Integrated LC–MS/MS Approach for the Quantitative Profiling of Sofosbuvir, Voxilaprevir, and Velpatasvir in Rat Plasma: Method Validation and Preclinical Insights

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 demonstrated acceptable accuracy, precision, and sensitivity for preclinical pharmacokinetic applications. The method was successfully applied to a rat pharmacokinetic study, indicating its suitability for exploratory bioanalytical investigations.