Separation of delta6- and delta9,11-estradiol: analytical method development, validation and practical application.

For estradiol (E2) the separation of the degradation products delta6- and delta9,11-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 6alpha-hydroxy-E2, 6beta-hydroxy-E2, 6-keto-E2, delta9,11-E2, beta-equilenol and delta6-E2. The method employs a Kinetex 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.