Factorial Design in Optimization of Chromatographic Separation of Ramipril and Its Impurities

In this paper optimization of chromatographic retention of ramipril and its five impurities employing factorial design is presented. On the basis of preliminary experiments three factors were chosen as inputs (acetonitrile content, pH of the mobile phase and buffer concentration) and retention factor as output. As optimal full factorial design 2³ was chosen, factors were examined at two different levels “low” and “high”. Three replications at zero level were added in order to check linearity and complete statistical tests. Relationship between inputs and output is presented in form of second order interaction model. Adequacy of model was explained using analysis of variance. After analysis of results optimal chromatographic conditions were set. Separations were conducted on a C₁₈ column with a mixture of acetonitrile and water phase (TEA in potassium dihydrogen phosphate) in ratio 23:77 v/v. Finally, the LC method was validated and applied for quality control analysis of commercially available tablets. The proposed method is simpler and faster as compared to existing official methods and therefore more adequate for routine control of ramipril during shelf life. Also a general approach which includes factorial design in method optimization offers a possibility for predicting and following the chromatographic behavior of such complex mixtures.