Separation of Δ6- and Δ9,11-estradiol: analytical method development, validation and practical application

For estradiol (E2) the separation of the degradation products 6- and 9,11-E2 is especially challenging due to their structural similarity. There is no method described in the literature yet which adequately addresses this problem. The present study describes a HPLC method for the separation and quantitation of E2 and its degradation products 6α-hydroxy-E2, 6β-hydroxy-E2, 6-keto-E2, Δ9,11-E2, β-equilenol and Δ6-E2. The method employs a KinetexTM PFP analytical column, using methanol and deionized water as mobile phases. Different UV- and fluorescence detection modes were used for maximal sensitivity and specificity. The applicability and capability of the method was demonstrated for Vagifem® tablets. Finally, the method was validated with respect to selectivity, sensitivity, linearity, precision and accuracy.