A quantitative LC-MS/MS analysis of Xylazine, p-Fluorofentanyl, Fentanyl and Fentanyl-Related compounds in postmortem blood

•4-ANPP, acetyl fentanyl, fentanyl, p-fluorofentanyl, norfentanyl, and xylazine quantitated.•O- and m-fluorofentanyl qualitatively identified.•Analytes extracted from postmortem blood using solid-phase extraction.•Analytes separated and detected by liquid chromatography tandem mass spectrometry.•Results provide valuable information to the forensic toxicology community. The purpose of this study was to develop and validate a method to quantitate the veterinary sedative xylazine as well as 4-anilino-N-phenethylpiperidine (4-ANPP), acetyl fentanyl, fentanyl, norfentanyl, and p-fluorofentanyl in blood utilizing liquid chromatography tandem mass spectrometry. This method also qualitatively monitors for the presence of o-fluorofentanyl and m-fluorofentanyl isomers. UCT Clean Screen® DAU extraction columns were utilized to isolate the analytes in postmortem blood samples. The extracts were eluted, evaporated, reconstituted, and then analyzed using a Waters Acquity™ UPLC coupled a triple quadrupole mass spectrometer. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes, except for xylazine (0.2 ng/mL). The upper limit of quantitation for all analytes was 100 ng/mL. No interferences from matrix, internal standard, or common drug analytes were observed. Bias (-13.1–4.6 %) and precision (-13.14–10.3 %) fell within the acceptable ± 20 % criteria range. Dilution integrity at x2, x10, and x100 was evaluated and all results were within ± 20 % of the target concentration. Processed extract stability was evaluated after 72 h and all results were within ± 20 % of the analyte initial concentration. Matrix effects were the most prominent with xylazine, but deemed acceptable as the deuterated internal standard also observed comparable enhancement. Analysis of 89 postmortem blood samples by this method resulted in positive results for fentanyl (0.27–66 ng/mL, n = 82), xylazine (0.24–958 ng/mL, n = 21), 4-ANPP (0.10–38 ng/mL, n = 72), acetyl fentanyl (0.18–1.5 ng/mL, n = 3), p-fluorofentanyl (0.11–33 ng/mL, n = 30), and norfentanyl (0.10–98 ng/mL, n = 73).