Fluorodensitometric determination of conjugated estrogens in raw material and in pharmaceutical preparations

A simple, rapid and reproducible fluorodensitometric method for the determination of conjugated estrogens has been developed. The proposed procedure includes the following steps: extraction, hydrolysis of sodium sulphate esters of estrone, equilin, equilenin and their 17--hydroxy derivatives, separation of the liberated 3-phenolic steroids and in situ measurement of fluorescence. The fluorescence emission was measured after spraying the spots of estrone and estradiol with 2,4-dinitrophenylhydrazine in sulphuric acid—ethanol medium and equilin and 17--dihydroequilin with phosphoric acid and sodium hydroxide solution, respectively. Equilenin and 17--dihydroequilenin were determined by measuring the native fluorescence. The method applied to the determination of raw material and tablets provided results which agreed well with the stated content and the requirements of USP XXI for conjugated estrogens.