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 0.005–2 ng/ml, ULOQ 3–2000 ng/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.