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.

Synthesis and characterization of barbarin, a possible source of unexplained aminorex identifications in forensic science.

Aminorex is a US DEA Schedule 1 controlled substance occasionally detected in racing horses. A number of aminorex identifications in sport horses were thought to have been caused by exposure to plant sources of aminorex. Glucobarbarin, found in plants of the Brassicaceae family, has been suggested as a potential proximate chemical source by being metabolized in the plant or the horse to aminorex. In Brassicaceae, glucobarbarin is hydrolyzed by myrosinase to yield barbarin, which serves as an insect repellant and/or attractant and is structurally related to aminorex. The synthesis, purification, and characterization of barbarin is now reported for use as a reference standard in aminorex related research concerning equine urinary identifications of aminorex and also for possible use in equine administration experiments. Synthesis of barbarin was performed via ring closure between phenylethanolamine and carbon disulfide in tetrahydrofuran with the catalyst pyridine under reflux. The reaction yielded a white crystalline substance that was purified and chemically characterized as barbarin for use as a Certified Reference Standard or for studies related to equine aminorex identification.

Enantioselective simultaneous analysis of selected pharmaceuticals in environmental samples by ultrahigh performance supercritical fluid based chromatography tandem mass spectrometry.

In order to assess the true impact of each single enantiomer of pharmacologically active compounds (PACs) in the environment, highly efficient, fast and sensitive analytical methods are needed. For the first time this paper focuses on the use of ultrahigh performance supercritical fluid based chromatography coupled to a triple quadrupole mass spectrometer to develop multi-residue enantioselective methods for chiral PACs in environmental matrices. This technique exploits the advantages of supercritical fluid chromatography, ultrahigh performance liquid chromatography and mass spectrometry. Two coated modified 2.5 µm-polysaccharide-based chiral stationary phases were investigated: an amylose tris-3,5-dimethylphenylcarbamate column and a cellulose tris-3-chloro-4-methylphenylcarbamate column. The effect of different chromatographic variables on chiral recognition is highlighted. This novel approach resulted in the baseline resolution of 13 enantiomers PACs (aminorex, carprofen, chloramphenicol, 3-N-dechloroethylifosfamide, flurbiprofen, 2-hydroxyibuprofen, ifosfamide, imazalil, naproxen, ofloxacin, omeprazole, praziquantel and tetramisole) and partial resolution of 2 enantiomers PACs (ibuprofen and indoprofen) under fast-gradient conditions (<10 min analysis time). The overall performance of the methods was satisfactory. The applicability of the methods was tested on influent and effluent wastewater samples. To the best of our knowledge, this is the first feasibility study on the simultaneous separation of chemically diverse chiral PACs in environmental matrices using ultrahigh performance supercritical fluid based chromatography coupled with tandem mass spectrometry.

Aminorex associated with possible idiopathic pulmonary hypertension in a cocaine user.

The conversion of levamisole to aminorex in horses was first described in 2009 and, for the first time, confirmed in humans two years later by our laboratory. Aminorex and levamisole interfere with serotonin metabolism and both are proven cause of potentially fatal idiopathic pulmonary hypertension (IPH). Because most of the world's seizures of illicit cocaine is now contaminated with levamisole, this raises the possibility that users of levamisole adulterated cocaine users may be at risk for IPH. Here we describe the first case of IPH in a user of levamisole-contaminated cocaine. Levamisole and aminorex were both identified and quantified in hair and other biological specimens by means gas chromatography/mass spectrometry system (levamisole: urine, 75.05ng/mL; blood, 15.05ng/mL; brain, >0.15ng/g; liver, >0.15ng/g; hair, 12.15ngmg; aminorex: urine, 38.62ng/mL; blood, 8.92ng/mL, brain >0.15ng/g; liver, 0.15ng/g; hair 7.35ng/mg; cocaine, benzoylecgonine, morphine, 6-acetylmorphine, methadone, 2-ethylidine-1, 5-dimetil-3, 3 diphenylpyrrolidine were also detected). Moreover histological changes associated with IPH were observed in the lung. As IPH produces relatively non-specific symptoms in its early stages, this index case may serve as a harbinger of many more cases to come. It should also alert clinicians to the possibility that their patient may be suffering from this relatively rare disorder.

Determination of levamisole, aminorex, and pemoline in plasma by means of liquid chromatography-mass spectrometry and application to a pharmacokinetic study of levamisole.

Levamisole is an anti-helminthic drug and gained forensic interest after it was found that it was used as a cocaine adulterant. A liquid chromatography-mass spectrometry (LC-MS) method for the determination of levamisole and its metabolite aminorex in human plasma is described. Selectivity is given; calibration curves were linear within a calibration range of 1 ng/mL-500 ng/mL. Limits of detection and quantification (LODs, LOQs) were 0.85 ng/mL for levamisole and 0.09 ng/mL, and 0.34 ng/mL for aminorex, respectively. Precision data was in accordance with the GTFCh guidelines. The validated method was successfully applied to study the pharmacokinetics of levamisole after administration of 100 mg of levamisole orally. Levamisole could be detected up to 36 h after ingestion in serum, while aminorex never exceeded the LOQ. A one-compartment model best described levamisole pharmacokinetics. The following parameters were calculated: ka = 1.2 [1/h], CL/F = 52 l/h, V/F = 347 l, f (renal) = 0.0005, t ½ = 2.0 h, AUC = 1923 ng/mL*h, cmax = 214 ng/mL, tmax = 1.98 h. Levamisole could be quantified in 42.5% of cocaine--positive plasma samples (2.2 to 224 ng/mL). Aminorex was positive in only 11.3% of the cases; however, it was never found higher than the LOQ. Pemoline, another stimulant detected in horse urine samples after administration of levamisole, was not found either in serum or in urine of this pharmacokinetic study. In post-mortem cases, levamisole and aminorex could be detected in femoral blood and the urine of cocaine users. Pemoline was not detected.

Aminorex and rexamino as metabolites of levamisole in the horse.

Administration studies of levamisole in horses were carried out using two different levamisole preparations, namely, levamisole hydrochloride oral bolus and levamisole phosphate injectable solution. These preparations were analysed in detail for the presence of aminorex-like impurities. Both levamisole preparations were found to contain 1-(2-mercaptoethyl)-4-phenyl-2-imidazolidinone (I) and 4-phenyl-2-imidazolidinone (II) as degradation impurities, but neither aminorex nor rexamino was detected in these preparations. After the administration of these preparations to horses, aminorex, rexamino, in addition to levamisole and compound II, were detected in post-administration urine and plasma samples, among which compound II was found to have the longest detection time. Administration study of compound II was then performed on another horse to investigate whether it could be a metabolic precursor of aminorex and/or rexamino. However, no aminorex and rexamino was detected in the post-administration samples, suggesting that compound II was not a metabolic precursor of aminorex or rexamino. A metabolite (III) of compound II, tentatively identified to be a hydrolysis product of compound II, was observed instead. It has been established unequivocally that the normal use of levamisole products in horses can lead to the presence of aminorex, rexamino and 4-phenyl-2-imidazolidinone (II) in their urine and blood samples. As compound II has the longest detection time, the detection of aminorex (and in some cases rexamino) in some of the official samples from racehorses can be ascribed to the use of levamisole products as long as compound II is also present as a marker. These findings should be of direct relevance to the investigation of some of the cases of aminorex detection in official doping control samples from racehorses.