In vitro metabolite profiling of ADB-FUBINACA, a new synthetic cannabinoid

Background: Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption.N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1Hindazole- 3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable.Methods: We aimed to determine optimal markers for identifying ADB-FUBINACA intake.Metabolic stability was evaluated with human liver microsome incubations.Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOFR, Sciex) and several data mining approaches (MetabolitePilot™, Sciex).Results: Metabolite separation was achieved on an Ultra Biphenyl column (RestekR); full-scan TOFMS and information-dependent acquisition MS/MS data were acquired.ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug).Twenty-three metabolites were identified.Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation.Conclusion: We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers.N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption.This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results