Clean Screen® DAU Featured in Method to Analyze Xylazine, p-Fluorofentanyl, Fentanyl, and Fentanyl-Related Compounds in Postmortem Blood

The presence of xylazine continues to be on the rise in samples containing fentanyl or fentanyl-related substances. A recent method published in the Journal of Chromatography B uses SPE and LC/MS-MS to quantitate xylazine, 4-ANPP, acetyl fentanyl, fentanyl, norfentanyl, and p-fluorofentanyl. Xylazine is rarely abused on its own, and research suggests it has effects comparable to heroin when used in conjunction with opioids. 4-ANPP is both a precursor and a metabolite to fentanyl, so it is often monitored in fentanyl-related analyses. Acetyl fentanyl and p-fluorofentanyl are fentanyl-related substances frequently observed in forensic casework.

The analysis method was fully validated based on the guidelines provided by the American Academy of Forensic Sciences (AAFS) Academy Standards Board (ASB) Standard Practices for Method Development in Forensic Toxicology. Samples were prepared by adding 2 mL pH 6 phosphate buffer to 1 mL of whole blood. The samples were mixed and centrifuged before loading onto pre-conditioned Clean Screen^® DAU cartridges. The cartridges were washed and dried before the analytes were eluted with a mixture of dichloromethane, isopropanol, and ammonium hydroxide (78:20:2). The final eluate was analyzed via LC-MS/MS.

This validated method was then used to evaluate 89 authentic postmortem samples. The concentration ranges for each analyte include fentanyl (0.27 – 66 ng/mL), xylazine (0.24 – 958 ng/mL), 4-ANPP (0.10 – 38 ng/mL), acetyl fentanyl (0.18 – 1.5 ng/mL), p-fluorofentanyl (0.11 – 33 ng/mL), and norfentanyl (0.10 – 98 ng/mL). Other common analytes seen in these cases include ethanol, methamphetamine, cocaine, and benzoylecgonine. Xylazine was detected in 23% of the cases evaluated. This validated method provides a means to monitor xylazine, fentanyl, and fentanyl-related substances to supply the forensic toxicology community with information and help improve public health.

Citation: M.T. Truver, S.C. Brogan, E.A. Jaeschke, A.M. Kinsey, J.L. Hoyer, C.W. Chronister, M.M. Crosby, B.A. Goldberger, A Quantitative LC-MS/MS Analysis of Xylazine, p-Fluorofentanyl, Fentanyl and Fentanyl-Related Compounds in Postmortem Blood, Journal of Chromatography B (2024), doi: https:// doi.org/10.1016/j.jchromb.2024.124059