Pitfalls and success of experimental design in the development of a Mixed Micellar ElectroKinetic Chromatography method for the analysis of budesonide and its impurities

A mixed MEKC method for the analysis of budesonide and its related substances is presented. The micelles were formed from sodium cholate (CHOL) and 3-(N,N-dimethylmyristylammonio) propanesulfonate (MAPS). A multivariate optimisation was carried out with the aim of obtaining a baseline separation of all compounds. The influence of voltage, borate concentration, cholate concentration, MAPS concentration and pH was evaluated on the responses, corresponding to critical resolution values. Problems with the investigated experimental design were encountered due to the complexity of the separation process. As a consequence, a first design was not sufficient to reach the optimal conditions, but was needed in order to obtain the necessary information to successfully plan a second in-depth study by means of response surface methodology. The optimal conditions were as follows: capillary total and effective lengths of 48.5 and 40.0 cm, respectively, with 50 mm id; 70mM borate buffer (pH 8.8) containing 65mM CHOL and 10mM MAPS; temperature 201C and voltage 16 kV. Separation of all the compounds, including R- and S-epimers of budesonide, was obtained in a reasonable time. Validation of the method was performed for both drug substances and drug product.