Kei Zaitsu

Metabolism of the recently encountered designer drug, methylone, in humans and rats

The urinary metabolites of methylone in humans and rats were investigated by analysing urine specimens from its abuser and after administrating to rats with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), using authentic standards. The time-course excretion profiles of methylone and its three metabolites in rats were further investigated after a single intraperitoneal dosing of 5 mg kg−1 methylone hydrochloride. Two major metabolic pathways were revealed for both humans and rats as follows: (1) side-chain degradation by N-demethylation to the corresponding primary amine methylenedioxycathinone (MDC), partly conjugated; and (2) demethylenation followed by O-methylation of either a 3- or 4-OH group on the benzene ring to produce 4-hydroxy-3-methoxymethcathinone (HMMC) or 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC), respectively, mostly conjugated. Of these metabolites, HMMC was the most abundant in humans and rats. The cumulative amount of urinary HMMC excreted within the first 48 h in rats was approximately 26% of the dose, and the amount of the parent methylone was not more than 3%. These results demonstrate that the analysis of HMMC will be indispensable for proof of the use of methylone in forensic urinalysis.

Determination of the metabolites of the new designer drugs bk-MBDB and bk-MDEA in human urine

This is the first report on identifying the specific metabolites of the new designer drugs 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB) and 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDEA) in human urine using synthesized standards. Based on GC/MS and LC/MS, we identified N-dealkylation, demethylenation followed by O-methylation, and beta-ketone reduction as their major metabolic pathways. The quantitative analyses by LC/MS revealed that both demethylenation followed by O-methylation and beta-ketone reduction were superior to N-dealkylation and that both bk-MBDB and bk-MDEA were mainly metabolized into their corresponding 4-hydroxy-3-methoxy metabolites (4-OH-3MeO metabolites). After hydrolysis, the concentrations of 4-OH-3MeO metabolites and 3-hydroxy-4-methoxy metabolites of both bk-MBDB and bk-MDEA dramatically increased, suggesting that the metabolites mainly exist as their conjugates.

Recently abused β-keto derivatives of 3,4-methylenedioxyphenylalkylamines: a review of their metabolisms and toxicological analysis

Abstractβ-Keto derivatives of 3,4-methylenedioxyphenylalkylamines (bk-MDPAs), especially 2-methylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (methylone), 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB), and 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDEA), are abused as substitutes for 3,4-methylenedioxyphenylalkylamines in some countries, causing increased social problems. With the widespread abuse of bk-MDPAs, the analysis of bk-MDPAs and their metabolites in human specimens is quite important in forensic and clinical toxicology. In this review, the metabolisms of bk-MDPAs and simultaneous analytical methods for bk-MDPAs and their metabolites by gas chromatography–mass spectrometry, liquid chromatography–mass spectrometry, and liquid chromatography–tandem mass spectrometry are presented.

Influences of methamphetamine-induced acute intoxication on urinary and plasma metabolic profiles in the rat.

Methamphetamine (MA) is an illicit psychostimulant, and its abuse has become an international public health problem. MA intoxication can cause life-threatening hyperthermia, renal and liver failure, cardiac arrhythmias, and neurological damage. To investigate the relationship between the underlying mechanism of such intoxication and metabolic networks, mass spectrometry-based metabolomics experiments were performed on Sprague-Dawley rats treated with MA at 10mgkg(-1)h(-1) for 4h. Using a combination of gas chromatography-time-of-flight mass spectrometry and capillary electrophoresis-tandem mass spectrometry, global and targeted analyses were performed on biological samples collected during 0-24 and 72-96h (for urine), and at 24 and 96h (for plasma) after the last drug administration. Body temperature and plasma biochemical parameters were also measured to detect abnormal reactions in neuronal and other several tissues. 5-Oxoproline, saccharic acid, uracil, 3-hydroxybutyrate (3-HB), adipic acid, glucose, glucose 6-phosphate, fructose 1,6-bisphosphate, and tricarboxylic acid (TCA) cycle intermediates, such as fumarate, were proposed as potential biomarkers related to MA-induced intoxications. In particular, the observation of decreased TCA cycle intermediates and 3-HB and increased glucose suggested that high doses of MA inhibit biogenic energy production by glycolysis, oxidative phosphorylation via the TCA cycle, and the beta-oxidation of fatty acids. These results may provide not only a clue to clarify the underlying mechanism of diverse intoxication effects, but also biological fluid-based diagnostic and forensic methods with which to objectively demonstrate intoxication without directly determining the drug.

Metabolism of the newly encountered designer drug α -pyrrolidinovalerophenone in humans: identification and quantitation of urinary metabolites

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.

Urinary excretion of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA), including the sulfate and glucuronide of 4-hydroxy-3-methoxymethamphetamine (HMMA), in humans and rats

The urinary concentrations of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy), specifically 4-hydroxy-3-methoxymethamphetamine sulfate (HMMA-Sul) and 4-hydroxy-3-methoxymethamphetamine glucuronide (HMMA-Glu), have been directly measured in both MDMA users and rats by an established liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) procedure. The concentrations of these conjugates in urine from MDMA users (n = 25) ranged from 6.5 to 202 µM (from 1.8 to 55.6 µg ml−1) for HMMA-Sul and from 1.3 to 87.0 µM (from 0.5 to 32.3 µg ml−1) for HMMA-Glu, and the ratio of HMMA-Sul to HMMA-Glu ranged from 1.6 to 9.9 (3.1 ± 1.8). These results demonstrate that the sulfation is quantitatively more significant than the glucuronidation for HMMA in humans. In rats, in contrast, almost all the conjugated HMMA (>99%) was excreted as the glucuronide. These findings indicate that hydrolysis should be carefully made in urine analysis by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) by using either an acid or an enzyme possessing both sulfatase and β-glucuronidase activities. It is concluded that a considerable interspecies variation exists in the conjugation of HMMA between humans and rats.

Recently abused synthetic cathinones, α-pyrrolidinophenone derivatives: a review of their pharmacology, acute toxicity, and metabolism

The aim of this review is to present the chemical aspects, pharmacology, acute toxicities, and metabolisms of α-pyrrolidinophenone derivatives, a new group of synthetic cathinones. Compared to other synthetic cathinones, α-pyrrolidinophenone derivatives have high lipophilicity due to the pyrrolidine ring substitution at the nitrogen atom, resulting in higher blood–brain barrier permeability. To date, some acute intoxication and fatal cases involving α-pyrrolidinophenone derivatives have been reported, and the symptoms induced by their high dosages are due to central nervous system and cardiovascular toxicities. Based on the previous metabolism studies, reduction of the β-ketone moiety to the corresponding alcohol metabolites and oxidation to the 2′′-oxo metabolites are the main metabolic pathways observed among α-pyrrolidinophenone derivatives. In addition to such pathways, specific metabolic pathways like hydroxylation followed by oxidation of the 4′-methyl group, O-demethylation of the 4′-methoxyl group, and demethylenation followed by O-methylation of the 3′,4′-methylenedioxy group can be observed for the corresponding ring-substituted compounds.

Urinary excretion and metabolism of the newly encountered designer drug 3,4-dimethylmethcathinone in humans

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.

METABOLISM OF THE PSYCHOTOMIMETIC TRYPTAMINE DERIVATIVE 5-METHOXY-N,N-DIISOPROPYLTRYPTAMINE IN HUMANS: IDENTIFICATION AND QUANTIFICATION OF ITS URINARY METABOLITES

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

Discrimination and identification of regioisomeric β-keto analogues of 3,4-methylenedioxyamphetamines by gas chromatography-mass spectrometry

Very recently, β-keto derivatives of 3,4-methylenedioxyamphetamines (MDAs) have appeared on the illicit drug market. In the present study, we synthesized three isomers of β-keto derivatives of MDAs, 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB), 2-ethylamino-1-(3,4-methylenedioxyphenyl) propan-1-one (bk-MDEA), and 2-dimethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDDMA), and measured their electron ionization mass spectra without and with trifluoroacetyl (TFA) derivatization using gas chromatography-mass spectrometry (GC-MS). Although the spectral profiles of the three isomers were very similar to each other in both the free and TFA-derivatized forms, there were characteristic peaks at m/z 44 and 140, for bk-MDEA without and with TFA derivatization, respectively; a peak at m/z 110 for bk-MBDB-TFA was also characteristic. These peaks are useful for discrimination of an isomer from others. All isomers could be well separated in both free and TFA-derivatized forms using a slightly polar fused-silica capillary GC column DB-5MS. The present data are likely to be very useful for actual identification and quantitation of β-keto analogues of MDAs by GC-MS, because abuse of these materials is expected to spread worldwide in the near future.