5-Methyl etodesnitazene human metabolism: LC-ESI±-HRMS/MS analysis (mono- and di-protonation) of human hepatocyte incubations and positive biospecimens.

The rapid emergence of new synthetic opioids (NSOs), especially potent 2-benzylbenzimidazole derivatives known as nitazenes, poses challenges to public health and toxicologists. Among these substances, 5-methyl etodesnitazene has recently appeared on the illicit market. This compound was shown to be approximately twice as potent as fentanyl at the µ-opioid receptor, and overdose may lead to potentially fatal respiratory depression. Detecting the drug and its metabolites is therefore critical for clinical and forensic investigations. This study aimed to characterize the metabolic profile of 5-methyl etodesnitazene in humans. Metabolic transformations were investigated through incubations with ten-donor pooled human hepatocytes and analysis of blood and urine samples from a postmortem case positive for 5-methyl etodesnitazene. Samples were analyzed using liquid chromatography-electrospray ionization-high-resolution tandem mass spectrometry (LC-ESI-HRMS/MS) in positive- and negative-ionization modes, and software-aided data mining. In positive mode, 5-methyl etodesnitazene produced an intense signal in its di-protonated form, which generated informative spectra, facilitating structural elucidation. Biotransformations included N- and O-deethylation, consistent with structural analogs, as well as ω-carboxylation specific to the methylated benzimidazole core; phase II metabolites were not detected. We suggest N-deethyl-5-methyl etodesnitazene, N-deethyl-5-carboxy etodesnitazene, and 5-carboxy etodesnitazene in blood, and N-deethyl-5-carboxy-4'-hydroxy etodesnitazene, 5-carboxy-4'-hydroxy etodesnitazene, and N-deethyl-5-carboxy etodesnitazene in urine, as metabolite biomarkers of consumption in clinical and forensic settings. Overall, metabolite profiles were consistent across in vitro and in vivo data, with minor matrix-specific differences. Considering that nitazene metabolites are often active and their metabolism is susceptible to genetic polymorphism, further research on the pharmacokinetics and pharmacodynamics of 5-methyl etodesnitazene is warranted.