Direct-to-Definitive Urine Drug Testing Panel with Addition of Xylazine and 4-Hydroxy Xylazine Identification and Quantitation

Significance of the Topic

Xylazine misuse is rising as a non-opioid CNS depressant appearing in illicit drug supplies, leading to increasing overdose fatalities and impaired driving incidents. Improved forensic screening for xylazine and its major metabolite in urine is critical to detect adulteration and monitor emerging public health threats.

Objectives and Study Overview

This work extends an existing direct-to-definitive UHPLC-MS/MS urine panel of 102 analytes by incorporating xylazine and 4-hydroxyxylazine. The study validates the updated method and demonstrates its application to forensic case samples.

Applied Instrumentation

• Waters ACQUITY UPLC I-Class PLUS System with flow-through-needle injector
• Waters Xevo TQD Triple Quadrupole Mass Spectrometer
• ACQUITY UPLC BEH Phenyl Column (1.7 µm, 2.1 × 50 mm)
• MassLynx and TargetLynx XS Software for data acquisition and quantification

Applied Methodology

Urine aliquots were analyzed using the Threshold Accurate Calibration (TAC) technique, with dual injections of each sample “neat” and spiked at known concentration. Samples underwent enzymatic hydrolysis (beta-glucuronidase) and were mixed with methapyrilene as an injection recovery control. Chromatographic separation used a gradient of 2 mM ammonium formate in water (with 0.1% formic acid in methanol) at 0.6 mL/min and 45 °C. Electrospray ionization in positive mode and optimized MRM transitions enabled detection of xylazine and 4-hydroxyxylazine. Calibration ranged from 38–500 ng/mL with quality controls at 38, 200, and 400 ng/mL.

Main Results and Discussion

Validation met ANSI/ASB and NY State guidelines: precision (%CV 6.8–10.1%) and bias (−11.5 to −2.2%) were acceptable. Calibration was linear (R² ≥ 0.994), carryover < 3%, and matrix effects showed 7–18% ion enhancement with ≤ 13% variability. The limit of detection was 10 ng/mL based on TAC ratio criteria. Process stability and dilution integrity met acceptance thresholds. Casework application identified urinary xylazine concentrations from 58 to 3860 ng/mL and 4-hydroxyxylazine from < 38 to 168 ng/mL, confirming significant glucuronide conjugation and co-occurrence with fentanyl.

Benefits and Practical Applications

• Matrix normalization without requiring individual isotopic standards for each analyte
• Simultaneous detection of over 100 drugs and metabolites, including emerging threats
• Streamlined workflow for direct-to-definitive forensic urine screening
• Enhanced sensitivity and reliable quantitation of xylazine and its metabolite

Future Trends and Opportunities

Broader adoption of TAC-based normalization may improve multi-analyte screening across diverse matrices. Automation of sample preparation and data processing can boost throughput. Expansion to additional novel psychoactive substances will strengthen forensic surveillance and public health monitoring.

Conclusion

The UHPLC-MS/MS panel has been successfully updated to include xylazine and 4-hydroxyxylazine with fully validated performance, enabling robust direct-to-definitive urine testing. This approach enhances forensic laboratories’ ability to detect emerging drug threats.

References

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• Canadian Centre on Substance Use and Addiction. CCENDU Drug Alert: Xylazine, 2022.
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• Teoh et al. Simultaneous Detection of Xylazine and Other Drugs in Beverages. Australian Journal of Forensic Sciences, 2022;55:708–719.
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• ANSI/ASB Standard 036. Standard Practices for Method Validation in Forensic Toxicology. 2019.
• New York State Clinical Laboratory Standards. Wadsworth Center, 2024.