Abstract

The study aimed to develop and validate a precise, reliable, and sensitive LC-MS/MS method for the simultaneous estimation of Sofosbuvir (SOF), Voxilaprevir (VOX), and Velpatasvir (VEL) in rat plasma for pharmacokinetic applications. Plasma samples were protein-precipitated and analyzed using an Agilent Eclipse XDB C18 column with an acetonitrile–ammonium formate (20:80, pH 3.0) mobile phase, and detection was carried out on a SCIEX QTRAP 5500 in MRM mode. Optimized mass transitions were m/z 530.4537 → 294.1257 for SOF, 869.9463 → 259.6483 for VOX, 884.0635 → 761.3207 for VEL, and 883.0154 → 587.4326 for the internal standard, Elbasvir. The method demonstrated excellent linearity (r² > 0.999), acceptable precision (CV ≤ 15%), and accuracy (85–115%). Recovery of all analytes ranged from 95.39% to 98.74%, with negligible matrix effects. The LLOQ values were 20 ng/mL for SOF, 5 ng/mL for VOX, and 5 ng/mL for VEL. The developed method is robust, sensitive, and suitable for pharmacokinetic evaluation, providing a dependable analytical tool for preclinical and clinical investigations of these antiviral agents.

Key words: LC-MS/MS, Sofosbuvir, Voxilaprevir, Velpatasvir, Pharmacokinetics, Bioanalytical Validation