Profiling and Identification of Novel Tiletamine Metabolites in Human Urine and Hair by Liquid Chromatography Coupled With Quadrupole‐Orbitrap Mass Spectrometry

ABSTRACT Rationale Tiletamine, an animal tranquilizer increasingly misused by humans, lacks comprehensive metabolic characterization in authentic biological matrices. To date, tiletamine phase II metabolism remains poorly characterized. This study investigated the metabolic fate of tiletamine in human urine and hair by identifying novel metabolites and elucidating biotransformation pathways relevant to forensic toxicological monitoring. Methods Authentic human urine ( n = 3) and hair ( n = 2) samples from individuals with documented tiletamine exposure were analyzed using liquid chromatography coupled with Q Extractive HF hybrid quadrupole‐Orbitrap high‐resolution mass spectrometry (LC‐QE‐HF‐MS). Metabolites were detected and identified based on full‐scan MS and data‐dependent MS/MS (ddMS 2 ) fragmentation patterns. Structural elucidation, including the assignment of hydroxylation sites, was achieved through a diagnostic fragment ion analysis. Results A total of 14 urinary metabolites were identified, 11 of which (4 phase I and 7 phase II) were previously unreported. Hydroxylation was the predominant phase I process, whereas glucuronidation predominated among phase II reactions. Methylation and glucuronidation were identified as previously unreported metabolic pathways. In hair samples, tiletamine and four metabolites, including three reduced metabolites and T1, were identified for the first time. In addition, three metabolic transformations, namely, reduction, hydroxylation, and methylation, were newly identified in this matrix. Notably, T1 exhibited substantially higher signal intensities than all other metabolites across all urine samples. Conclusions This study substantially expands the known urinary metabolome by identifying novel phase I and II metabolites. The consistently high abundance of T1 in urine suggests that it is a promising urinary biomarker for monitoring tiletamine use. These findings broaden the range of analytical targets available for toxicological screening and confirmational analysis, thereby improving the detection and monitoring of tiletamine exposure.