Liquid chromatography–tandem mass spectrometric analysis of ten estrogen metabolites at sub-picogram levels in breast cancer women

• Improved LC–ESI-MS method for determination of estrogens and estrogen metabolites in plasma.
• Detection of sub-picogram levels of catechol estrogens in women plasma.
• ESI-ionization suppression due to reagent was minimized by reacting the excess BMP with methanol.
• The method could be applicable for diagnosis of women candidate to breast cancer.
• The proposed procedure is more sensitive and showed less ESI-ionization suppression effect, than commonly applied dansylation method.

The measurement of estrogens at sub-picogram levels is essential for research on breast cancer and postmenopausal plasma. Heretofore, these concentration levels have rarely been achieved. However, it is possible through derivatization but still represent problems for monitoring catechol estrogens and 16α-hydroxyestrone (16α-OH-E1). Estrogens possess poor ionization efficiency in MS/MS, which results in insufficient sensitivity for analyzing samples at trace concentrations. The method presented here was used to extract ten estrogen metabolites (EMs) with a derivatization step involving a new adduct. The electrospray ionization (ESI) MS/MS sensitivity for the EMs was enhanced by derivatization with 3-bromomethyl-propyphenazone (BMP). The lower limits of quantification (LLOQ) of the EMs were 12–100 femtogram on-column, equivalent to 0.3–3.6 pg/mL plasma, and the limits of detection (LOD) were 0.1–0.8 pg/mL plasma. The percentage coefficient of variation (CV%) at the LLOQ was <20 for all investigated EMs. Ionization suppression was minimized by reacting the excess reagent, BMP, with methanol. The method was successfully applied for the determination of ten EMs in the plasma of fifty healthy postmenopausal and fifty ductal breast cancer women aged 47–65 years old. 16α-OH-E1 and three catechol estrogen metabolites, 4-OH-E1, 2-OH-E2 and 4-OH-E2, were successfully measured in the plasma of healthy and breast cancer women. The methyl-propyphenazone-EM derivatives exhibited better sensitivity in ESI-MS (7.5-fold) compared to the commonly used dansylation procedure.