title: In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic Opioids Acetylfentanyl, Acrylfentanyl, Furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl.
abstract: New fentanyl analogs have recently emerged as new psychoactive substances and have caused numerous fatalities worldwide. To determine if the new analogs follow the same metabolic pathways elucidated for fentanyl and known fentanyl analogs, we performed in vitro and in vivo metabolite identification studies for acetylfentanyl, acrylfentanyl, 4-fluoro-isobutyrylfentanyl, and furanylfentanyl. All compounds were incubated at 10 μM with pooled human hepatocytes for up to 5 h. For each compound, four or five authentic human urine samples from autopsy cases with and without enzymatic hydrolysis were analyzed. Data acquisition was performed in data-dependent acquisition mode during liquid chromatography high-resolution mass spectrometry analyses. Data was analyzed (1) manually based on predicted biotransformations and (2) with MetaSense software using data-driven search algorithms. Acetylfentanyl, acrylfentanyl, and 4-fluoro-isobutyrylfentanyl were predominantly metabolized by N-dealkylation, cleaving off the phenethyl moiety, monohydroxylation at the ethyl linker and piperidine ring, as well as hydroxylation/methoxylation at the phenyl ring. In contrast, furanylfentanyl's major metabolites were generated by amide hydrolysis and dihydrodiol formation, while the nor-metabolite was minor or not detected in case samples at all. In general, in vitro results matched the in vivo findings well, showing identical biotransformations in each system. Phase II conjugation was observed, particularly for acetylfentanyl. Based on our results, we suggest the following specific and abundant metabolites as analytical targets in urine: a hydroxymethoxy and monohydroxylated metabolite for acetylfentanyl, a monohydroxy and dihydroxy metabolite for acrylfentanyl, two monohydroxy metabolites and a hydroxymethoxy metabolite for 4-fluoro-isobutyrylfentanyl, and a dihydrodiol metabolite and the amide hydrolysis metabolite for furanylfentanyl.
authors.affiliation: Centre for Forensic Science, School of Mathematical and Physical Sciences, University of Technology Sydney (UTS), PO Box 123, Broadway, Sydney, NSW, 2007, Australia.
authors.forename: Shimpei
authors.initials: S
authors.lastname: Watanabe
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: Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185, Linköping, Sweden.|Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758, Linköping, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Green
authors.affiliation: Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185, Linköping, Sweden.|Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758, Linköping, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Kronstrand
authors.affiliation: Division of Drug Research, Department of Medical Health Sciences, Linköping University, 58185, Linköping, Sweden. ariane.wohlfarth@rmv.se.|Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758, Linköping, Sweden. ariane.wohlfarth@rmv.se.
authors.forename: Ariane
authors.initials: A
authors.lastname: Wohlfarth
mesh_terms: D000465:Algorithms; D001711:Biotransformation; D002478:Cells, Cultured; D005283:Fentanyl / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D006868:Hydrolysis; D006900:Hydroxylation; D066298:In Vitro Techniques; D012984:Software
chemical_list: D005283:Fentanyl
keywords: LC-HRMS; authentic human urine samples; fentanyl analogs; human hepatocytes; metabolite identification