Rapid bioanalytical LC-MS/MS method for the simultaneous determination of sofosbuvir and velpatasvir in human plasma-application to a pharmacokinetic study in Egyptian volunteers

Abstract

A novel, rapid and validated LC-MS/MS bioanalytical method has been developed for the extraction and determination of sofosbuvir and velpatasvir simultaneously in human plasma using ledipasvir as an internal standard (IS). The simple and reproducible protein precipitation technique with acetonitrile was successfully used for the deproteinization and extraction of the analytes from human plasma matrix. The developed method achieved consistent recoveries over different concentrations with average extraction recoveries of 81.72% and 80.46% for sofosbuvir and velpatasvir, respectively. The chromatographic separation was performed within only 2.80 min as a run time by an isocratic elution through C18 Zorbox eclipse plus (100 × 4.6 mm, 5 μm). Optimum mobile phase consisted of 0.1% formic acid in water: acetonitrile: methanol (30:60:10, v/v/v) pumped at a flow rate of 0.55 mL min−1 and injection volume was 10 μL. LC-MS/MS detection was done by multiple reaction monitoring transitions operating at positive ionization mode for both analytes and IS. Bioanalytical method validation as per EMA guidelines was carried out where the proposed method revealed linearity over the concentration range of 5–5000 and 10–1500 ng mL−1 for sofosbuvir and velpatasvir, respectively. After validation, the method was applied to the analysis of the two drugs after a single oral administration of Epclusa 400/100 mg tablets to Egyptian healthy volunteers.