Multivariate and univariate optimization studies of liquid-chromatographic separation of steroid mixtures.
We developed a high-performance liquid-chromatographic separation of five steroids (estriol, estradiol, cortisol, progesterone, and testosterone), eluting with a water-acetonitrile gradient from a reversed-phase (C18) column. By applying a simplex search algorithm to maximize a chromatographic-response function, we sought to optimize the original conditions of the chromatographic analysis, which did not separate two pairs of overlapping peaks. Our chromatographic-response function incorporated both peak separation and total time of analysis. Three factors were varied simultaneously to maximize this function: flow rate, column temperature, and gradient shape. From the simplex optimization, we selected a flow rate of 1.50 mL/min, a temperature of 52 degrees C, and a linear gradient for our analysis. Subsequent univariate studies of the initial mobile phase composition showed that acetonitrile/water (20/80 by vol) gave an adequate separation.
D000704:Analysis of Variance; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004958:Estradiol / Q000302:isolation & purification; D004964:Estriol / Q000302:isolation & purification; D006854:Hydrocortisone / Q000302:isolation & purification; D011374:Progesterone / Q000302:isolation & purification; D013256:Steroids / Q000032:analysis*; D013696:Temperature; D013739:Testosterone / Q000302:isolation & purification
D013256:Steroids; D013739:Testosterone; D011374:Progesterone; D004958:Estradiol; D004964:Estriol; D006854:Hydrocortisone