In vitro and in vivo metabolism of the synthetic cannabinoid FUB-144: comprehensive phase I and II profiling using UHPLC–HRMS

Abstract

Synthetic cannabinoids (SCs), including FUB-144, are emerging psychoactive substances; however, a lack of metabolic information limits toxicological risk assessments. Owing to ethical constraints in obtaining human biological samples, in vitro studies using human liver microsomes (HLMs) and animal experiments are essential to elucidate metabolic pathways. In this study, we investigated SC metabolism using HLM cultures treated with 30 µM FUB-144 and a mouse model orally administered 20 mg/kg FUB-144. Metabolites were analyzed by UHPLC-HRMS. In total, 12 phase I and 2 phase II metabolites were identified (referred to as M1–M14). Phase I metabolism was dominated by hydroxylation, primarily at the indole nucleus and tetramethyl cyclopropyl moiety, followed by secondary reactions, such as dehydrogenation and further oxidation. Phase II metabolism produced two glucuronide conjugates (M4 and M10), generated by conjugation at oxidized sites. The in vitro findings were largely consistent with in vivo data, as most metabolites observed in microsomal assays were confirmed in mouse serum and urine samples. Among these, M8—formed through sequential hydroxylation and oxidation—was sustained at relatively high concentrations for up to 24 h, suggesting its diagnostic value. A time-course analysis revealed a rapid decline of the parent compound in serum, paralleled by a pronounced rise in metabolites, indicating the fast metabolic turnover of FUB-144. Taken together, this study provides a comprehensive description of phase I and II metabolic pathways and establishes a critical foundation for toxicological and forensic monitoring of FUB-144.