Electrochemical Simulation of 25B-NBOMe Phase I Metabolism and Metabolite Profiling by HPLC-QTOF-MS

This is the first report on the electrochemical simulation of phase I metabolism of 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe), a relatively new psychoactive substance available on the illicit drug market. The electrochemical approach enables fast generation and characterization of potential in vivo metabolites, and thus, can assist in the preliminary assessment of xenobiotic activity and toxicity profiles in humans. Phase I oxidation reactions of 25B-NBOMe were simulated in a three-electrode thin-layer electrochemical flow cell. Electrochemically generated products were directly analyzed by high-resolution mass spectrometry. To verify relevance to human metabolism, they were compared with those detected in biological samples taken from individuals severely intoxicated with 25B-NBOMe. The electrochemical conversion of 25B-NBOMe yielded key phase I metabolites-hydroxylated and N-desalkylated-along with their corresponding dehydrogenated products. O-Desmethylated and bis-O,O-desmethylated drug derivatives were also formed electrochemically, though in lower amounts. The former was confirmed in gastric contents, blood, and urine samples. Furthermore, phase II metabolites, such as O-desmethyl-25B-NBOMe glucuronide and sulfonate, were detected exclusively in some biological specimens, highlighting the complementary role of in vivo analysis. Our findings demonstrate that the electrochemical method provides a promising platform for the rapid and straightforward evaluation of 25B-NBOMe phase I metabolism. The partial overlap with authentic human metabolites supports its relevance as a screening and hypothesis-generating tool. The electrochemical approach, although not fully consistent with data from biological samples, can complement conventional in vitro and in vivo models, aiding in the identification of potential biomarkers and the evaluation of toxicological risk associated with novel psychoactive substances.