Determination of xylazine, xylazine-4-hydroxy, and 2,6-dimethylaniline in canine urine: veterinary and forensic applications in two suspected poisoning cases.

PURPOSE: Xylazine, an α2-adrenergic agonist, is widely used in veterinary medicine for anesthesia, sedation, and analgesia. However, non-therapeutic use, overdose, and deliberate administration may pose serious health risks to animals, necessitating reliable detection methods for both clinical and forensic purposes. METHODS: In this study, a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed and fully validated for the determination of xylazine and its metabolites in canine urine. RESULTS: The method exhibited excellent selectivity and linearity over the range of 0.05-100 ng/mL, with coefficients of determination (R²) ≥ 0.9989. Recovery values ranged from 70.3% to 93.4%, with matrix effect values between 78.6% and 95.6%, and process efficiency values ranging from 74.9% to 94.3%. Precision and accuracy values were within ± 15% at all quality control levels, and the analytes were stable under all tested conditions (+ 8 °C for 3 days, + 4 °C and - 20 °C for 5 days). In two forensic cases, urine samples collected from canines presenting with unconsciousness contained xylazine and its metabolites, while no other sedative or toxic agents were detected. CONCLUSION: The presence of both 2,6-dimethylaniline (DMA) and 4-hydroxyxylazine supports the necessity for including metabolite analysis in exposure assessment. This study highlights the applicability of LC-HRMS for the detection of xylazine-related compounds in canine urine and its potential use in veterinary and forensic toxicology contexts.