B-278 Xylazine Detection in Clinical Urine Specimens Ordered for Comprehensive Drug Screening

Abstract Background Xylazine, a non-opioid tranquilizer, is not approved for human use but has been detected in the US illegal drug supply. Fentanyl adulterated with xylazine was declared an emerging threat by the White House Office of National Drug Control Policy in April 2023. According to the DEA’s laboratory system, between 2020 and 2021 the northeast census area had the highest number of instances of exhibits involving xylazine in the US. In 2024, we added xylazine to our urine comprehensive drug screen offering. The objectives of the current retrospective study were to review data for specimens received for testing in 2024 and 1. Determine the number positive for xylazine and identify other drugs present in these specimens and 2. Determine the contribution of xylazine to the total number of positives specimens for these other drugs. Methods Data for this study were from deidentified urine specimens from New England that were consecutively analyzed using a qualitative comprehensive urine drug screen by a CLIA-validated gas chromatography mass spectrometry (GC/MS) method. Specimens were from medical centers, including urgent care, community health centers, and primary care providers. Specimens were subjected to liquid-liquid extraction with pH adjustment to 8-10, clomipramine as internal standard and dichloromethane as organic solvent. After dry down at 40°C under nitrogen, extracts were reconstituted with ethyl acetate and injected onto an Agilent 7890 GC with 5975 MS using a HP1, 0.2mm ID, 12m, 0.33µfilm, cross linked methyl silicone column. A ramped temperature program from 70° C to 280° C was used with the MS operating in full scan mode. Positive and negative quality control samples were assayed with each batch. This assay was validated for the detection of 99 drugs and metabolites. Compounds were identified by a relative retention time ±0.03 of a library reference match and a mass spectral match of at least 80%. Results During the study 52 specimens were positive for xylazine. The most common other drugs present in these specimens were fentanyl (75%); cocaine and/or metabolites (including cocaethylene, [56%]); ketamine and norketamine (25%); diphenhydramine (19%) and methadone and/or metabolite (15%). Other less prevalent drugs detected included lidocaine, acetaminophen, amphetamine and methamphetamine. Evaluation of all the xylazine-positive specimen showed a positivity of 25.6% for fentanyl positives, 16.7% for cocaine/ metabolites, 13.8% for ketamine/norketamine, 4.9% for diphenhydramine, and 6.5% for methadone/metabolite. Conclusion These data suggest that xylazine may be present in illicit drug sources other than fentanyl. Xylazine has adverse side effects, including hypotension, bradycardia, respiratory depression, physical dependence, and potential soft tissue injuries when injected that may cause necrotic tissue. Therefore, laboratories who perform drug testing should consider adding xylazine to their testing menu.