title: 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 (CLH) 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 CLHof 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.
authors.affiliation: Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, 3, DK-2100, Denmark.
authors.forename: Carolina
authors.initials: C
authors.lastname: Noble
authors.affiliation: Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, 3, DK-2100, Denmark.
authors.forename: Marie
authors.initials: M
authors.lastname: Mardal
authors.affiliation: Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, 3, DK-2100, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Bjerre Holm
authors.affiliation: Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, 3, DK-2100, Denmark.
authors.forename: Sys
authors.initials: S
authors.lastname: Stybe Johansen
authors.affiliation: Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Frederik V's Vej 11, 3, DK-2100, Denmark.
authors.forename: Kristian
authors.initials: K
authors.lastname: Linnet
mesh_terms: D001569:Benzodiazepines / Q000097:blood / Q000378:metabolism*; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015198:Designer Drugs / Q000378:metabolism* / Q000493:pharmacokinetics; D005554:Forensic Medicine; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000378:metabolism*; D011485:Protein Binding; D011619:Psychotropic Drugs / Q000097:blood / Q000378:metabolism*; D015813:Substance Abuse Detection; D053719:Tandem Mass Spectrometry
chemical_list: D015198:Designer Drugs; D011619:Psychotropic Drugs; D001569:Benzodiazepines; D003577:Cytochrome P-450 Enzyme System
keywords: clearance; designer benzodiazepines; high-resolution mass spectrometry; metabolism; microsomes; new psychoactive substances