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The current study describes the use of central composite design for multivariate optimization of resolution and retention time, taking into account different critical method parameters like organic phase, pH, flow rate, and wavelength for risk assessment. The chromatographic method for the assay of the most effective anti-viral regimen (EPCLUSA, DARVONI, and HARVONI) was developed. An experimental design was presented by sequential investigation of four independent parameters. The method was developed using XTERRA C18 (250 mm × 4.6 mm, 5 μm particle size) column in isocratic mode using potassium dihydrogen phosphate buffer (pH adjusted to 5) and acetonitrile (50:50 % v/v) as mobile phase at a flow rate of 1.0 ml/min and UV detection wavelength of 260 nm. The separation of four drugs with fine resolution and preferable retention times was achieved. Retention times of four drugs were found to be 2.96, 3.91, 7.15, and 11.94 min for daclatasvir, sofosbuvir, velpatasvir, and ledipasvir, respectively. The percentage accuracy of labelled claim was in the range of 99–102%, and the pooled %RSD for repeatability, precision, and accuracy was less than 2%. The suggested method was applied for quantification and identification of studied drugs in tablets; the results agreed with the label claim and were validated according to the ICH guidelines. The optimized method can be used for pharmacokinetic and quality control studies.

Multivariate optimization and evaluation of quaternary mixture in bulk and co-formulated dosage forms by central composite design