Characterization, optimization, and selection of identification criteria for LC–QTOF–MS

The establishment of stringent identification criteria is essential for accurate reporting of toxicological drug testing, particularly in forensic settings involving medico-legal cases. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is widely employed for its broad analyte coverage and high mass accuracy, yet limited published and validated identification criteria pose significant challenges for its use beyond presumptive screening in low case volume settings. This study characterized, optimized and selected LC-QTOF-MS identification criteria, assessing the influence of concentration, matrix and drug class on their performance. In addition to standard identification parameters, an effective combined weight score (CWS) threshold that emphasized library score and mass error was established. Higher analyte concentrations improved spectral reproducibility, while urine matrices introduced variability in isotope ratios and library scores. Authentic casework demonstrated 99.9% efficiency, 98.9% sensitivity, and 100% specificity, indicating a highly reliable method that achieves excellent accuracy, minimizes false positives as required for confirmatory techniques, and maintains sufficient sensitivity for effective screening of casework, thereby supporting robust and defensible forensic toxicology workflows. These findings also highlight the importance of refining LC-QTOF-MS specific identification criteria to enhance consistency and reliability in forensic toxicology reporting, and allows for reproducibility across other instrumentation, workflows, and fields.