Abstract: | An efficient, isocratic, reversed-phase high performance liquid chromatography method was developed, optimized, and validated to separate nine antihypertensive drugs by experimental design methodology and the Crossed D-Optimal process. In order to find the most suitable separation conditions, twenty-three experiments were carried out, based on the simultaneous effects of three solvent (methanol, acetonitrile, and water) compositions in combination with different pH values of the mobile phase. The components were cross-combined with a pH factor (quadratic x quadratic process), whereas optimal adjusted models were used for the eight individually chosen responses. The optimal mobile phase consisted of methanol, acetonitrile, and 0.05 M aqueous ammonium acetate at pH 3.1 (18:26: 56 v/v/v, pH adjustment with formic acid). Analysis was carried out on a C18 column (150 × 4.6 mm, 5 µm) at 40 °C using photodiode array detection at 242 nm. The system was found to produce sharp and well resolved peaks for all analytes while the retention times ranged from 2.3 to 31 min. The method was linear (r2 > 0.999) and reliable since the accuracy (recovery = 100 ± 2.9) and the precision (relative standard deviation < 2%) met International Conference on Harmonization guidelines. The technique was shown to be a useful tool for separating complex mixtures using experimental design methodology. |