Simultaneous determination of deflazacort metabolites II and III, cortisol, cortisone, prednisolone and prednisone in human serum by reversed-phase high-performance liquid chromatography.

A new high-performance liquid chromatographic method for the analysis of human serum from subjects administered prednisolone or deflazacort has been established. Two deflazacort metabolites (metabolites II and III), prednisolone and its metabolite (prednisone), as well as cortisol and cortisone in human serum were analyzed simultaneously. These six compounds and the internal standard, betamethasone, were extracted from 1 ml of human serum using a Sep-Pak Plus Environmental C18 column and then separated on a Hypersil ODS (25 x 0.46 cm I.D.) column using a gradient system. The lower limit of quantitation for each compound was 10 ng/ml based on signal-to-noise ratio of 5 and calibration curve linearity. Within-day and between-day validations gave a coefficient of variation of less than 10% and a relative error of ca. 10% (n = 11).