Identifying Metabolites of Meclonazepam by High-Resolution Mass Spectrometry Using Human Liver Microsomes, Hepatocytes, a Mouse Model, and Authentic Urine Samples

Abstract

Abstract.Meclonazepam is a benzodiazepine patented in 1977 to treat parasitic worms, which recently appeared as a designer benzodiazepine and drug of abuse. The aim of this study was to identify metabolites suitable as biomarkers of drug intake in urine using highresolution mass spectrometry, authentic urine samples, and different model systems including human liver microsomes, cryopreserved hepatocytes, and a mice model. The main metabolites of meclonazepam found in human urine were amino-meclonazepam and acetamido-meclonazepam; also, minor peaks for meclonazepam were observed in three of four urine samples. These observations are consistent with meclonazepam having a metabolism similar to that of other nitro containing benzodiazepines such as clonazepam, flunitrazepam, and nitrazepam. Both metabolites were produced by the hepatocytes and in the mice model, but the human liver microsomes were only capable of producing minor amounts of the amino metabolite. However, under nitrogen, the amount of aminomeclonazepam produced increased 140 times. This study comprehensively elucidated meclonazepam metabolism and also illustrates that careful selection of in vitro model systems for drug metabolism is needed, always taking into account the expected metabolism of the tested drug.KEY WORDS: benzodiazepine; in vitro metabolism; LC-MS/MS; new psychoactive substance; toxicokinetics.

Detection of the designer benzodiazepine flunitrazolam in urine and preliminary data on its metabolism

“…Based on the chromatographic peak areas determined from the urine samples using LC–HRMS (Figure ), 7‐acetamido‐FNTZ, and mainly 7‐amino‐FNTZ were deemed the main urinary metabolites which remained detectable for up 37 hours after FNTZ intake. These observations are consistent with flunitrazepam and meclonazepam metabolic pattern. However, the window of detection of FNTZ is largely lower that the one observed for the two other benzodiazepines.…”

Designer Benzodiazepines: A Review of Published Data and Public Health Significance

“…7,[12][13][14][15] Some are metabolized in vivo to traditional BZs. 7,[12][13][14][15] Some are metabolized in vivo to traditional BZs.…”

“…Metabolic profiles of a number of the designer BZs have been characterized by mass spectrometric analysis of human urine samples. 7,[12][13][14][15] Some are metabolized in vivo to traditional BZs. For example, the designer BZ diclazepam is metabolized to lorazepam, and the designer compound cloniprazepam is metabolized to clonazepam.…”

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4'‐chlorodiazepam (Ro5–4864)

“…In this context, a study by Pettersson Bergstrand et al showed the presence of designer benzodiazepines in 40% of urine samples from a routine drug testing laboratory in Sweden that tested positive for benzodiazepines on a cloned enzyme donor immune assay (CEDIA) and negative for 'prescription benzodiazepines' by liquid chromatography tandem-mass spectrometry (LC-MS/ MS) analysis. 10 Whereas in vivo metabolism data of some designer benzodiazepines has been published by various authors, 2,4,[11][12][13][14][15] data on newer compounds are scarce. 16 For timely method development, in vitro metabolism studies using pooled human liver microsomes (pHLM) can be utilized to get spectral data of potential phase I metabolites.…”