title: Quantitative analysis of estrogens and estrogen metabolites in endogenous MCF-7 breast cancer cells by liquid chromatography-tandem mass spectrometry.
abstract: 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); 17β-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.
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Huang
authors.forename: Pei-Hua
authors.initials: PH
authors.lastname: Chiang
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Chen
mesh_terms: D001943:Breast Neoplasms / Q000737:chemistry* / Q000378:metabolism*; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media; D004963:Estrenes / Q000032:analysis* / Q000378:metabolism*; D005260:Female; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002457:Cell Extracts; D003470:Culture Media; D004963:Estrenes