Optimization of the Separation of Ephedrine, Pseudoephedrine, Phenylephrine, and Synephrine by Hydrophilic Interaction Liquid Chromatography Employing Experimental Design Methodology

Abstract

Hydrophilic interaction liquid chromatography (HILIC) has emerged as an alternative separation technique for small polar compounds. Design of Experiments (DoE) methodology in the analysis of the retention behavior and method development have been shown to be successful. This article addresses the retention behavior and optimization of chromatographic separation of a model mixture consisting of four sympathomimetic drugs (ephedrine, pseudoephedrine, phenylephrine, synephrine), on a bare silica column by HILIC. The acetonitrile content, pH of the aqueous phase, and concentration of ammonium acetate in aqueous phase were optimized according to the matrix of Box-Behnken design, and retention data were fitted to second-order polynomial models. The obtained coefficients of determination R-2 were higher than 0.990 and adjusted R-2 exceeded 0.972, while the lowest value for Predicted R-2 was 0.845. Beside of statistical analysis, graphical estimation of retention behavior was performed. In order to optimize the separation, indirect modeling of selectivity factors of critical peak pairs (the diastereoisomers ephedrine/pseudoephedrine and the positional isomers phenylephrine/synephrine) and grid point search approach were employed. After the development of the grids, optimal chromatographic conditions were selected (acetonitrile content 91%, pH of the aqueous phase 4.5 and ammonium acetate concetration 35mmolL(-1)), providing a total analysis time of 14min.