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Clinical Chemistry 28: 444-448, 1982;
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Clinical Chemistry, Vol 28, 444-448, Copyright © 1982 by American Association for Clinical Chemistry

Multivariate and univariate optimization studies of liquid- chromatographic separation of steroid mixtures

DM Fast, PH Culbreth and EJ Sampson

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.




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Copyright © 1982 by the American Association for Clinical Chemistry.