LC-MS/MS Method Detects Six Opioids Simultaneously in Plasma and Saliva

The global opioid crisis, responsible for approximately 600,000 deaths in 2019 alone — with nearly 80% opioid-related — has created an urgent need for analytical tools capable of reliably detecting and quantifying multiple opioids across biological matrices. In the United States, synthetic opioids like fentanyl contributed to roughly 75,000 deaths in a single year, while Europe faces growing concerns around diversion and misuse of prescription opioids for pain management. Accurate, multi-analyte detection methods are essential both for clinical therapeutic drug monitoring and forensic investigation.

This study developed and validated an LC-MS/MS method targeting six clinically and forensically relevant opioids — fentanyl, buprenorphine, oxycodone, morphine, tramadol, and tapentadol — simultaneously in both plasma and oral fluid. The analytical platform used a Waters Acquity UPLC system with an HSS T3 column (100 × 2.1 mm, 1.8 μm) at 45°C, coupled to a triple quadrupole mass spectrometer operating in positive ESI mode via multiple reaction monitoring (MRM). Two deuterated internal standards, morphine-d3 and methadone-d3, were employed to correct for matrix effects and extraction variability. The total chromatographic runtime was 14 minutes, with retention times ranging from 1.33 minutes (morphine) to 6.06 minutes (methadone-d3).

Sample preparation used a simple protein precipitation approach: 0.1 mL of sample was diluted with 0.1 mL methanol:water (1:1, v/v), making the protocol accessible for routine laboratory workflows. Validation followed FDA bioanalytical method guidelines and assessed selectivity, linearity, intra-day and inter-day precision, accuracy, matrix effects, extraction efficiency, stability, carryover, and dilution integrity. Lower limits of quantification (LLOQs) were set at 0.1 ng/mL for fentanyl, 1.2 ng/mL for tramadol, and 0.6 ng/mL for the remaining four opioids — demonstrating sensitivity adequate for therapeutic and toxicological concentrations. The calibration range spanned 0.1 to 300 ng/mL across all analytes.

Precision and accuracy results were excellent, with coefficients of variation (CV) consistently below 15% for intra-day, inter-day, and intermediate precision analyses across all analytes in both matrices. Extraction efficiencies exceeded 90% for the majority of compounds, and matrix effects remained within acceptable regulatory limits. Stability testing confirmed that samples stored at -20°C maintained analyte integrity through multiple freeze-thaw cycles and extended storage. Carryover was negligible, and dilution integrity was confirmed, supporting the method's applicability across a wide concentration range encountered in real-world samples.

The method was applied to authentic plasma and oral fluid specimens collected from pain management patients at centers in Spain. All six target opioids were detectable in oral fluid, validating the practical utility of this non-invasive matrix. However, plasma-to-oral-fluid concentration ratios showed compound-dependent variability, reflecting differences in drug physicochemical properties, protein binding, salivary pH, and flow rate. This variability underscores that while oral fluid is a valuable complementary matrix, direct plasma-oral fluid concentration equivalence cannot be assumed for all opioids. The authors note this is the first published method simultaneously validating all six of these opioids in both matrices, representing a meaningful advance for integrated clinical and forensic monitoring programs.