Innovative Thin-Layer Chromatography/Fluorescence Detection Approach for Sensitive and Specific Determination of...

An innovative thin-layer chromatography method coupled with the fluorescence detection was developed for a specific estimation of ledipasvir. The separation was achieved on plates of silica gel 60 F254 using ethylacetate: hexane: acetonitrile: triethylamine; (6: 3.5: 1.5: 0.5, $\mathrm{v}/\mathrm{v}/\mathrm{v}/\mathrm{v}$) as a mobile phase system. The method involved the exposure of the developed thin-layer chromatography plate of ledipasvir to strong ultraviolet irradiation, resulting in a great enhancement in the fluorescence properties of ledipasvir. The irradiated plates were scanned after the excitation at 315 $\mathrm{nm}$. The method provided a sufficient separation of ledipasvir from sofosbuvir with ${R}_F$values of 0.28 and 0.36 for ledipasvir and sofosbuvir, respectively. The developed procedures were validated based on guidelines from the International Conference on Harmonization and Food and Drug Administration guidelines. The calibration curve was linear over the range of 5-50 $\mathrm{ng}/\mathrm{band}$. The excellent analytical features of the proposed method allow to the specific determination of ledipasvir in pharmaceutical tablets without interference from sofosbuvir or excipients. As the main elimination route for ledipasvir is via the fecal excretion (86%), the method was applied for the estimation of ledipasvir in fecal specimens with adequate recovery. In addition, the proposed method was applied for testing the content uniformity of ledipasvir in the pharmaceutical tablets.