Metabolism of N-ethylhexedrone and buphedrone: An in vivo study in mice using HPLC-MS/MS.

N-ethylhexedrone (NEH) and buphedrone (BUPH) are synthetic drugs structurally related to natural cathinone. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS), which have caused major concern in scientific and forensic communities over the past years, due to their widespread consume. Thus, there is a constant need for monitoring the use of these new substances and gather knowledge on their metabolism and excretion profiles, in order to try to identify markers of NPS consumption. This study aimed at the identification and quantification of NEH, BUPH and selected phase I metabolites using HPLC-MS/MS. NEH, BUPH and some related metabolites were synthesized in-house and quantified in 24 h mice urine, following single dose administration of each drug (64 mg kg-1, i.p.). NEH and BUPH were quantified in mice urine at 58.3 ± 14.4 and 146.2 ± 14.9 µg mL-1, respectively. Similar metabolic pathways were observed for both drugs. Among the metabolites studied, the most excreted ones derived from N-dealkylation of either NEH or BUPH (at around 80 µg mL-1 of urine). Other metabolites resulting from ketone reduction and ketone reduction combined with N-dealkylation or 4-aryl hydroxylation (detected for the first time in non-ring substituted SC) were also identified and quantified. Urine samples were screened using liquid chromatography-high resolution mass spectrometry and various phase II metabolites, including N-acetylated, glucuronides and dicarboxylic acid conjugates were tentatively identified, some of them for the first time. This work is a contribution to the identification of metabolites from SC that can become potential markers to estimate drug consumption.

The in vivo and in vitro metabolism and the detectability in urine of 3',4'-methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a new pyrrolidinophenone-type designer drug, studied by GC-MS and LC-MS(n.).

3',4'-Methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a designer drug of the pyrrolidinophenone-type, was first seized in Germany in 2009. It was also identified in 'legal high' samples investigated in the UK. Therefore, the aim of the presented work was to identify its in vivo and in vitro phase I and II metabolites using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ion trap mass spectrometry (LC-MS(n) ). Furthermore, detectability of MDPBP in rat and human urine using standard urine screening approaches (SUSA) by GC-MS and LC-MS(n) was studied. The metabolites were isolated either directly or after enzymatic cleavage of conjugates by solid-phase extraction (C18, HCX). The metabolites were then analyzed and structures proposed after GC-MS (phase I) and LC-MS(n) (phase II). Based on these identified metabolites, the following main metabolic steps could be proposed: demethylenation followed by methylation of one hydroxy group, aromatic and side chain hydroxylation, oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Furthermore, in rat urine after a typical user's dose as well as in human urine, mainly the metabolites could be detected using the authors' SUSA by GC-MS and LC-MS(n) . Thus, it should be possible to monitor an application of MDPBP assuming similar toxicokinetics in humans. Finally, CYP2C19 and CYP2D6 could be identified as the isoenzymes mainly responsible for demethylenation.

The effect of CYP2J2, CYP3A4, CYP3A5 and the MDR1 polymorphisms and gender on the urinary excretion of the metabolites of the H-receptor antihistamine ebastine: a pilot study.

AIMS: To determine the effect of gender and the genetic polymorphisms of CYP2J2, CYP3A4, CYP3A5 and MDR1 on the urinary excretion of the H(1) antihistamine ebastine in healthy subjects. METHODS: Eighty-nine Caucasians were studied. The presence of polymorphisms in genes known to be involved in ebastine metabolism and transport (CYP2J2*2,*3,*4,*6,*7, CYP3A4*1B, CYP3A5*3, *6 and MDR1(ABCB1)(C3435T)) was assessed by means of PCR-restriction fragment length polymorphism and sequencing methods. Genotype was correlated with the urinary excretion of the main ebastine metabolites (desalkylebastine and carebastine) under basal conditions and after administration of grapefruit juice. RESULTS: Women excreted statistically greater amounts of desalkylebastine in urine (mean +/- SD (95% confidence intervals, 95% CI), 23.0 +/- 19.5 (18.1, 27.9) micromol) than men (12.4 +/- 11.0 (7.9, 16.9)), (mean difference: 10.6 (2.4, 18.7), P < 0.005). The CYP2J2, CYP3A4 and CYP3A5 analysed polymorphisms did not greatly affect ebastine metabolite excretion. The MDR1(C3435T) polymorphism was found to affect both the urinary excretion of the active metabolite carebastine (32.3 +/- 18.3 (23.1, 41.4), 22.8 +/- 14.7 (18.6, 27.0) and 21.5 +/- 15.3 (14.7, 28.3) for CC, CT and TT carriers, respectively; P < 0.05) and the grapefruit juice-induced inhibition of its transport/formation (mean fold-decrease +/- SD (95% CI), 1.5 +/- 0.8 (1.0, 2.0), 1.1 +/- 0.9 (0.7, 1.4) and 0.9 +/- 0.4 (0.6, 1.2) for CC, CT and TT carriers, respectively; P = 0.01). CONCLUSIONS: Gender and the presence of the MDR1(C3435T) polymorphism both influence the excretion of ebastine metabolites in urine.

Studies on the metabolism and toxicological detection of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using gas chromatography-mass spectrometry.

The aim of the presented study was to identify the metabolites of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using GC-MS techniques. After enzymatic hydrolysis, extraction and various derivatizations, seven metabolites of MPBP could be identified suggesting the following metabolic steps: oxidation of the 4'-methyl group to the corresponding alcohol and further oxidation to the respective carboxy compound, hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or reduction of the keto group to the 1-dihydro compound. A previously published GC-MS-based screening procedure for pyrrolidinophenones involving enzymatic hydrolysis and mixed-mode solid-phase extraction of urine samples allowed detection of MPBP metabolites. Assuming similar metabolism and dosages in humans, an intake of MPBP should be detectable via its metabolites in urine.

Pharmacokinetics of the H1-receptor antagonist ebastine and its active metabolite carebastine in healthy subjects.

Pharmacokinetics of ebastine (CAS 90729-43-4), a histamine H1-receptor antagonist, was evaluated in healthy male volunteers. The subjects were given single oral doses of 5, 10, 20 and 40 mg of ebastine (5 or 6 subjects) and repeated oral doses of 20 mg once daily for 7 days (6 subjects). Administration of ebastine resulted in a negligible level of the unchanged drug in plasma and urine. Mean plasma concentration of carebastine (CAS 90729-42-3), an active carboxylated metabolite, reached maximum levels of 40, 112, 195 and 388 ng/ml at 4-6 h after single oral administration of ebastine at doses of 5, 10, 20 and 40 mg, respectively. Plasma levels of carebastine showed a first-order decrease with apparent half-lives of 13.8 to 15.3 h. The Cmax and AUC of carebastine increased in proportion to the dose. Urinary excretion of carebastine during 72 h after single administration accounted for 1.3-1.8% of the dose. Food intake did not affect the pharmacokinetics and gastrointestinal absorption of ebastine. Repeated administrations of ebastine once daily for 7 days did not cause any change in the pharmacokinetics of ebastine and carebastine. Plasma concentration of carebastine reached the steady state on day 4. The Cmax (360-396 ng/ml) was 1.6- to 1.7-fold greater than that after the first administration (229 ng/ml). These results strongly suggest that carebastine is responsible for the antihistamine activity after administration of ebastine.

Determination of some butyrophenones in body fluids by gas chromatography with surface ionization detection.

Haloperidol, moperone, pipamperone and bromperidol were found measurable with high sensitivity by gas chromatography (GC) with surface ionization detection (SID). The calibration curves using moperone as internal standard were linear in the range of 0.4-4 pmol on column for haloperidol, pipamperone, and bromperidol. The detection limit of the drugs was about 0.1 pmol on column. For their actual determination with forensic samples, a detailed procedure for their extraction from human whole blood and urine was established with Sep-Pak C18 cartridges. The recovery of the 4 drugs, which had been added to whole blood and urine, was more than 90%. Haloperidol in whole blood and urine of a schizophrenic patient, who had been administered with this drug (3 mg/day p.o.), could be quantitated by the present GC-SID, and the levels were 7.18 and 43.2 pmol/ml, respectively.

Positive- and negative-ion mass spectrometry of butyrophenones.

Positive-ion electron-impact (EI), positive-ion chemical ionization (CI) and medium-pressure negative-ion CI mass spectra of ten butyrophenones are presented. Low-pressure (0.01 Torr) negative CI spectra are also presented for some compounds. In the positive EI mode, a peak at m/z 42, which corresponded to the propyl group, appeared for all compounds; a peak at m/z 123 was also common to all compounds except for the two with a bis(fluorophenyl) group. Molecular ions were generally very small or missing in the positive EI mode. In the positive CI mode, strong [M+H]+ quasi-molecular peaks generally appeared together with [M+C2H5]+ peaks; [M-F]+ peaks appeared in many compounds and [M-OH]+ peaks also appeared for compounds having a hydroxypiperidinyl group. In the negative CI mode with a 1 Torr chamber pressure, their spectra were generally simple with [M-H]- quasi-molecular ions; anions of liberated halogens were not observed except for bromine at this pressure. In the negative CI mode at low pressure (0.01 Torr), some fragment peaks in the lower mass range appeared in addition to the quasi-molecular ions; halogen peaks (m/z 19 or 35) and anions at m/z 95, which corresponded to the fluorophenyl group, appeared in most spectra recorded at this pressure. An procedure for the extraction of butyrophenones from human urine and plasma and their separation by gas chromatography was also developed to serve for their identification in forensic science practice.

The metabolism of the neuroleptic agent 1 (4'-fluorophenyl)-4-(cyclohexyl-1'-piperzinyl-4'-carboxylated)-butan-1-one hydrochloride in rats and man.

1. An oral dose the neuroleptic agent 1-(4'-fluorophenyl)-4-(cyclohexyl-1'-(14C)piperazinyl-4'-carboxylate)butan-1-one was mainly eliminated in the urine within 12 h by rats and man. During 5 days, 63-6% and 83-3% was eliminated in the urine of rats and man respectively. 2. Plasma concentrations in man related a maximum during 30 min to 1 h, representing 1-43 microgram equiv./ml. The proportion of unchanged drug in plasma decreased from 48% at 15 min to less than 10% after 1 h. 3. Seven major radioactive components were detected in the chloroform extract of basified rat urine and five major components in similar extracts of human urine. The major rat metabolites were isolated and identified by mass spectrometry as components resulting from mono- and dihydroxylation in the cyclohexane ring, reduction of the keto group to a secondary alcohol and hydrolysis and decarboxylation of the cyclohexylcarbamoyl group. The major metabolite in the rat urine extract was the dihydroxylated secondary alcohol derivative while the major human metabolite was the monohydroxylated secondary alcohol derivative. The metabolites were also partly eliminated as conjugates.

Identification of basic metabolites of 4-[4-(p-chlo- robenzoyl)piperidino]-4'-fluorobutyrophenone, an experimental neuroleptic agent.

Two metabolites of 4-[4-(p-(chlorobenzoyl)piperidino]-4'-fluorobutyrophenone (RMI 9901) as well as unchanged drug, have been identified in the urine of rats following oral administration of the drug. Analysis of basic urine extracts by combined gas chromatography-mass spectrometry was employed for metabolite identification. N-dealkylation appears to be a major metabolic pathway and results in formation of 4-(p-chlorobenzoyl)piperidine (I). Subsequent oxidation of this metabolite results in the formation of 4-(p-chlorobenzoyl)-2-piperidone (II), and represents rather unusual metabolic pathway for compounds of this chemical class.