HPLC determination of glucocorticoid alcohols, their phosphates and hydrocortisone in aqueous solutions and biological fluids

High-performance liquid chromatographic (HPLC) assays are described for the determination of dexamethasone phosphate, dexamethasone and hydrocortisone and for the determination of triamcinolone, triamcinolone acetonide, triamcinolone acetonide phosphate and hydrocortisone in aqueous solutions and biological fluids. These assays allow quantification of the glucocorticoids in plasma down to a concentration of 100 ng ml(-1) (dexamethasone phosphate 300 ng ml(-1), hydrocortisone 40 ng ml(-1)). During the development of optimum extraction procedures the pK(a) values of the esters were determined by extractions performed at different pH values. The stability in vitro of the phosphate esters in ampoules, plasma and blood was studied. The esters are stable in their formulated ampoules after long-term storage, whereas the half-life in vitro of dexamethasone phosphate in plasma at 37 degrees C is 5 h and that of triamcinolone acetonide phosphate is 3.5 h. Determination by HPLC of endogenous hydrocortisone in samples from patients who received either dexamethasone phosphate or triamcinolone acetonide phosphate gave results identical with those obtained by radio-immunoassay (RIA).