Desirability Based Optimization and Validation of New RP-HPLC Method for Simultaneous Determination of Bisoprolol Fumarate, Hydrochlorothiazide and Their Impurities

Abstract

In this paper optimization and validation of a new RP-HPLC method for simultaneous determination of bisoprolol fumarate, hydrochlorothiazide and impurities A, L, K and B are presented. Factors which could have significant influence on separation were selected: percentage of mobile phase B [acetonitrile—ammonium dihydrogen phosphate/orthophosphoric acid buffer solution (80:20, v/v)] at defined t 1 and t 2 of the gradient, ammonium dihydrogen phosphate concentration and pH value of the buffer solution (mobile phase A). Selected factors were varied according to Box-Behnken plan of experiments and their influence on critical pairs separation was investigated. Derringer´s desirability function was used for selection of optimal chromatographic conditions. Finally, optimal values of investigated factors were 34.4 % (t 1) and 38.0 % (t 2) of mobile phase B, 46.5 mM ammonium dihydrogen phosphate concentration and pH 3.35. The method was successfully validated according to ICH guidelines acceptance criteria for robustness, selectivity, linearity, accuracy and precision. Small variations of selected chromatographic parameters did not affect qualitative and quantitative system responses significantly, which proved the method’s robustness. Limits of quantification (LOQ) and detection (LOD) for impurities were determined and LOQ values were the first points in linearity testing. Linearity was confirmed by r ≥ 0.9945 (impurities) and r = 0.9998 (active substances). Accuracy was confirmed by calculated recoveries [93.5–107.1 % (impurities) and 98.1–101.9 % (active substances)]. Precision was tested at three levels: injection repeatability, analysis repeatability and intermediate precision. Calculated relative standard deviations were less than 1, 2 and 3 %, respectively. Finally, the method was applied to real sample analysis.