Estriol-3 [BSA]

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, 17 beta-estradiol, estriol (16 alpha-OH-17 beta-estradiol), estrone-3-sulfate, 17 beta-estradiol-3-((beta-D-glucuronide), 17 beta-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. (C) 2017 Elsevier B.V. All rights reserved.

The release of a multitude of pollutants from untreated municipal wastewater (UMWW) to surface waters may have adverse effects on aquatic wildlife including endocrine disruption. For effect-directed analysis (EDA), a Danube river water sample downstream of emission of UMWW in Novi Sad, Serbia was extracted on-site and after processing in the lab was subjected to reporter gene assays which revealed pronounced estrogenic (ER alpha), androgenic (AR) and oxidative stress response (OSR). The sample was fractionated with reversed-phase high performance liquid chromatography (RP-HPLC) collecting thirty fractions at two-minute intervals. Biological analysis identified 5 ER alpha- and 3 AR-active fractions while none of the fractions showed considerable activity with regards to OSR. It appeared that OSR of parent sample (PS) distributed over all fractions. Chemical analysis of active fractions by LC-MS/MS and LC-HRMS/MS found female reproductive hormones (estrone (E1), estradiol (E2), estriol (E3)) as cause of ER alpha activity while male reproductive hormones (testosterone, dihydrotestosterone (DHT)) and gestagens (progesterone and medroxyprogesterone) were active in the AR bioassay. Designed chemical mixtures in concentration ratios detected in the active fractions were tested with the bioassays. The identified chemicals quantitatively explained the observed bioactivity with no substantial contribution attributable to xenobiotics. In terms of bioanalytical equivalent concentrations (BEQs), detected chemicals explained 5-159% of ER alpha-active fraction's biological effect and 31-147% for AR-active fractions. Estradiol and dihydrotestosterone were the compounds dominating the most of the effect in this study. In summary, androgenic compounds were found to be as potent as estrogenic compounds while OSR was found to be the cumulative effect of the mixture of many compounds present in the sample rather than the mixture effect dominated by individual chemicals. The obtained results stress the importance of wastewater treatment plant (WWTP) to minimize the pollutant load from UMWW in order to reduce the risk of endocrine disruption to the aquatic life as well as to improve the status of receiving freshwater ecosystem. (C) 2017 Elsevier B.V. All rights reserved.