High-performance liquid chromatographic separation of potential hydroxylated metabolites of estradiol 17-sulfate by female rat liver microsomes.

(ES) by hepatic microsomes from male rats, we found the formation of two metabolites, and identified them as 2and 4hydroxyestradiol 17-sulfates (2and 4-OH-ES, Fig. 1) by comparisons with authentic specimens.1 In female rats, in contrast, at least three metabolites in addition to the abovementioned two catechol metabolites were detected during HPLC.1 The identification of structure-unknown metabolites in female rats could not be carried out because there are no available authentic compounds. Reluctantly, we solvolyzed the metabolites to free steroids, which were estimated as 6-, 7-, and 16α-hydroxyestradiol by their chromatographic behaviors on HPLC.2 Recently, we reported a synthetic method for four ES derivatives having a hydroxy group at the C6α, C6β, C7α, or C7β positions as authentic specimens for identifying these structure-unknown metabolites.3 They were 6α-, 6β-, 7α-, and 7β-hydroxyestradiol 17-sulfates (6α-, 6β-, 7α-, and 7β-OH-ES, respectively). This time, we investigated the simultaneous separation conditions for these steroids, including 16αhydroxyestradiol 17-sulfate (16α-OH-ES), in order to establish the analytical method of ES metabolites by female rat liver microsomes. We now describe the details under which these steroidal conjugates were successfully separated using a mobile phase containing γ -cyclodextrin (CD).