The study investigates the human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene. Using human hepatocyte incubations and postmortem samples, the researchers identified key metabolites and their biomarker potential for clinical and forensic use. The main metabolic pathways included N-deethylation, O-dealkylation, and O-glucuronidation. Isotonitazene and metonitazene shared 10 O-dealkyl metabolites, while etodesnitazene and metodesnitazene shared 9. The study confirmed that the 10-donor-pooled human hepatocyte model accurately predicts human metabolism. Metabolites such as N-deethyl-isotonitazene and O-dealkyl metabolites were identified as potential biomarkers in urine after glucuronide hydrolysis, while N-deethyl metabolites were suggested as additional biomarkers in blood. The findings highlight the importance of metabolite detection in toxicology for documenting opioid use and improving forensic analysis. The study also emphasizes the need for further research to understand the pharmacokinetics and metabolism of these new synthetic opioids.The study investigates the human metabolism of four synthetic benzimidazole opioids: isotonitazene, metonitazene, etodesnitazene, and metodesnitazene. Using human hepatocyte incubations and postmortem samples, the researchers identified key metabolites and their biomarker potential for clinical and forensic use. The main metabolic pathways included N-deethylation, O-dealkylation, and O-glucuronidation. Isotonitazene and metonitazene shared 10 O-dealkyl metabolites, while etodesnitazene and metodesnitazene shared 9. The study confirmed that the 10-donor-pooled human hepatocyte model accurately predicts human metabolism. Metabolites such as N-deethyl-isotonitazene and O-dealkyl metabolites were identified as potential biomarkers in urine after glucuronide hydrolysis, while N-deethyl metabolites were suggested as additional biomarkers in blood. The findings highlight the importance of metabolite detection in toxicology for documenting opioid use and improving forensic analysis. The study also emphasizes the need for further research to understand the pharmacokinetics and metabolism of these new synthetic opioids.