Development and Validation of an Oral Fluid LC–MS/MS Method for Screening Emerging Synthetic Opioids Including Fentanyl Analogues, Nitazenes, and Xylazine

The emergence of highly potent synthetic opioids, including fentanyl analogues, nitazenes, and xylazine-associated drug mixtures, has created significant analytical challenges for forensic and toxicological laboratories. The aim of this study was to develop and validate a qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the screening of emerging synthetic opioids in oral fluid. Sample preparation was based on direct extraction of paper swabs with 500 μL of methanol containing 1% formic acid, followed by vortex mixing, ultrasonication, centrifugation, and direct LC-MS/MS analysis without evaporation or reconstitution. Method validation included selectivity and specificity, detection capability, matrix effects, inter-individual matrix variability, processed-sample stability, carryover, and intra-day precision. All target analytes fulfilled the established identification criteria at 1 ng/mL, the lowest concentration evaluated. Intra-day precision was acceptable, with RSD values ranging from 0.6% to 14.8%. Matrix effects were within 80-120% for the majority of analytes at both evaluated concentration levels, although compound-dependent ion suppression or enhancement was observed. Processed samples met the predefined stability criterion for 48 h at 4 °C and 72 h at - 20 °C, and carryover remained below the predefined acceptance criterion. Limited inter-individual variability was observed among the five donor matrices for the subset of analytes evaluated. The developed method provides a simple qualitative screening approach for an expanded panel of emerging synthetic opioids in oral fluid. Evaluation using authentic positive specimens is required to further establish its performance in forensic and DUID applications.