Application of Box–Behnken Design and Green Analytical Chemistry Principles in Development of HPLC Method to Simultaneously Analyze Combination of Chlorthalidone, Bisoprolol Fumarate, and Cilnidipine

ABSTRACT High‐performance liquid chromatography method for the estimation of chlorthalidone, cilnidipine, and bisoprolol fumarate in synthetic mixture simultaneously was developed and validated using analytical quality by design. The mobile phase was optimized using Box–Behnken design by selecting three factors for optimization of chromatographic conditions: flow rate, acetonitrile volume, and pH of buffer. International Council on Harmonisation Q2(R1) guideline was used for method validation. Linearity for chlorthalidone, bisoprolol fumarate, and cilnidipine was obtained in the range of 12.5–62.5, 10–50, and 10–50 µg/mL, respectively. Hypersil C 18 column was used as the stationary phase. Good resolution was obtained using the mobile phase comprising acetonitrile:potassium dihydrogen orthophosphate buffer at pH 5.7 (63:37 %v/v). The flow rate of the mobile phase and detection wavelength were 1.15 mL/min and 227 nm. Retention times for chlorthalidone, bisoprolol fumarate, and cilnidipine were 2.418, 3.485, and 7.985 min, respectively. Among factors, volume of acetonitrile showed greater influence on responses, retention time, and resolution of drugs. Percentage relative standard deviation for validation parameters of method was not greater than 2, indicating that the developed method is specific, reliable, precise, and is applicable in routine analysis. In addition, the method was environmentally friendly based on evaluation of analytical method's greenness, applying principles of green analytical chemistry.