In Vitro Metabolism of a Benzofuran-Substituted Nitazene: Ethyleneoxynitazene

<b>Background/Objectives</b>: New synthetic opioids (NSOs) like nitazenes pose significant public health risks due to their high potency and increasing prevalence. Ethyleneoxynitazene, a benzofuran-containing nitazene, recently emerged on the illicit market and was identified in seizures in Europe. Although no intoxications have been reported to date, its µ-opioid receptor activity raises concern. This study investigated the metabolism of ethyleneoxynitazene to better understand its pharmacological profile, toxicity, and detectability in clinical and forensic contexts. <b>Methods</b>: Ethyleneoxynitazene was incubated with cryopreserved human hepatocytes pooled from 10 donors. Metabolites were detected by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and identified using Compound Discoverer (Thermo Scientific; Waltham, MA, USA); detection and identification were assisted by in silico metabolite predictions with BioTransformer. <b>Results</b>: Sixteen metabolites were identified, with major biotransformations including <i>N</i>-deethylation at the <i>N</i>,<i>N</i>-diethylethanamine chain, hydroxylation at the dihydrofuran ring, and dihydrofuran ring opening via oxidative cleavage, leading to the formation of the corresponding ethanoic acid. <b>Conclusions</b>: This study provides the first characterization of the metabolism of a nitazene without an alkoxyphenyl moiety; the absence of this particular group reflects significant differences in the pharmacokinetic and pharmacodynamic profile compared to other nitazenes. We propose <i>N</i>-deethyl-3'-ethanoic acid-4'-hydroxy ethyleneoxynitazene, <i>N</i>-deethyl-hydroxy ethyleneoxynitazene, 3'-ethanoic acid-4'-hydroxy ethyleneoxynitazene, hydroxy ethyleneoxynitazene, and <i>N</i>-deethyl ethyleneoxynitazene as metabolite biomarkers of ethyleneoxynitazene consumption in clinical and forensic toxicology. Given the potential activity of some metabolites and interindividual variability in metabolic pathways, further studies are warranted to refine these findings through the analysis of biological samples from multiple ethyleneoxynitazene-positive cases.