Quantification of Sofosbuvir and Velpatasvir in Human Plasma using LCMS/MS Technique -Application to Pharmacokinetic Study

Objective: An effective high-performance Liquid Chromatography-tandem Mass Spectrophotometric (LCMS/MS) method was developed for the simultaneous quantitation of sofosbuvir (SOFOS) and Velpatasvir (VELPA) in spiked human plasma. Methods: The extraction of both analytes from plasma was performed by Liquid-Liquid Extraction (LLE) technique. Separation was achieved on a Zorbax C18 Stable Bond (SB), C18 (4.6mm id x 50 mm) analytical column using acetonitrile: 1% formic acid (50:50) v/v with a flow rate of 600µl/min. The MS/MS analysis was performed in Multiple Reaction Monitoring (MRM) to obtain the product ion m/z 530→242.3 for SOFOS, m/z 883.8 → 643.0 for VELPA and m/z 889.5 →732.6 for internal standard (ledipasvir). Results: The calibration curve was found linear over the range of 0.5→5000 ng/ml for SOFOS and 1.5→2000 ng/ml for VELPA. Intra and inerday accuracy (% nominal 98 → 102%), precision (% CV ≤3.8%) was excellent. Matrix effect (matrix factor 1.340 for SOFOS and 1.004 for VELPA), selectivity (% interference = 0) with a extraction recovery of 96.70% →98.30%. The stability (% nominal 95.85→98.90 %) of all types were within acceptable limit. Conclusion: The proposed method was applied successfully for the pharmacokinetic study of marketed dosage form in rabbit blood samples with single oral human equivalents dose. The developed method has further applied during clinical and preclinical trials in human and other experimental animals.