The high-performance liquid chromatography of nineteen hormonal steroids with special respect to its suitability for routine purification of these steroids from crude, organic extracts of biological fluids prior to final quantitation by immunoassay has been studied. In all systems the gradient elution technique was applied. Separation of steroids has been investigated using different stationary phases chemically coated with non-polar, hydroxyl, NO2 and CN groups. Reproducibility of retention times was studied on a stationary phase coated with hydroxyl groups (DIOL column) using different organic eluents. Coefficients of variation range from 0.76 to 8.16%. Reproducibility was shown to be unequivocally better in the gradient part than in the isocratic part of the chromatographic run. In contrast to the other steroids, 18-hydroxylated steroids were more or less unstable in certain systems studied. As to resolution and reproducibility, the DIOL column run with an n-hexane-dioxane gradient has been shown to be superior to the other systems studied.
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