Biotransformation of 5-Methoxy-N-isopropyl-N-methyltryptamine by zebrafish and human liver microsome with high-resolution mass spectrometry

To explore the metabolites of 5-Methoxy-N-isopropyl-N-methyltryptamine (5-MeO-MiPT) and unveil its toxicological effects, we examined its metabolic profiles using zebrafish and human liver microsome models. Employing ultra-high-performance liquid chromatography Q Exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-QE-HRMS), we analyzed samples from intoxicated zebrafish and human liver microsomes. In the zebrafish model, we identified a total of six metabolites. Primary phase I metabolic pathways involved N-Demethylation and Indole-hydroxylation reactions, while phase II metabolism included Glucoside conjugation directly, Glucoside conjugation after Indole-hydroxylation, and Sulfonation following Indole-hydroxylation. In the human liver microsome model, nine metabolites were generated. Major phase I metabolic pathways encompassed N-Demethylation, 5-O-Demethylation, and N-Depropylation, N-Oxidation, Indole-hydroxylation, N-Demethylation combined with Indole-hydroxylation, and 5-O‐Methylation-carboxylation. Phase II metabolism involved Glucoside conjugation after Indole-hydroxylation, as well as Glucoside conjugation after 5-O-Demethylation. Proposed phase I metabolites, such as 5-MeO-MiPT-N-Demethylation (5-MeO-NiPT) and 5-MeO-MiPT-Indole-hydroxylation, alongside the phase II metabolite OH&Glucoside conjugation-5-MeO-MiPT, were identified as effective markers for screening 5-MeO-MiPT intake. This study systematically delineates the phase I and II metabolites of 5-MeO-MiPT, confirming their pathways through in vivo and in vitro extrapolation. Additionally, inclusion of the parent drug itself and OH&Glucoside conjugation-5-MeO-MiPT could serve as valuable confirmation tools.