Separation and quantitative determination of progesterone and pregnenolone.

In the course of experiments on the enzymatic transformation of steroids we were confronted with the problem of determining quantitatively small amounts of progesterone and pregnenolone (A6-pregnene-3@-ol-20-one). While progesterone can be easily estimated by its absorption maximum at 240 rnp, no spectrophotometric method has been known thus far for the determination of steroids with a ketone group not conjugated with a double bond. In a search for suitable derivatives of pregnenolone the semicarbazone (1) was investigated first. It showed a maximum at 228 mp which was rather unsatisfactory because many impurities were found to absorb ultraviolet light in the same region. Similar difficulties were encountered with the acethydrazone which exhibits maximum absorption at 235 rnp. Moreover, acethydrazide showed signs of decomposition when kept at room temperature, and the products interfered strongly with this maximum. Benzhydrazide proved to be more stable than acethydrazide, but it displayed maximum absorption at 225’ rnp, which interfered with the maximum of pregnenolone benzhydrazone at 260 rnp. Removal of the excess benzhydrazide by distribution of the reaction mixture between water and ether gave unsatisfactory results. Pregnenolone dinitrophenylhydrazone is known to have an absorption maximum at 368 mp in chloroform (2), while 2,4-dinitrophenylhydrazine shows a maximum at 343.5 rnp (3). Mixtures of both substances gave maxima between the two values. Separation by chromatography on alumina was incomplete. It was found possible, however, to remove the excess dinitrophenylhydrazine by oxidizing it with either Fehling’s solution or Benedict’s reagent. The reaction product was m-dinitrobenzene which was identified by melting point and mixed melting point. This compound does not show any significant absorption between 330 and 400 ml*. Moreover, it can be removed easily by chromatography, since it