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- Opioid detection and quantification in plasma and oral fluid by LC-MS/MS.Publication . Rosendo, Luana M; Costa, Suzel; Simões, Susana; Franco, João; Serrano Gadea, Noelia; Escorial, Mónica; Toboso Ortega, Francisco Javier; Jiménez-García, Segundo; Peiró, Ana M; Duque, Isabel; Rosado, Tiago; Barroso, Mário; Gallardo, EugeniaThe opioid crisis remains a significant public health concern, necessitating the development of sensitive and reliable analytical methods for drug detection. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of fentanyl, buprenorphine, oxycodone, morphine, tramadol, and tapentadol in plasma and oral fluid. The method was validated according to FDA guidelines, assessing selectivity, linearity, precision, accuracy, matrix effect, extraction efficiency, stability, carryover, and dilution integrity. The lower limits of quantification (LLOQs) were established at 0.1 ng/mL for fentanyl, 1.2 ng/mL for tramadol, and 0.6 ng/mL for the remaining opioids, demonstrating high sensitivity. The method exhibited excellent precision and accuracy, with coefficients of variation below 15% for intra-day, inter-day, and intermediate precision analyses. Extraction efficiencies exceeded 90% for most analytes, and matrix effects remained within acceptable limits. Real-world application to authentic plasma and oral fluid samples confirmed the method's robustness and reliability. Oral fluid concentrations were detectable across all target opioids, although plasma-oral fluid ratios showed some compound-dependent variability. These findings highlight the potential of oral fluid as a non-invasive complementary matrix to plasma for opioid monitoring, with relevant implications for forensic toxicology and clinical drug monitoring.