Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of ‘nitazenes’. We examined the in-vivo metabolism of etazene through analysis of a human (overdose) urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking an NPS cocktail purchased online. Next to etazene, N-ethylpentedrone, deschloroketamine and flubromazepam were also taken by the patient.

The urine sample was analyzed on a Q-Exactive high resolution mass spectrometer (HRMS) operating in full scan mode (range 70-700m/z) with data-dependent acquisition based on an inclusion list. Etazene metabolites were predicted with the BioTransformer 3.0 software and the exact masses were added into the inclusion list. The urine sample was analyzed both with simple dilution of the sample (addition of internal standard and dilution with aqueous mobile phase) and after β-glucuronidase treatment (addition of internal standard and Abalonase® to the sample with subsequent incubation at 37°C for 30min).

Six possible metabolites were identified in the urine sample of the patient. The parent compound was less abundant than some metabolites. In line with previous findings for other nitazene opioids, N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene) and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites (M5: hydroxylated M2 and M6: hydroxylated M1) and etazene (M4) were also found. Hydroxylation is presumed at the benzimidazole ring. For M5 and M6, different chromatographic peaks with the same MS² spectra were detected, suggesting the presence of positional isomers of the hydroxyl-group at the benzimidazole ring. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found.

A rapid and straightforward HRMS method was developed to study the in-vivo metabolites of etazene. N- and O-deethylated products were the predominant urinary metabolites. Detection of these metabolites in urine can be useful to demonstrate etazene exposure. Our findings are comparable to results obtained in a metabolism study performed in rats.