A Quantitative Color Reaction for Cortisone and Related 17,21-dihydroxy-20-ketosteroids By

The numerous combinations of functional groups occurring in the steroid hormones render difficult the development of a specific test for individual compounds. However, reactions which reveal certain groupings are available and may be applied singly or in combination to elucidate the characteristics of pure steroids or of compounds in a mixture. The ketol side chain, located at carbon atom 17 in certain corticosteroids, has reducing properties and also yields formaldehyde upon oxidation with periodic acid. These reactions have been applied in the analysis of partially purified steroid mixtures (14). The reaction of active carbonyl groups in steroids with phenylhydrazines is the basis of histochemical methods (5). Production of colored and fluorescent solutions in sulfuric acid (6) and reaction with antimony trichloride (7), dinitrobenzene (8), and other reagents (9) have also been employed. To this group may now be added another test which appears to be specific for 17,21-dihydroxy-20-ketosteroids. The method is based upon the appearance of a yellow color in solutions containing the steroid, phenylhydrazine, and sulfuric acid.