Application of dansyl derivatization to the high pressure liquid chromatographic identification of equine estrogens

The HPLC qualitative analysis of conjugated estrogens is accomplished by a two-step procedure involving the formation of the corresponding dansyl derivatives. The first step involves the acid hydrolysis of the conjugated estrogens, followed by dansyl derivatization and HPLC separation of these derivatives on a liChrosorb Si-60 column with 50% (v/v) chloroform-n-heptane as the mobile phase. All of the dansyl estrogens are well separated except for the 17-keto estrogens, estrone, equilin, and equilenin. The second step, designed to detect the three 17-keto estrogens, begins with the selective sodium borohydride reduction of the conjugated 17-keto estrogens to the corresponding 17-hydroxyl compounds (the beta-epimer being formed in vast predominance over the alpha-epimer), followed by acid hydrolysis, dansyl derivatization, and HPLC separation of the derivatives as in the first step. Detection of the 17-keto estrogens is possible by determining differences in peak heights between the chromatograms of the first and second analyses. The The proposed method is sensitive, the dansyl derivatives stable, and nine different estrogens can be readily identified.