Quantitative determination and metabolic profiling of synthetic cathinone eutylone in vitro and in urine samples by liquid chromatography tandem quadrupole time-of-flight mass spectrometry

In Taiwan, synthetic cathinones are the most prevalent new psychoactive substances, and their use is growing continuously. Urine samples are currently analysed to determine drug abuse, but the metabolic profiles and metabolites of these compounds are not widely reported. Given that cases of eutylone abuse have been growing since 2020, this study established a method employing supported liquid extraction combined with liquid chromatography tandem quadrupole time-of-flight mass spectrometry to identify and quantify eutylone and its metabolites in urine samples. Method validation was performed, and eight authentic samples were analysed. Moreover, in vitro metabolism experiments were conducted, and metabolites were generated by incubating eutylone with human liver microsomes and cytosol. Metabolite characterisation was achieved by confirming the accurate mass and product ions in full MS/MS spectra. Five metabolites were identified in in vitro experiments; they resulted from eutylone N-dealkylation, β-ketone reduction, demethylenation, aliphatic hydroxylation and sequential demethylenation and O-methylation. The metabolic profile was obtained evaluating the metabolites at different incubation times: Demethylenation occurred first, followed by N-dealkylation, β-ketone reduction and aliphatic hydroxylation. Three additional metabolites were identified in authentic samples. Based on in vitro and in vivo evidence, we propose that the demethylenation and O-methylation metabolite, the β-ketone reduction metabolite, and the β-ketone reduction, demethylenation and O-methylation metabolite are the most appropriate biomarkers of eutylone consumption. Using these markers can help expand the eutylone detection window and provide information for toxicology research.