A Quantitative LC-MS/MS Method for Xylazine and Metabolites in Urine
Authors: Orahoske Cody , Lynch Kara , Halifax John 
Published: October 2024

Abstract
The prevalence of xylazine in fatal overdoses is on the rise across the United States, as reported by the Center for Disease Control and Prevention (CDC). Xylazine, a nonopioid sedative commonly used to adulterate illicitly manufactured fentanyl (IMF), poses severe health risks, including soft tissue wounds and increased overdose potential. Originally emerging in the eastern United States, its presence became widespread in Philadelphia overdose cases by 2019. Recently, there has been a notable increase in xylazine infiltrating the drug supply on the West Coast, as evidenced by a surveillance program in San Francisco in June and July 2023. Existing literature quantifying xylazine concentrations in human urine faces limitations due to the high variability in the drug supply regionally and temporally, hindering generalizability to West Coast patients. To address this gap, we developed and validated a quantitative LC-MS/MS method following Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines to establish baseline urine concentrations in West Coast patients. The primary objective of this study was to develop a quantitative method for analyzing xylazine and two metabolites in the urine of exposed patients to establish expected concentrations. Chromatographic separation utilized a Shimadzu Prominence LC-20 series liquid chromatography system and a Phenomenex C-18 2.3µm 100 Å 50x3mm Kinetex column. Data acquisition employed a Sciex Triple Quad 4500. Mobile phases included Honeywell Water LC-MS grade and Honeywell Acetonitrile LC-MS grade. Xylazine and metabolites were detected using multiple reaction monitoring with the following transitions: xylazine 221.1/164.1 and 221.1/90.0,4-hydroxy-xylazine 237.1/137.1 and 231.1/136.1,2,6-dimethylaniline 121.9/105.1 and 121.9/77.0. Validation followed SWGTOX guidelines, and de-identified urine samples (n = 46) from the San Francisco Opiate Treatment Outpatient Program were analyzed.The method was linear from 1 to 10,000 ng/ml, established through triplicate analyses over five days, with an R2 value of 0.9972. The lower limit of quantification for xylazine was determined to be 1 ng/ml, with a coefficient of variation (CV) of 10.01% across 20 samples. Intra-day and inter-day precision was assessed using three different spiked pools representing low, medium, and high values, yielding CV values of 8.71%,7.45%, and 5.99%, respectively for inter-day precision. The intra-day CV ranged from 0.14% to 11.86%,0.82%to 5.37%, and 0.92% to 7.05% over the course of five days. In patients, the concentration of xylazine ranged from 4 to 3,789 ng/ml, with only one patient sample showing the presence of the metabolite 4-hydroxy-xylazine, while there was no detection of 2,6-dimethylaniline in this sample set. The LC-MS/MS method, validated according to SWGTOX guidelines, is suitable for quantifying xylazine in urine samples. This study, the largest on West Coast patients to date, provides essential baseline information for local clinicians dealing with the health implications of xylazine exposure, expanding knowledge beyond the East Coast-focused literature.