Multilevel metabolic profiling of synthetic cannabinoid 5F-ADB: identifying definitive biomarkers for forensic source tracking and ecotoxicological risk assessment

Methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl]amino}-3,3-dimethylbutanoate (5F-ADB), a potent synthetic cannabinoid, induces intense euphoria, hallucinations, and addiction, posing significant risks to human health. Current drug surveillance efforts lack data to identify drug abuse, and the environmental impacts of 5F-ADB entering aquatic systems via synthesis or use remain uncharacterized. To address these gaps, a multilevel assessment system (in vitro-invertebrate-vertebrate) was established to elucidate 5F-ADB metabolic pathways and identify robust biomarkers. Human liver microsomes (HLMs), Daphnia magna, and zebrafish were exposed to 5F-ADB, with metabolites profiled by high-performance liquid chromatography coupled with mass spectrometry. Metabolic pathways were inferred, and metabolite toxicity was evaluated. Results revealed 9, 11, and 22 metabolites in HLMs, D. magna, and zebrafish models, respectively. Dominant pathways in HLMs and zebrafish included ester hydrolysis, defluorinated hydroxylation, and combined ester hydrolysis/defluorinated hydroxylation. Daphnia magna metabolism primarily featured defluorinated hydroxylation, depentylation, and ester hydrolysis coupled with hydroxylation. Glucuronidation metabolites were exclusive to zebrafish. Based on abundance and stability, H-M4 (ester hydrolysis), D-M1 (ester hydrolysis/depentylation), and Z-M15 (ester hydrolysis/condensation) were identified as key biomarkers for HLMs, D. magna, and zebrafish, respectively. Toxicity assessments indicated reduced toxicity for most metabolites versus 5F-ADB. However, H-M7, D-M7, D-M11, and Z-M15 (products of ester hydrolysis/condensation or defluorinated hydroxylation/oxidation) exhibited comparable toxicity to the parent compound. Critically, D-M7 (defluorinated hydroxylation/oxidation) demonstrated heightened hydrophilicity and potentially elevated ecotoxicity in D. magna, warranting further ecological risk investigation. This study provides the first multitrophic metabolic characterization of 5F-ADB, delivering critical data for tracing illicit synthesis, monitoring drug use distribution, and evaluating environmental hazards of synthetic cannabinoids.