MSACL 2024 Abstract

Introduction: The most common cause of drug overdose fatality in the United State involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl adulterated or associated with xylazine as an emerging threat to the United States, and a national response plan will include xylazine testing. Xylazine, is a clonidine analog and is not FDA-approved for use in humans, but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers an effective way to detect xylazine in patient samples with high sensitivity and specificity.

Objectives: The objectives of this study are to develop a LC-MS/MS method to detect xylazine metabolites in urine, and to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing.

Methods: Remnant urine samples were used for this study under IRB approval with waiver of informed consent. Samples were identified when they tested positive for xylazine during routine clinical testing by LC-MS/MS. Remnant urine samples were diluted with xylazine-D6 internal standard (Cayman Chemical), and hydrolyzed using B-One β-glucuronidase (KURA biotech). Hydrolysis efficiency was quantified using codeine-6-glucuronide standard (Cerilliant) prepared in negative urine at 5,000 ng/mL. Liquid chromatographic separation utilized a 100 mm x 2.1 mm, 2.6 μm Kinetex C18 column (Phenomenex) with a 4 minute gradient ramping from 2% to 98% mobile phase B (A: 0.1% formic acid and 2mM ammonium acetate in water; B: 0.1% formic acid and 2mM ammonium acetate in methanol) with a flow rate of 400 μl/min. The column temperature was held at 50°C. Xylazine and 4-OH xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards obtained from Sigma Adrich, and/or Cayman Chemical (xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137). Xylazine and 4-OH xylazine were quantified using calibration curves prepared at 5, 50, 500, and 5000 ng/mL in negative urine. Product ion scanning was used to detect four additional xylazine metabolites, based on spectral matching to previously published MS/MS spectra, using remnant urines with high concentrations of xylazine.1 Based on the collected product ion scanning experiments, the highest abundance fragment ions were used to perform multiple reaction monitoring for the proposed sulfone xylazine (253>181, 253>147), oxo xylazine (235>122, 235>114), OH-sulfone xylazine (269>197, 269>163), and OH-oxo xylazine (251>197, 251>148) metabolites in all remnant urines using chromatography and retention times consistent with the product ion scanning experiments. All experiments were performed using an Aquity HPLC coupled to a TQS-micro triple quadrupole mass spectrometer (Waters).

Results: During the study period, 138 urines screened positive for fentanyl by immunoassay which were reflexed to confirmatory testing by LC-MS/MS. Xylazine was detected at >=1 ng/mL in 38% of these urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested for xylazine. Xylazine was detected in only 1 urine which did not screen positive for fentanyl but contained methamphetamine and amphetamine.
Xylazine concentrations quantified in remnant urines ranged from <5 ng/mL to 10,729 ng/mL. 4-OH xylazine concentrations ranged from <5 ng/mL to 219 ng/mL. We observed five previously reported xylazine metabolites in urines containing xylazine: 4-OH xylazine, oxo xylazine, sulfone xylazine, OH-oxo xylazine, OH-sulfone xylazine Hydrolysis of urines with glucuronidase increased the peak areas of xylazine metabolites containing hydroxyl function groups: 4-OH xylazine, OH-oxo xylazine, OH-sulfone xylazine. The mean concentration ratio of 4-OH xylazine-to-xylazine was 0.2. The mean peak area ratios of metabolite-to-xylazine for sulfone xylazine, OH-oxo xylazine, OH-sulfone xylazine, and oxo xylazine were 3.1, 1.4, 0.5, and <0.1, respectively. The proposed sulfone xylazine metabolite was observed to have the most abundant signal of all detected metabolites in the majority of urines, although there is currently no standard available to enable quantification or confirmation of this metabolite structure.

Conclusions: Xylazine and 4-OH xylazine were quantitated in patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in urine and serum and association of xylazine with clinical and demographic variables.

References:
1.Meyer GMJ, Maurer HH. Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MSn, and LC–HR-MSn. Anal Bioanal Chem. 2013;405(30):9779-9789. doi:10.1007/s00216-013-7419-7.