Optimization of Chromatographic Separation of Acetylsalicylic Acid, Amlodipine, Impurity a of Amlodipine, and Atenolol in Hydrophilic Interaction Liquid Chromatography Employing DoE Methodology

Abstract

The retention behavior of substances in hydrophilic interaction liquid chromatography (HILIC) is difficult to predict. Mixture investigated in this paper consists of acetylsalicylic acid, amlodipine, impurity A of amlodipine, and atenolol, a very often used combination in treatment of some cardiovascular diseases. Retention behavior dependence on the most influential chromatographic factors is described by mathematical models, with the special emphasis on pH of the mobile phase. D-optimal design is applied to generate more complex models and to obtain more accurate results. Comparison of Predicted R-2 values of quadratic and cubic model for pH dependence (0.847 and 0.934, respectively) shows that the cubic model has significantly better prediction ability than quadratic in the investigated system. After describing retention behavior, chemometrical tools (indirect modeling of complex chromatographic responses and grid point search optimization) are used to locate the optimal conditions for analyzed mixture in terms of satisfactory separation and minimal analysis duration. The optimal conditions are Column: Kinetex HILIC 100 A (100mmx4.5mm, 2.6 mu m particle size); injection volume: 5 mu L; flow rate: 1mLmin(-1); column temperature: 30 degrees C; detection wavelength: 254nm; mobile phase: acetonitrile-water phase (75mM ammonium acetate, pH 5.3) (91:9V/V).