Simultaneous quantification of estrogens, their precursors and conjugated metabolites in human breast cancer cells by LC-HRMS without derivatization.

Liquid chromatography-mass spectrometry (LC-MS) is the state of the art technique for quantification of steroid hormones. Currently used methods are typically limited by the need of pre-column derivatization to increase ionization efficiency; however, this causes hydrolysis of conjugated metabolites. Our newly established LC-HRMS method is able to simultaneously quantify conjugated and unconjugated steroids without prior derivatization using deuterated internal standards and solid-phase extraction. This assay was validated according to ICH Q2(R1) guidelines for the analysis of the 10 main steroids of the estrogenic pathway, namely 4-androstene-3,17-dione, dehydroepiandrosterone (DHEA), DHEA-3-sulfate, estrone, 17β-estradiol, estriol (16α-OH-17β-estradiol), estrone-3-sulfate, 17β-estradiol-3-(β-d-glucuronide), 17β-estradiol-3-sulfate and testosterone. Assay performance characteristics were excellent with results for accuracy (98.8-101.2%), precision (mean: 2.05%, all ≤2.80%), stability over five freeze-thaw-cycles (95.7-100.4%) and SPE accuracy (96.9-102.0%), as well as suitable lower and upper limits of quantification for cell culture experiments (LLOQ 0.005-2ng/ml, ULOQ 3-2000ng/ml). Furthermore, we demonstrated the functionality of our method for the monitoring of steroid levels in the human breast cancer cell line MCF-7. This sensitive assay allows for the first time detailed investigations on estrogen metabolomics in breast cancer cells and may also apply to other estrogen-dependent tumor entities.