In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Abstract

Flubromazolam is a triazole benzodiazepine with high potency and long-lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)-enzymes. To confirm those metabolites detected in vitro, authentic samples obtained from two forensic cases were also analyzed by LC-HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CL ) using well-stirred and parallel-tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate-fortified pHLM with or without uridine 5'-diphosphoglucuronic acid and incubations with CYP-enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α- and/or 4-position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α-hydroxy-flubromazolam and its corresponding glucuronide were detected in vivo together with the N-glucuronide of flubromazolam. The predicted CL of flubromazolam using the well-stirred and parallel-tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein-binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α-hydroxy-flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd.

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.…”

“…The suggested structure of the product ions of m/z 292.1119 and 223.0666 agreed with those previously reported for flubromazolam, except for the ion of m/z 343.0115. As suggested for the single parent glucuronide found in previous studies, both flubromazolam glucuronides were assumed to be N‐glucuronides, similar to what has been reported for the structurally related midazolam . The [M + H + ] precursor ions of the 2 parent glucuronides were only detected in full scan mode, and their identification was further supported by the presence of the [M + H + ] ion of flubromazolam and its product ions in the respective product ion mass spectra.…”

“…In the case of flubromazolam, the introduction of the constructing chiral glucuronic acid in N 2 position would lead to the formation of diastereomers which thereby could be separated in our LC system. The reason previous studies only found 1 parent glucuronide for flubromazolam might be that they were not chromatographically separated. 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.…”

“…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 …”

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.…”