecision data < 15%. Overall, 134 plasma samples of police cases from the German regions of Franconia and Northrhine-Westphalia were analyzed for the enantiomers of methamphetamine and amphetamine. In 28 cases, the intake of racemic illicit amphetamine could be demonstrated; (R)-(-) / (S)-(+)-amphetamine concentration ratios in these cases were between 1.38 and 4.50 with most of the ratios being < 2.0. These ratios were compared to a subgroup of 25 consumers with a co-consumption of (S)-(+)-methamphetamine and racemic amphetamine detected by the qualitative proof of (R)-(-)-amphetamine but also by (R)-(-) / (S)-(+)-amphetamine concentration ratios (< 1 in 11 of 25 cases). Within our collective of 106 plasma samples after methamphetamine use, 25 samples showed co-consumption with amphetamine which shows that co-consumption of both stimulants is not a rare scenario. Furthermore, we could show that if non-stereoselective methods are used and the concentration ratio of total methamphetamine/total amphetamine is determined, a reliable estimation of co-consumption is not possible.

Chromatographic separation of R/S-enantiomers of amphetamine and methamphetamine: Pathways of methamphetamine synthesis and detection in blood samples by qualitative enantioselective LC-MS/MS analysis.

Methamphetamine can be synthesized either enantiopure or in its racemic form. We separated (R)- and (S)-enantiomers of methamphetamine and amphetamine by a fast LC-MS/MS-method using a Lux® 3µm AMP 150×3.0mm analytical column after simple protein precipitation with methanol. Sufficient resolution could be achieved. Method validation for qualitative detection showed limits of quantification <5ng/mL while only little (maximum 14.5%) ion suppression could be shown. Stability in the processed sample could be achieved using isotopically labelled internal standards. Plasma samples of police cases from the german regions of Franconia and Northrhine revealed that in the majority of 106 tested samples (>99%) only (S)-methamphetamine was detected which leads to the conclusion that, in Germany, predominantly enantiopure (S)-methamphetamine is consumed which is synthesized via (1R,2S)-ephedrine or (1S,2S)-pseudoephedrine. However, racemic methamphetamine seems also to be on the market.