Characterization and identification of eight designer benzodiazepine metabolites by incubation with human liver microsomes and analysis by a triple quadrupole mass spectrometer

Abstract

Designer benzodiazepines (DBZDs) have become of particular importance in the past few years. The metabolite monitoring of DBZD in biological fluids could be of great interest in clinical and forensic toxicology. However, DBZD metabolites are not known or not commercially available. The identification of some DBZD metabolites has been mostly explored by self-administration studies or by in vitro studies followed by high-resolution mass spectrometry. The question arose whether a unit resolution instrument could be efficient enough to allow the identification of DBZD metabolites. In this study, we used an in vitro experiment where eight DBZDs (diclazepam, flubromazepam, etizolam, deschloroetizolam, flubromazolam, nifoxipam, meclonazepam and clonazolam) were incubated with human liver microsomes (HLMs) and metabolite identification was carried out by using a UHPLC coupled to a QTRAP triple quadrupole linear iontrap tandem mass spectrometer system. Post-mortem samples obtained from a real poisoning case, involving deschloroetizolam and diclazepam, were also analysed and discussed. Our study using HLM allowed the identification of 26 metabolites of the 8 DBZDs. These were denitro-, mono- or di-hydroxylated and desmethyl metabolites. In the forensic case, diclazepam was not detected whereas its metabolites (lormetazepam and lorazepam) were present at high concentrations in urine. We also identified hydroxy-deschloroetizolam in urine, while the parent compound was not detected in this matrix. This supports the approach that LC coupled to a simple QTRAP could be used by laboratories to identify other not-known/not-commercialized new psychoactive substance (NPS) metabolites.

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry

“…However, since only 1 of the 2 flubromazolam glucuronide isomers (isomer‐1) was detected in all patient samples, separation of these analytes is not that important since the preferred analytical target metabolite should be isomer‐1.The product ion spectrum for flubromazolam hydroxy metabolite isomer‐1 did not include a water loss from the [M + H + ] precursor ion. Previous studies have suggested that hydroxylation of midazolam and flubromazolam takes place on the methyl part of the triazole ring (α‐hydroxy) and the diazepine ring (4‐hydroxy). This in combination with the proposed structure of the product ions with m/z 333.0033 and 317.0084 suggest that hydroxylation of flubromazolam isomer‐1 most likely occurred on the diazepine ring.…”

“…To improve the detection of designer benzodiazepines, several studies have been undertaken to determine their metabolic patterns both in vitro and in vivo . Accordingly, studies have elucidated the metabolic profiles of flubromazolam, whereas no metabolites have been reported for pyrazolam . Nevertheless, in a bioanalytical study of designer benzodiazepines, a higher immunoassay test response than that expected from the concentration of parent compound was found for urine samples collected in pyrazolam intoxication cases, suggesting cross‐reactivity of the antibodies with metabolites of pyrazolam …”

Metabolism of novel opioid agonists U‐47700 and U‐49900 using human liver microsomes with confirmation in authentic urine specimens from drug users

“…Metabolic profile determination using human liver microsomes (HLM) is now routinely employed for studies involving various emerging therapeutic and novel psychoactive substances, including designer opioids . Understanding of metabolic profiles allows for increased windows of detection through the identification of primary and secondary metabolites.…”

The emergence of new psychoactive substance (NPS) benzodiazepines: A review

“…The metabolic pathways for benzodiazepines are fairly similar (Figures 3 and 4) and this allows metabolites to be predicted and actively searched for when analyzing samples using techniques such as LC-MS. 157,162 As a result of the aforementioned similar metabolic pathways, care must be taken when interpreting the apparent presence of a metabolite. For example, diclazepam is metabolised to lorazepam, lormetazepam, and delorazepam which are all prescription drugs.…”