Chemical assay of diethylstilbestrol.

HE CHEMICAL ASSAY of diethylstilbestrol in extremely small amounts has not been previously accomplished in a satisfactory manner. The ultraviolet absorption of diethyistilbestrol has been proposed as a method (1), but this fails in the presence of other absorbing substances in tissue extracts. In addition, the absorption curve lacks any sharply defined band. Measurement of the phenolic function (2) suffers from a similar lack of specificity, while methods based on nitration (3, 4, 5) lack sensitivity and specificity. Dingemanse (6) was the first to propose the use of antimony pentachioride as a reagent for dliethyistilbestrol, and this reagent has been adopted by Warren, Goulden, and Robinson (7) and by Jones and Deatherage (8). A sensitivity of 0.002 mg. has been claimed for the antimony pentachioride reaction but our experience with it has not been as fortunate, owing to rather high blank optical densities produced by unknown chromogens and to a day-to-day variation of optical densities of standard solutions. We have therefore sought a different approach to this problem. Diethylstilbestrol reacts with bromine under various conditions to form colored compounds or complexes (9, 10), similar to the behavior of certain 17-a-hydroxysteroids (11). Cocking (12) recently described two reactions of this type, involving diethyistilbestrol, which appeared to be of promise in the analytic sense. We have examined one of these in some detail, and present herewith a chemical assay based on Cocking’s observations. The proposed method seems to be quite specific, fairly sensitive, and applicable to the assay of diethyistilbestrol in tissue extracts.