Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the simultaneous determination of prednisone, prednisolone and their major hydroxylated metabolites, viz., 20β-hydroxyprednisone, 6β-, 20α- and 20β-hydroxyprednisolone, in human urine is presented. The retention times were 6.5, 11.4, 18.1, 24.2, 31.6 and 35.3 min, respectively. The technique employs betamethasone as the internal standard. Samples are extracted with ethyl acetate using a diatomaceous earth extraction column, and the extract was dried and injected onto a silica gel column with ultraviolet detection at 254 nm. The calibration curve is linear within the studied range 50-1500 ng/ml for prednisolone and 50-750 ng/ml for the other steroids. The intra-day and inter-day coefficients of variation are less than 10% for prednisone and prednisolone but higher for the metabolites. The assay was used to study the excretion rate profile of each of these steroids in the urine of a normal male subject receiving a 49.3-mg intravenous dose of prednisolone. The results indicate that prednisone, 6β-, 20α- and 20β-hydroxyprednisolone may be the major unconjugated metabolites of prednisolone while 20β-hydroxyprednisone may be a minor metabolite.