Metabolic profiling of 2C-EF in human liver microsomes: Identification of major metabolites and biotransformation pathways

2C-EF (4-(2-fluoroethyl)-2,5-dimethoxyphenethylamine) is a synthetic psychoactive compound belonging to the 2C family of substituted phenethylamines. Despite its recent emergence on the recreational drug market, data on its pharmacological profile, toxicity, and metabolism is extremely limited. The growing popularity of novel psychoactive substances poses a serious public health concern. These compounds are often consumed without knowledge of their potency, toxicity, or interactions, leading to unpredictable physiological and psychological effects. In this context, the absence of toxicological and metabolic data on 2C-EF highlights the need for further investigation. To date, no study have explored its metabolic fate in biological systems. The metabolism of 2C-EF was investigated in vitro using pooled human liver microsomes (HLMs) and liquid-chromatography coupled to a high-resolution mass spectrometry system (HRMS). Eight metabolites were identified, resulting from phase I and II transformations. The identified metabolites suggest that 2C-EF undergoes demethylation at position 2 or 5, hydroxylation, and deamination, followed by either oxidation to the corresponding carboxylic acid or reduction to the corresponding alcohol, via an intermediate aldehyde. Additionally, metabolites resulting from acetylation and glucuronidation were also identified. The metabolic profile of 2C-EF mirrors that of structurally related 2C compounds, indicating conserved enzymatic pathways despite the presence of a fluoroethyl substituent. A comprehensive characterization of its metabolism is key to evaluating health risks and improving analytical identification in clinical and forensic contexts; nonetheless, additional in viv o investigations are needed to fully elucidate its metabolic and toxicological profile.