Mathematical Modeling for HPLC Separation and Quantification of an Anti hypertensive Combination by Response Surface Methodology

Background Determination of a multi-component mixture by HPLC requires many preliminary runs for method development which is both time-consuming and expensive due to the usage of large solvent volumes. Objective In the present study, the main objective was to reduce the preliminary runs that are required for optimizing the method conditions and also shorten the run time of analysis to be suitable for quality control laboratories where there are a large number of samples to be analyzed. Methods That was achieved using a two-factor, three-level response surface experiment which is a multivariate design that predicts the significant factors for optimizing the studied responses. Results The response surface design suggests that both acetonitrile ratio and flow rate are significant factors for full resolution of the studied mixture: atenolol, amiloride, and hydrochlorothiazide. The studied mixture was fully separated and determined in less than 5 min with perfect resolution. Conclusion Experimental design is a very beneficial tool for optimization of the method conditions in HPLC, especially if the studied mixture ingredients have overlapping peaks. For atenolol, amiloride, and hydrochlorothiazide, acetonitrile and flow rate were found to be the significant factors that affected the resolution of the studied mixture.