Design of Experiments in Optimization and Validation of a Hydrophilic Interaction Liquid Chromatography Method for Determination of Amlodipine Besylate and Bisoprolol Fumarate

Abstract

In this paper chemometrically-assisted optimization and validation of hydrophilic interaction liquid chromatographic (HILIC) method for the quantitative analysis of amlodipine besylate (AB) and bisoprolol fumarate (BF) in pharmaceuticals are presented. In this study, the development of HILIC method for AB and BF separation was performed by a systematic approach which included Design of Experiments (DoE) methodology. First, in method optimization, a central composite design was applied. Influence of the acetonitrile content in the mobile phase, pH of the aqueous phase, and concentration of ammonium acetate on selected chromatographic responses was investigated. Optimal chromatographic conditions in terms of adequate separation and minimal analysis duration were set by graphical optimization overlapping the contour plots as: acetonitrile–water phase (10 mM ammonium acetate, pH 4.0 adjusted with glacial acetic acid) (92:8, v/v), column Luna HILIC 200 A, 150 mm × 4.6 mm, 5 µm particle size, column temperature 30°C, flow rate 1 mL min−1, and UV detection at 230 nm. After this step, method robustness was tested using a 25-1 fractional factorial design. Statistical and graphical evaluation of results confirmed robustness of the method and then the method was fully validated. Finally, the proposed method was successfully applied for AB and BF determination in pharmaceuticals.