MSACL 2024 Abstract

A Quantitative LC-MS/MS Method for Xylazine and Metabolites in Urine

Cody Orahoske(1,2),John Halifax(1,3),Kara Lynch(1,2)
(1) University of California, San Francisco, Department of Laboratory Medicine (2) Zuckerberg San Francisco General Hospital, (3) University of California, Berkeley, Department of Epidemiology

Cody Orahoske, Ph.D. (Presenter) University of California San Francisco

Presenter Bio: I’m a Clinical Chemistry Postdoctoral Fellow at the University of California, San Francisco, with a focus on clinical mass spectrometry, toxicology, and laboratory medicine. My work includes developing LC-MS/MS assays and improving test utilization to help bridge the gap between research and patient care. Outside the lab, I’m a husband and proud father, which has deepened my commitment to advancing healthcare. Before my career in science, I was a semi-professional boxer—an experience that taught me resilience and discipline, which I carry into my work today. I’m also an active member of MSACL, ADLM, and ASCP.

Relevant Financial Disclosures (within past 24 months, reported on Mar 19, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: The Center for Disease Control and Prevention （CDC） reports increasing xylazine prevalence in fatal overdoses nationally in the United States. Xylazine, a nonopioid sedative often used to adulterate illicitly manufactured fentanyl （IMF）, is associated with severe soft tissue wounds and increased overdose risk. The drug initially emerged in eastern United States, with widespread detection in Philadelphia overdose cases by 2019. Recently, xylazine has penetrated the drug supply on the West Coast, with a substantial increase noted through a surveillance program in San Francisco in June and July 2023. While existing literature has quantitated xylazine concentrations in human urine, the high variability of the drug supply regionally and temporally inhibits generalizability of these expected concentrations to patients on the West Coast. To establish a baseline range for urine concentrations in West Coast patients, we developed and validated a quantitative LC-MS/MS method with a wide dynamic range according to Scientific Working Group for Forensic Toxicology （SWGTOX） guidelines.

Objectives: The primary objective of this study was to develop a quantitative method for xylazine and two xylazine metabolites in the urine of exposed patients to establish expected concentrations in human urine.

Methods: The method was developed to quantitate xylazine and two of its metabolites,4-hydroxy-xylazine and 2,6-dimethylaniline. Briefly, chromatographic separation was achieved with a six-minute gradient elution from 20% to 90% mobile phase B using a using a Shimadzu Prominence LC-20 series liquid chromatography system and a Phenomenex C-18 2.3µm 100 Å50x 3mm Kinetex column. Data acquisition was performed using a Sciex Triple Quad 4500, operated in positive mode with multiple reaction monitoring. The mobile phases consisted of Honeywell Water LC-MS grade with 0.05% formic acid and 5mM ammonium formate as mobile phase A, and Honeywell Acetonitrile LC-MS grade with 0.05% formic acid as mobile phase B. The method validation adhered to SWGTOX guidelines 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.
De-identified urine aliquots from intake patients at the San Francisco Opiate Treatment Outpatient Program （OTOP） were analyzed using a clinically validated comprehensive drug test by high resolution mass spectrometry. Samples positive for xylazine （n=46） were thawed, vortexed, centrifuged, and then diluted 10-fold into a 96-well plate before being injected into the LC-MS/MS system for analysis.

Results: 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.

Conclusion: This method, validated in accordance with SWGTOX guidelines, is suitable for quantifying xylazine in urine samples. To date, other published studies of xylazine concentrations in human urine have been limited to the East Coast; this analysis of xylazine in human urine is the largest to date on West Coast patients, providing crucial baseline context to local clinicians as they grapple with uncertain health outcomes in xylazine-exposed patients.