THE IDENTIFICATION OF RELATED SUBSTANCES IN 9-ALPHA-FLUOROPREDNISOLONE-21 ACETATE BY MEANS OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH DIODE-ARRAY DETECTOR AND MASS-SPECTROMETRY

A method for the analysis and identification of the principal related substances in 9-alpha-fluoroprednisolone acetate is described. This compound has been chosen as a model for the investigation of related substances which can be originated in the general procedure for introducing a fluorine substituent at position 9 of a corticosteroid molecule. HPLC procedures, both in reversed and in normal phase were used; a rapid scanning UV detector which allows direct spectrophotometric data to be obtained on chromatographic peaks, proved to be a tool of great importance. Thus, after reversed-phase chromatographic separation and observation of the UV spectra and their respective second derivatives, it was possible to characterize some of the principal effective and potential related substances such as 9-alpha-fluorohydrocortisone acetate, 9-alpha-bromoprednisolone acetate, 9-beta, 11-beta-epoxyprednisolone acetate and 9(11)-dehydro-prednisolone acetate, emerging as chromatographic peaks. Identification of 9-alpha-bromoprednisolone acetate and of 9-alpha-fluorohydrocortisone acetate which proved to be the most significant impurities, was confirmed by means of an exhaustive study of the mass spectra of these substances conveniently isolated by normal-phase HPLC. The chromatographic, spectrophotometric and mass-spectrometric characteristics of the studied compounds are reported.