Optimization of High-Performance Liquid Chromatographic Method Using Central Composite Design for the Simultaneous...

A simple, rapid, precise and accurate stability indicating high-performance liquid chromatographic (HPLC) method was developed for simultaneous quantification of Ciprofloxacin (CIP) and Loteprednol (LOT) along with their forced degradation products using central composite design (CCD) approach. CCD was prepared with three independent variables in a gradient HPLC method. In gradient program (GP) the ratio of phosphate buffer in the mobile phase was 70%, 75%, 80%, 85% and 90%, the pH of phosphate buffer was 2.6, 2.8, 3.0, 3.2 and 3.4, flow rate was 0.8, 0.9, 1.0, 1.1 and 1.2 mL/min. Resolution, tailing factor (CIP) and tailing factor (LOT) were selected as response factor. The effective separation of LOT and CIP was achieved on Phenomenex EVO-C18 column (4.6 mm × 250 mm × 5 μm particle size) opting gradient mode of elution. The mobile phase composed of 10 mM Phosphate buffer pH 3.2 with ortho-phosphoric acid and the organic phase composed of mixture of acetonitrile and methanol in 50:50% v/v with flow rate of 1 mL/min and diode array detection at 258 nm. The optimized variables found were flow rate of 1.0 mL/min, ratio of phosphate buffer in GP 80% at pH 3.0. The method was validated as per ICH guidelines and applied for analysis of stability samples.