Thin-layer chromatography/fluorescence detection approach for sensitive and selective determination of hepatitis C...

A validated thin-layer chromatography (TLC) method combined with fluorescence detection mode was developed for the selective determination of a recently approved anti-hepatitis C virus (HCV) drug (velpatasvir). The separation was performed on silica gel 60 F₂₅₄ plates using ethylacetate:methanol:triethylamine (48:1.5:1.0, v/v/v) as a mobile phase. Plates were scanned in the fluorescence mode after excitation at 335 nm. This method provided an excellent separation of velpatasvir from sofosbuvir with R_F values of 0.22 and 0.46 for velpatasvir and sofosbuvir, respectively, after scanning the developed plates in the ultraviolet detection mode at 335 nm. The calibration curve was linear over the range 4-40 ng/band with a correlation coefficient of 0.9994. The developed procedure was validated according to ICH guidelines with a detection limit of 1.30 ng/band and quantitation limit of 3.95 ng/band. The suggested method could selectively determine velpatasvir with high sensitivity in a synthetic tablet powder containing a co-formulated anti-HCV drug (sofosbuvir) without any interference from excipients or sofosbuvir. In addition, the method was successfully applied for determination of velpatasvir in spiked human plasma with adequate % recovery.