Quantification of Sofosbuvir in Human Plasma: RP-HPLC Method Development and Validation

Abstract

Sofosbuvir, a potential inhibitor of the nucleotide analog of hepatitis C virus (HCV) NS5B polymerase, is used to treat hepatitis C and marketed under brand name Sovaldi. This study was aimed to develop and validate a simple, sensitive and precise RP-HPLC method for the estimation of sofosbuvir in human blood plasma using quinine sulfate as internal standard (ISTD). Separation was achieved using Shimadzu-HPLC system equipped with Gracesmart RP-C₁₈ (250 mm × 4.6 mm, 5 μm) column eluted at a flow rate of 0.5 mL/min and detected at 260 nm. The mobile-phase composition was optimized to be ammonium formate (pH 3.0; 10 mM) and acetonitrile (67: 33, % v/v). The retention times of sofosbuvir and quinine sulfate were found to be 13.60 and 8.39 min, respectively. The proposed method was linear over a range from 20 to 1200 ng/mL. Liquid–liquid extraction technique was applied for the extraction of drug and ISTD from human plasma using TBME – DCM (80: 20, % v/v) mixture. The proposed method was validated as per bioanalytical method validation guidelines for industry.