Quantitative analysis of estrogens and estrogen metabolites in endogenous MCF-7 breast cancer cells by liquid chromatography-tandem mass spectrometry.

To study the roles of estrogens and estrogen metabolites (EMs) in breast carcinogenesis, we reported a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing selective reaction mode (SRM) to analyze estrogens and EMs in the extracellular and intracellular compartments of endogenous MCF-7 breast cancer cells through simple ethyl acetate (EA) extraction and dansyl chloride derivatization. Under a 35-min LC gradient elution on a reversed phase C18 column, the method was shown to simultaneously quantify 12 estrogens and EMs: estrone (E1) and its 2-, 4-, 16α-hydroxy derivatives (2-OHE1, 4-OHE1, 16α-OHE1), and 2-, 4-methoxy derivatives (2-MeOE1, 4-MeOE1); 17ß-estradiol (E2) and its 2-, 4-hydroxy derivative (2-OHE2, 4-OHE2) and 2- and 4-methoxy derivatives (2-MeOE2 and 4-MeOE2); and estriol (E3), using ethinylestradiol (EE2) as the internal standard (IS). Using a calibration curve-standard addition hybrid method, we were able to determine the amount of estrogens and EMs in not only the treated cells but also the non-treated cells. The limits of quantification (LOQs) were determined to range from 0.05-80 pg on column with an inter-batch accuracy around 72-123% and precision around 1-10%. Results indicated that trace amounts (<0.9 fg/cell) of E1 and E2 were present in both the extra- and intra-cellular compartments under non-treated condition but DMSO could induce E1 and E2 as well as trace amounts (<2.25 fg/cell) of EMs in the cell. E2 treatment substantially increased not only E1 and E2 in the intra-cellular (60 fg/cell) and extra-cellular (3000 fg/cell) compartment but also substantially induced EMs primarily in the extracellular compartment (0.6-25 fg/cell). These data implied that EMs could be quickly generated and distributed to the extracellular compartment by E2 within 24h of treatment and DMSO solvent could potentially induce slight estrogen effects.