title: Looking at flubromazolam metabolism from four different angles: Metabolite profiling in human liver microsomes, human hepatocytes, mice and authentic human urine samples with liquid chromatography high-resolution mass spectrometry.
abstract: Flubromazolam is a triazolam benzodiazepine that recently emerged as a new psychoactive substance. Since metabolism data are scarce and good analytical targets besides the parent are unknown, we investigated flubromazolam metabolism in vitro and in vivo. 10μmol/L flubromazolam was incubated with human liver microsomes for 1h and with cryopreserved human hepatocytes for 5h. Mice were administered 0.5 or 1.0mg flubromazolam/kg body weight intraperitoneally, urine was collected for 24h. All samples, together with six authentic forensic human case specimens, were analyzed (with or without hydrolysis, in case it was urine) by UHPLC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF. Data mining was performed manually and with MassMetasite software (Molecular Discovery). A total of nine metabolites were found, all generated by hydroxylation and/or glucuronidation. Besides O-glucuronidation, flubromazolam formed an N+-glucuronide. Flubromazolam was not metabolized extensively in vitro, as only two monohydroxy metabolites were detected in low intensity in hepatocytes. In the mice samples, seven metabolites were identified, which mostly matched the metabolites in the human samples. However, less flubromazolam N+-glucuronide and an additional hydroxy metabolite were observed. The six human urine specimens showed different extent of metabolism: some samples had an intense flubromazolam peak next to a minute signal for a monohydroxy metabolite, others showed the whole variety of hydroxylated and glucuronidated metabolites. Overall, the most abundant metabolite was a monohydroxy metabolite, which we propose as α-hydroxyflubromazolam based on MSMS fragmentation. These metabolism data will assist in interpretation and analytical method development.
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden; Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185 Linköping, Sweden. Electronic address: ariane.wohlfarth@rmv.se.
authors.forename: Ariane
authors.initials: A
authors.lastname: Wohlfarth
authors.affiliation: Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185 Linköping, Sweden.
authors.forename: Svante
authors.initials: S
authors.lastname: Vikingsson
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
authors.forename: Markus
authors.initials: M
authors.lastname: Roman
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
authors.forename: Mikael
authors.initials: M
authors.lastname: Andersson
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden; Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185 Linköping, Sweden.
authors.forename: Fredrik C
authors.initials: FC
authors.lastname: Kugelberg
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden; Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185 Linköping, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Green
authors.affiliation: Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden; Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185 Linköping, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Kronstrand
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000493:pharmacokinetics*; D002853:Chromatography, Liquid; D015198:Designer Drugs / Q000493:pharmacokinetics*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000378:metabolism; D011619:Psychotropic Drugs / Q000493:pharmacokinetics*
chemical_list: D015198:Designer Drugs; D011619:Psychotropic Drugs; D001569:Benzodiazepines
keywords: Designer benzodiazepine; Flubromazolam; High resolution mass spectrometry; Metabolism