Kaewkhao N.Tarning J.Blessborn D.Mahidol University2025-11-292025-11-292025-01-01International Journal of Analytical Chemistry Vol.2025 No.1 (2025)16878760https://repository.li.mahidol.ac.th/handle/123456789/113300Nirmatrelvir, a key antiviral agent in the treatment of COVID-19, requires accurate and reliable monitoring of drug levels to optimize therapeutic efficacy. In this study, we developed and validated a sensitive and specific LC-MS/MS method for the quantification of nirmatrelvir in human plasma. The method includes nirmatrelvir-D9 as an internal standard, with quantification achieved using selected reaction monitoring in positive electrospray ionization mode, targeting m/z 500.3 ⟶ 110.1 for nirmatrelvir and m/z 509.3 ⟶ 110.1 for nirmatrelvir-D9. Sample preparation involved a simple phospholipid removal step using 96-well plate and automated liquid handler, which improved efficiency in a high-throughput process. The validated method, following international bioanalytical guidelines, demonstrated a linear range from 10.9 to 3013 ng/mL. Intra- and interassay precisions were both below 15%. All validation tests meet the criteria for matrix effect, carryover, dilution integrity, and stability. The method offers a rapid analysis time of 2 min per sample and provides highly accurate, reproducible results, making it a valuable tool for evaluating the pharmacokinetics of nirmatrelvir in clinical settings. Trial Registration: ClinicalTrials.gov identifier: NCT05041907.ChemistryArticleSCOPUS10.1155/ianc/66258332-s2.0-10502259339816878779