B-268 Detection of xylazine in fentanyl-positive urine drug screen specimens using a quantitative LCMS/MS method
Authors: Mills C , Dryja P , Babic N 
Published: October 2024

Abstract

Background
Fentanyl, a synthetic opioid used clinically for severe pain management, sedation, and as a perioperative analgesic, has been at the forefront of fatal drug-involved overdoses for almost a decade. Xylazine, an α-2 adrenergic receptor agonist, is an increasingly common adulterant in illicitly manufactured fentanyl products and analogs. Given its class, xylazine is non-responsive to naloxone, an opioid receptor antagonist used to reverse opioid overdose. Importantly, the U.S. Drug Enforcement Administration reported xylazine in 23% of seized fentanyl powders and 7% of fentanyl pills; additionally, a recent study by Kariisa et. al. identified an increase of xylazine presence in fatal fentanyl-involved overdoses (2.9% in January 2019 to 10.9% in June 2022) across twenty states.1 Collectively, these trends indicate a need for increased surveillance of xylazine in overdose-presenting patients to inform appropriate clinical responses. In this study, we developed a sensitive LC-MS/MS method for xylazine quantitation and used it to determine xylazine presence in our fentanyl positive patients.

Methods
An LC-MS/MS method for quantitation of xylazine from urine samples was developed using a Waters Xevo TQ-XS and reversed-phase CORTECS C8 2.7 µm column. MRMs and optimal MS tuning parameters were generated for xylazine and xylazine-d6 via IntelliStart, with transition ions of 221.18→90.04 (Quan) and 221.18→136.31 (Qual) selected for xylazine quantitation against a 227.23→89.98 xylazine-d6 transition as an internal standard. An LC method was developed using water (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) and methanol (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) gradient for a 4-minute runtime. Calibrators were prepared by addition of the pure drug to water, while QCs were prepared using healthy volunteer urine that was confirmed negative in a routine urine drug screen (UDS). Calibrators were value-assigned using a reference laboratory. A simple processing method consisting of the addition of 200 µL methanol containing 25 ng/mL xylazine-d6 to 50 µL of standards, QCs, or urine was used for sample preparation. Remnant fentanyl-positive patient samples (determined by ARK Fentanyl II Immunoassay - Abbott Architect C16000 analyzer) submitted for routine UDS evaluation were collected for xylazine analysis.

Results
Using custom xylazine standards, the method yielded a linear range from 2 ng/mL to 500 ng/mL with an average R2 of 0.9995 across runs (n = 12). The LLoQ for this assay was 2 ng/mL with a CV of 18.1%. The intra-assay CVs were &lt; 5.0% and inter-assay CVs were &lt; 15.0%. Of preliminary patient samples tested (n = 25), xylazine was detected in 32% of fentanyl-positive samples, ranging in concentration from 2.8 ng/mL to 192.8 ng/mL with an average of 64.8 ng/mL.

Conclusions
Here we report a simple LC-MS/MS method for quantitation of xylazine in remnant fentanyl-positive UDS specimens by LC-MS/MS. Preliminary testing revealed a xylazine positivity rate of 32% in fentanyl-positive patients. Uncomplicated sample work-up makes this method applicable to xylazine surveillance in all UDS-positive patient samples in high-throughput clinical laboratories.
Reference:
1. Kariisa M, et. al. “Illicitly Manufactured Fentanyl-Involved Overdose Deaths with Detected Xylazine.” MMWR Morb Mortal Wkly Rep 2023;72:721-727.