Development and Validation of the Chiral HPLC Method for Daclatasvir in Gradient Elution Mode on Amylose-Based Immobilized Chiral Stationary Phase

A selective and specific high-performance liquid chromatography method for the determination of daclatasvir enantiomers has been developed and validated. Various immobilized polysaccharide-based chiral stationary phases were used to define a separation strategy utilizing normal-phase and polar organic chromatography modes. Excellent resolution between daclatasvir and its enantiomer was achieved on amylose tris (3-chlorophenylcarbamate) stationary phase, namely CHIRALPAK ID-3, using binary gradient containing acetonitrile:diethylamine and methanol:diethylamine as the mobile phase. The flow rate of the mobile phases was maintained at 1.0 mL min −1 while the column oven temperature was maintained at 40 °C. The column effluent was monitored by UV detection at 315 nm. In comparison with isocratic method, the binary gradient method offered excellent peak shape and improved resolution between daclatasvir and its enantiomer while maintaining the specificity with diastereomers. The method was found to be precise, accurate, and linear ( R 2 > 0.999). Limit of detection and limit of quantitation of the enantiomer were found to be 0.083 µg mL −1 as and 0.25 µg mL −1 , respectively. Recovery of the enantiomer was found to be in the range of 90 to 112 %.