Metabolic Profile Analysis of Designer Benzodiazepine Etizolam in Zebrafish and Human Liver Microsomes

As one of the most widely abused designer benzodiazepines worldwide, Etizolam is characterized by its high addiction potential, low production cost, and difficulty in detection. Due to the rapid metabolism of Etizolam in the human body, the probability of detecting the Etizolam parent drug in actual case samples by forensic personnel is low. Therefore, without detecting the parent drug, analysis of Etizolam metabolites can help forensic personnel provide references and suggestions on whether the suspect has taken Etizolam. This study simulates the objective metabolic process of the human body. It establishes a zebrafish in vivo metabolism model and a human liver microsome in vitro metabolism model to analyze the metabolic characteristics of Etizolam. A total of 28 metabolites were detected in the experiment, including 13 produced in zebrafish, 28 produced in zebrafish urine and feces, and 17 produced in human liver microsomes. The UPLC-Q-Exactive-MS technology was used to analyze the structures and related metabolic pathways of Etizolam metabolites in zebrafish and human liver microsomes, and a total of 9 metabolic pathways were identified, including monohydroxylation, dihydroxylation, hydration, desaturation, methylation, oxidative deamination to alcohol, oxidation, reduction acetylation, and glucuronidation. Among them, metabolites involving hydroxylation reactions (including monohydroxylation and dihydroxylation) accounted for 57.1% of the total number of potential metabolites, indicating that hydroxylation may be the major metabolic pathway of Etizolam. Based on the response values of each metabolite, monohydroxylation (M1), desaturation (M19), and hydration (M16) were recommended as potential biomarkers for Etizolam metabolism. The experimental results provide reference and guidance for forensic personnel in identifying Etizolam use in suspects.