Effect of low concentration silanization agent and enzyme on the immobilization of lipase on a silica-based support material

Three new methods were developed for the simultaneous determination of velpatasvir (VEL) and sofosbuvir (SOF) in bulk powder and in their pharmaceutical dosage form. Two spectrophotometric methods included first derivative of ratio spectra (1DD) and ratio difference spectrophotometry (RD). In 1DD method, the signals of the first derivative of ratio spectra at 287.4 nm (using 10 μg/mL SOF as a divisor) and 245.8 nm (using 10 μg/mL VEL as a divisor) were selected to determine VEL and SOF, respectively. The RD method based on determination of VEL using 10 μg/mL SOF as a divisor to generate the ratio spectra then the peak to trough amplitude between 231.4 and 253.5 nm were plotted against VEL concentration. Similarly, by using 10 μg/mL VEL as a divisor, the peak to trough amplitudes between 253.5 and 238.2 nm were proportional to the concentration of SOF. The linearity ranges were 5–45 μg/mL and 5–50 μg/mL for VEL and SOF, respectively for both 1DD and RD methods. The third method as a new isocratic UPLC method. The chromatographic separation of SOF and VEL was achieved on Waters Acquity C18 (150 × 2.1 mm, 1.7 μm) column using a mobile phase consisting of a mixture of diammonium phosphate buffer pH 6 ± 0.02:acetonitrile (40:60, V/V) at flow rate 0.1 mL/min. Detection was set at 280 nm. The linearity ranges were 5–90 μg/mL and 5–240 μg/mL for VEL and SOF, respectively. Robustness evaluation of UPLC method was performed by means of Youden's test. The proposed methods was validated as per ICH and applied for the determination of cited drugs in dosage form. The statistical comparison using t-test and F-test showed that there is no significant difference between the proposed methods and the reported one regarding both accuracy and precision.