A validated bioanalytical LC-MS/MS method for the simultaneous determination of molnupiravir, N-hydroxycytidine, and baricitinib in human plasma.
A sensitive and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of the anti-COVID-19 agents molnupiravir (MPV), its active metabolite N-hydroxycytidine (NHC), and baricitinib (BARI) in human plasma. As MPV is rapidly converted to NHC in vivo, whereas BARI is predominantly eliminated unchanged, NHC was generated in vitro by alkaline hydrolysis to enable accurate determination of the active metabolite. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column using methanol and 0.1% formic acid in water (90:10, v/v). Escitalopram was selected as the internal standard owing to its consistent extraction recovery, and satisfactory chromatographic performance. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate, yielding recoveries of 87.5-91.6% for MPV, 87.5-89.4% for NHC, and 87.9-94.9% for BARI. The method was validated according to FDA bioanalytical guidelines over concentration ranges of 20-500, 20-10.000, and 20-500 ng/mL for MPV, NHC, and BARI, respectively. Accuracy, precision, stability, recovery, carryover, and matrix effect met the acceptance criteria. Normalized matrix factors for MPV, NHC, and BARI ranged from 0.948-0.984, 0.900-0.902, and 0.912-1.014, respectively, with CV values below 1%, indicating negligible matrix interference. The proposed method provides a reliable analytical tool for future pharmacokinetic and therapeutic drug monitoring studies.