Reversed- and normal-phase high-performance liquid chromatography of 18-hydroxylated steroids and their derivatives : Comparison of selectivity, efficiency and recovery from biological samples

Abstract

The chromatographic behaviour of adrenal 18-hydroxysteroids, and a series of derivatives thereof, has been studied in reversed-phase and normal-phase high-performance liquid chromatography (HPLC). Both 18-hydroxycorticosterone and 18-hydroxy-11-deoxycorticosterone exhibited a marked loss of chromatographic efficiency when separated on incompletely-covered C 18 reversed-phase packings with methanol-water gradient elution. Maximum-coverage reversed-phase supports (as determined by methyl red adsorption) showed no such effect, nor was it seen when aprotic solvents, such as dioxane, were used. This phenomenon is unique among the wide range of adrenal and testicular steroids that we have studied and affords a useful test, applicable under aqueous conditions, of coverage by the alkylsilane reversed phase, a factor of considerable importance in the successful resolution of complex mixtures of adrenal steroids by reversed-phase HPLC. The retention times of the 20-methoxy, 20-ethoxy, 21-acetoxy, etiolactone, 11β, 18-ether and dimeric derivatives of the naturally-occurring 18-hydroxysteroids have been determined in relation to major adrenal steroids. Procedures for extraction and reversed-phase HPLC of 18-hydroxysteroids from tissues without the formation of these less polar forms, which can complicate their separation by other chromatographic techniques, are illustrated with respect to a human malignant adrenocortical tumour which caused hypermineralocorticism.