Search

Purpose N-tert-Butoxycarbonylmethamphetamine (BocMA), a masked derivative of methamphetamine (MA), converts into MA under acidic condition and potentially acts as a precursor to MA following ingestion. To investigate the metabolism and excretion of BocMA, metabolism tests were conducted using human liver microsomes (HLM), rat liver microsomes (RLM) and rat. Methods BocMA metabolites were analyzed after 1000-ng/mL BocMA incubation with microsomes for 3, 8, 13, 20, 30, and 60 min. Rats were administered intraperitoneal injections (20 mg/kg) of BocMA and their urine was collected in intervals for 72 h. Metabolites were detected by liquid chromatography–tandem mass spectrometry with five authentic standards. Results Several metabolites including 4-hydroxy-BocMA, N-tert-butoxycarbonylephedrine and N-tert-butoxycarbonyl-cathinone were detected for HLM and RLM. In the administration test, three glucuronides of hydroxylated metabolites were detected. The total recovery values of BocMA and the metabolites during the first 72 h accounted for only 0.3% of the administered dose. Throughout the microsomal and administration experiments, MAs were not detected. Conclusion Hydroxylation, carbonylation and N-demethylation were proposed as metabolic pathways. However, BocMA and phase I metabolites were hardly detected in urine. This study provides useful information to interpret the possibility of BocMA intake as the cause of MA detection in biological sample.

The influence of lipophilicity and functional groups of synthetic cannabinoids (SCs) on their blood concentrations and urinary excretion has been studied by analyzing blood and urine specimens sampled from drivers who were involved in a car crashes under the influence of SCs. A total of 58 specimens (26 urine and 31 blood specimens), sampled within 13h of the occurrence, were analyzed by liquid chromatography-tandem mass spectrometry. Fifteen SCs were detected in those specimens; the SCs detected were categorized as follows: Class 1, Naphthoyl/Benzoyl indole (EAM2201 and three other analogs); Class 2, Indole-3-carboxylate/carboxamide containing naphthol/quinol (5F-PB-22 and four other analogs); and Class 3, Indazole-3-carboxamide containing valine/tert-leucine derivative (5F-AMB and five other analogs). The calculated lipophilicity index log P, the octanol/water participation coefficient, of those SCs in Classes 1, 2, and 3 ranged between 5.01-8.14, 5.80-6.74 and 2.29-3.81, respectively. Class 3 SCs were detectable in 12 out of 13 urine specimens, but those in Classes 1 and 2 were not detected in urine. Our analytical results indicated that the boundary line for their detectability in urine lies between log P 4 and 5. The blood concentrations of Class 3 SCs varied widely (0.0036-31ng/ml) depending on their log P, while much smaller variation was observed among those in Class 2 (0.10-5.0ng/ml).

Little information is available on the amounts of hypnotics incorporated into hair after a single administration and about effective analytical procedures to document cases like drug-facilitated sexual assaults. To obtain basic information, single-hair specimens from a volunteer who took a single dose of 10-mg zolpidem (ZP) were analyzed by a newly established liquid chromatography–tandem mass spectrometry procedure, using a one-pot pulverization extraction method. The detection limit of ZP was 50 fg/2-cm single hair, and ZP in each segment was determined for the single black hair specimens (n = 15). ZP was detectable in 14 hairs (positive for all the proximal 0–2 cm segments, negative for all 2–4 cm segments), but was not detected in a single hair (probably in the telogen stage). The amounts of ZP detected in each positive 2-cm segment of single hair ranged from 27 to 63 pg (average 43 pg). The estimated total incorporation of ZP in the scalp hair (black hair ~110,000 strands) was about 4.7 μg, which corresponds to about 0.06 % of the single 10-mg dose (8.03 mg as free ZP). In addition, the direct detection of single-dose ZP incorporated in hair and its imaging were successfully achieved by matrix-assisted laser desorption ionization-mass spectrometry. It is suggested that a combination of these methodologies will provide the highest-level evidence to document such exposure to a hypnotic drug.

Urinary metabolites of α-pyrrolidinovalerophenone (α-PVP) in humans were investigated by analyzing urine specimens obtained from abusers. Unambiguous identification and accurate quantification of major metabolites were realized using gas chromatography–mass spectrometry and liquid chromatography-tandem mass spectrometry with newly synthesized authentic standards. Two major metabolic pathways were revealed: (1) the reduction of the β-keto moiety to 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP, diastereomers) partly followed by conjugation to its glucuronide, and (2) the oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)valerophenone (2″-oxo-α-PVP) via the putative intermediate α-(2″-hydroxypyrrolidino)valerophenone (2″-OH-α-PVP). Of the metabolites retaining the structural characteristics of the parent drug, OH-α-PVP was most abundant in most of the specimens examined.

Mass spectrometric differentiation of the six isomers of mono-methoxyethylamphetamines (MeO-EAs) and mono-methoxydimethylamphetamines (MeO-DMAs) by gas chromatography–electron ionization–tandem mass spectrometry (GC–EI–MS–MS) was investigated. Based on their EI-mass spectra, the fragment ions at m/z 121 and 72 were selected as precursor ions for their regioisomeric and structurally isomeric differentiation, respectively. Collision-induced dissociation provides intensity differences in product ions among the isomers, enabling mass spectrometric differentiation of the isomers. Furthermore, high reproducibility of the product ion spectra at the optimized collision energy was confirmed, demonstrating the reliability of the method. To our knowledge, this is the first report on mass spectrometric differentiation of the six isomers of MeO-EAs and MeO-DMAs by GC–EI–MS–MS. Isomeric differentiation by GC–EI–MS–MS has a high potential to discriminate isomers of newly encountered designer drugs, making GC–MS–MS a powerful tool in the forensic toxicology field.

Cathinone-derived designer drugs have recently grown to be popular as drugs of abuse. 3,4-Dimethylmethcathinone (DMMC) has recently been abused as one of the alternatives to controlled cathinones. In the present study, DMMC and its major metabolites, 3,4-dimethylcathinone (DMC), 1-(3,4-dimethylphenyl)-2-methylaminopropan-1-ol (β-OH-DMMC, diastereomers), and 2-amino-1-(3,4-dimethylphenyl)propan-1-ol (β-OH-DMC, diastereomers), have been identified and quantified in a DMMC user’s urine by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry using newly synthesized authentic standards. Other putative metabolites including oxidative metabolites of the xylyl group and conjugated metabolites have also been detected in urine. The identified and putative phase I metabolites indicated that the metabolic pathways of DMMC include its reduction of the ketone group to the corresponding alcohols, N-demethylation to the primary amine, oxidation of the xylyl group to the corresponding alcohol and carboxylate forms, and combination of these steps. Concentrations of the identified metabolites were found to increase slightly after enzymatic hydrolysis, suggesting that these compounds are partially metabolized to the respective conjugates.

In order to investigate the incorporation of drugs into hair, matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MS/MS) imaging was performed on the longitudinal sections of single scalp hair shafts sampled from volunteers after a single oral administration of methoxyphenamine (MOP), a noncontrolled analogue of methamphetamine. Hair specimens were collected by plucking out with the roots intact, and these specimens were prepped by an optimized procedure based on freeze-sectioning to detect the drug inside the hair shaft and hair root. Time-course changes in the imaging results, with confirmatory quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for each 1-mm segment of single hair strands, revealed a substantial concentration of the drug first onto the hair bulbs after ingestion, while only a small portion appeared to be incorporated into the hair matrix, forming a 2-3 mm distinctive drug band with tailing. Comparable amount of the drug also appeared to be incorporated into the keratinized hair shaft in the upper dermis zone, forming another distinct drug band of about 2 mm, which both moved toward the distal side, following the strand's growth rate. These findings provide forensically crucial information: there are two major drug incorporation sites, at least for MOP, which cause overlap of the recordings and deteriorates its chronological resolution down to about 11 days or perhaps longer.

Urinary phase I metabolites of α-pyrrolidinobutiophenone (α-PBP) in humans were investigated by analyzing urine specimens obtained from drug abusers. Unequivocal identification and accurate quantification of major metabolites were realized using gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry with newly synthesized authentic standards. Two major phase I metabolic pathways were revealed: (1) reduction of the ketone group to 1-phenyl-2-(pyrrolidin-1-yl)butan-1-ol (OH-α-PBP, diastereomers) partly followed by conjugation to its glucuronide and (2) oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)butiophenone (2″-oxo-α-PBP) via the putative intermediate α-(2″-hydroxypyrrolidino)butiophenone (2″-OH-α-PBP). Of the phase I metabolites retaining the structural characteristics of the parent drug, OH-α-PBP was the most abundant in all specimens examined. Comparison of the phase I metabolism of α-PBP and α-pyrrolidinovalerophenone (α-PVP) suggested a relationship between the aliphatic side chain length and the metabolic pathways in α-pyrrolidinophenones: the shorter aliphatic side chain (1) led to more extensive metabolism via reduction of the ketone group than via the oxidation at the 2″-position of the pyrrolidine ring and (2) influenced the isomeric ratio of a pair of diastereomers.

A rapid and convenient extraction method has been developed for the determination of various drugs and metabolites of forensic interest in blood by modifying the dispersive solid-phase extraction method "QuEChERS". The following 13 analytes with various chemical properties were used for the method development and its validation: amphetamine, methamphetamine, zolpidem, the carboxylate-form major metabolite of zolpidem M-1, flunitrazepam, 7-aminoflunitrazepam, phenobarbital, triazolam, α-hydroxytriazolam, brotizolam, α-hydroxybrotizolam, chlorpromazine, and promethazine. The modification of the QuEChERS method includes the use of relatively large amounts of inorganic salts in order to coagulate blood, which allows easy isolation of the organic extract phase. A combination of 100 mg anhydrous magnesium sulfate as a dehydrating agent, 50mg sodium chloride as a salting-out agent, and 500 μL acetonitrile containing 0.2% acetic acid as the organic solvent provided the optimum conditions for processing a 100 μL whole blood sample. The recoveries of the analytes spiked into whole blood at 0.5 μg/mL ranged between 59% and 93%. Although the addition of the graphitized carbon Envi-carb for cleanup decreased the recoveries of zolpidem and its carboxylate-form metabolite M-1, it was very effective in avoiding interferences by cholesterol. The present method can provide a rapid, effective, user-friendly, and relatively hygienic method for the simultaneous extraction of a wide range of drugs and metabolites in whole blood specimens.