Solvent optimization of reversed-phase high-performance liquid chromatography for polar adrenal steroids using...

Computer-aided optimization of a mobile phase has been applied to the isocratic reversed-phase separation of ten polar adrenocortical steroids, including aldosterone and reduced metabolites of cortisol and cortisone. A method based on a seven-step procedure for calculation of the Chromatographic Optimization Function (COF) has been used. Logarithmically transformed retention indices were used for computing multiple polynomial regressions for the retention times of compounds as a function of solvent composition, with the resultant COF as a dependent parameter used for selection of the better mobile phase. Peak crossovers and overlaps are accommodated in this method and the maximum acceptable analysis time factor is incorporated as well as weighting factors for priority separations. The utility of this procedure for complex mixtures of closely eluting compounds is discussed with respect to the Overlapping Resolution Map method and with the COF method of Glajch and Kirkland as used for automated optimization. Its application to aldosterone-containing samples from human adrenocortical tumours is illustrated.