Development of a quantitative LC-MS/MS method for assessment of xylazine in urine

Xylazine, an alpha 2 adrenergic agonist approved for veterinary use, has become an increasingly common adulterant in the illicit drug supply and is frequently detected with fentanyl and other substances, exacerbating opioid epidemic. Because xylazine is not included in many routine toxicology screens, a practical quantitative urine method is needed for clinical testing and surveillance. We developed and validated a quantitative dilute and shoot method for xylazine in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with water (containing 0.1% formic acid) and methanol (containing 0.1% formic acid) as the aqueous and organic mobile phase, respectively. The assay was linear from 20 to 5000 μg/L, with administratively set limit of quantitation (LOQ) of 20 μg/L and cutoff of 40 μg/L. In 94 authentic urine samples, 11 were above the LOQ (two of which were below the cutoff), with a maximum concentration of 4298 μg/L. The validated method provides robust quantitative measurement of xylazine in urine across clinically relevant concentrations and is well suited for high throughput clinical toxicology and surveillance testing.