The determination of 17,21-dihydroxy-20-ketosteroids in urine and plasma.

In 1950 (l), the authors described a color reaction between 17,21-dihydroxy-20-ketosteroids and phenylhydrazine and showed that, after addition of 1 r or more of cortisone per ml. of urine or plasma, about 88 per cent could be detected in chloroform extracts. Nelson and Samuels (2) improved the procedure, primarily by employing chromatographic separation and micro cuvettes, so that they were able to measure the concentration of hydrocortisone in human plasma. For routine clinical use, however, it seemed desirable to develop a relatively simple procedure which could be completed within a working day. This communication is concerned with a description of such a procedure, which involves four steps : (1) a 15 second extraction with 5 volumes of chloroform, (2) a 10 second washing with dilute alkali, (3) a 15 second extraction of an aliquot of the chloroform extract with the alcoholic phenylhydrazine-sulfuric acid reagent, and (4) measurement of optical density in micro cuvettes in the Beckman spectrophotometer after heating the samples to 60” for 30 minutes.