Xylazine and major urinary metabolites detected in patients positive for fentanyl and xylazine

Xylazine in an anesthetic drug used for the sedation of animals that is increasingly appearing as an adulterant in uncontrolled drug supplies, primarily illicit fentanyl. The ability to detect xylazine exposure by urine drug testing may improve monitoring of this drug trend and our understanding of the effects and risks associated with xylazine exposure. Currently, limited information is available regarding the elimination of xylazine or its metabolites in humans. In this study we report quantification of xylazine and 4-hydroxy-xylazine (4-OH-x) in hydrolyzed urine specimens collected from 109 patients testing positive for fentanyl and xylazine using liquid chromatography tandem mass spectrometry (LC-MS/MS). 4-OH-x was a minor urinary metabolite in most patients with a median metabolite-to-xylazine (MR) concentration ratio 0.09. Additional urinary metabolites were identified including oxo-xylazine (oxo-x), OH-oxo-xylazine (OH-oxo-x), OH-sulfone-xylazine (OH-sulfone-x), and sulfone-xylazine (sulfone-x), with median MR peak area ratios of < 0.01, 0.60, 0.30, and 1.60, respectively. Sulfone-x signal exceeded that of xylazine in more than 70% of urine specimens. Sulfone-x is not glucuronidated and does not appear to form positional isomers. Additional studies are needed to examine whether detection of xylazine metabolites may improve the sensitivity and/or extend the detection time window for xylazine exposure.