HYDROLYSIS OF 17-KETOSTEROID CONJUGATES WITH A PROCEDURE BASED ON SIMULTANEOUS BENZENE EXTRACTION AND ACID HYDROLYSIS

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Abstract
A comparison of a hydrolytic method based on simultaneous benzene extraction and acid hydrolysis with methods using acid hydrolysis followed by extraction with an organic solvent has confirmed the superiority claimed for the simultaneous benzene extraction by other investigators. Less transformation, artefact formation and destruction was found with the simultaneous extraction method. A fractionated hydrolytic procedure performed at different pH values is proposed for the simultaneous benzene extraction technique. This procedure gives best protection to the acid and heat labile 17-ketosteroids and highest yields of individual 17-ketosteroids. The usefulness of this procedure in itself and in combination with [beta]-glucuronidase hydrolysis has been explored. It was found that a method employing simultaneous benzene extraction and acid hydrolysis after [beta]-glucuronidase hydrolysis gave slightly higher yields overall and for some individual 17-ketosteroids than any other method examined. It is suggested that the simple hydrolytic method based on simultaneous benzene extraction and acid hydrolysis alone may well be the preferred method for clinical assay at the present time. Its speed, simplicity and inexpensiveness together with its independence of enzyme inhibitors and other factors affecting the enzyme assay may for such an application outweigh the lower yield obtained for 11-oxy 17-ketosteroids.