MSACL 2024 Abstract

Development of LC-MS/MS Xylazine Assay for Investigation of Xylazine Prevalence in New Haven, CT Region Leah Militello, Gina Cassella-Mclane, Myles Mcgowan, Rebecca Anson, Joe M. El-Khoury Yale University Leah Militello, MD MBA (Presenter) Yale University >> POSTER (PDF)

Presenter Bio: I am a board-certified clinical pathologist currently completing a chemical pathology fellowship at Yale University. Next year I will be taking on the roles of Director of Clinical Chemistry, Director of Point of Care Testing, and Medical Director of the MLS Training Program at Rochester General Hospital. My interests include toxicology, LC-MS/MS, and quality improvement. With my background as a former accountant and an MBA with a concentration in health care administration, I consider it my life goal to help the clinical laboratory become a more visible part of healthcare and be recognized as the profit center that it is.

Introduction: Xylazine is a veterinary tranquilizer not approved for human use that has been implicated in an increasing number of overdose deaths nationwide. It is used as an adulterant in recreational drugs of abuse, including heroin, cocaine and fentanyl, and many users are exposed unknowingly. As an α2-adrenergic agonist, its physiologic effects include sedation, muscle relaxation and decreased perception of pain. Taken in combination with other drugs, especially other central nervous system depressants like alcohol or benzodiazepines, the effects can be lethal. Xylazine was involved in 10% of all drug overdose deaths in Connecticut in 2020. Typical drug abuse screening panels do not include an assay capable of xylazine detection, as no immunoassay designed to run on an automated analyzer has been FDA-approved thus far. There is increasing demand from providers in the emergency department for an assay to determine the presence of xylazine in intoxicated patients with unclear clinical presentation. Here we describe a laboratory-developed xylazine assay and investigate the presence of xylazine in patient samples that screened positive for other drugs of abuse within the geographic region served by our hospital system. This method also examined the impact of measuring the xylazine metabolite, 2,6-dimethylaniline, on assay sensitivity.

Objectives: Develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for detection of xylazine and use that assay to examine the prevalence of xylazine in the New Haven, CT region.

Methods: A total of 37 patient urine samples that screened positive by immunoassay for cocaine, opiates and/or fentanyl between October and November 2023 were selected for the study and subject to retrospective analysis. Sample preparation consisted of a centrifugation step, addition of the internal standard, methadone-d9, followed by vortexing and another centrifugation step. Mobile phase A was 0.1 % formic acid in water and mobile phase B was 0.1 % formic acid in acetonitrile. Liquid chromatography (LC) was performed using an Acquity UPLC HSS column (T31.8 µm, 2.1 x 100 mm) at 50°C. MS/MS used positive electrospray ionization and monitored m/z transitions of 221 > 164 for xylazine (primary), 221 > 90 for xylazine (qualifier), and 319 > 105 for methadone-d9 (IS). The measurement of the xylazine metabolite 2,6-dimethylaniline had m/z transitions of 122 > 105 (primary) and 122 > 107 (qualifier). Retention time was 1.94 minutes for xylazine, 2.26 minutes for 2,6-dimethylaniline, and 2.16 minutes for the methadone-d9 internal standard. Linearity was established between 1-1000 ng/mL.

Results: Xylazine was detected a total of 9 out of 37 patient samples, or 24%. Of these samples, one was positive exclusively for opiates and three were positive exclusively for cocaine. Two samples were positive for both cocaine and fentanyl, two were positive for both cocaine and opiates, and one was positive for both fentanyl and opiates. All but one of the opiate positive specimens confirmed positive for 6-MAM, indicating heroin use. The quantity of xylazine ranged from 6 ng/mL to greater than 1000 ng/mL, with a mean value of 215 ng/mL and a median of 24 ng/mL. The results of 2,6-dimethylaniline were all significantly lower than xylazine and it was not found to add value in addition to xylazine.

Conclusions: Analysis by LC-MS/MS showed a high prevalence (24%) of xylazine in patient urine samples that screened positive for cocaine, opiates, fentanyl or a combination of these drugs. This is consistent with the Center for Disease Control and Prevention (CDC) data reporting that around 30% of fentanyl overdoses have also been found to contain xylazine. This may indicate a high level of adulteration by xylazine in the illegal drug supply in New Haven. Measurement of the metabolite 2,6-dimethylaniline did not add value, so laboratories looking to develop assays for xylazine do not need to measure this metabolite.