In order to undertake an epidemiologic study relating levels of parent estrogens (estrone and estradiol) and estrogen metabolites (EMs) to other breast cancer risk factors, we have optimized methods for EM quantification with ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). A two-step approach was adopted; the first step comprised method development and evaluation of the method performance. The second step consisted of applying this method to quantify estrogens in postmenopausal women and determine if the observed patterns are consistent with the existing literature and prior knowledge of estrogen metabolism. First, 1-methylimidazole-2-sulfonyl chloride (MIS) was used to derivatize endogenous estrogens and estrogen metabolites in urine from study participants. Since C18 reversed phase columns have not been able to separate all the structurally related EMs, we used a C18-pentafluorophenyl (PFP) column. The parent estrogens and EMs were baseline resolved with distinct retention times on this C18-PFP column using a 30 minute gradient. This method was used to quantify the parent estrogens and 13 EMs in urine samples collected in an initial pilot study involving males as well as pre- and peri-menopausal females to assess a range of EM levels in urine samples and enable comparison to the previous literature for assay evaluation. Detection limits ranged from 1 - 20 pg/mL depending on the EM. We evaluated matrix effects and interference as well as the intra- and inter-batch reproducibility including hydrolysis, extraction, derivatization and LC-MS analysis using charcoal-stripped human urine as a matrix. Methods were then applied to the measurement of estrogens in urine samples from 169 postmenopausal women enrolled in an epidemiological study to examine relationships between breast cancer risk, the intestinal microbiome, and urinary EMs. The results from our cohort are comparable to previous reports on urinary EMs in postmenopausal women and enabled thorough evaluation of the method.

为了开展将母体雌激素（雌酮和雌二醇）和雌激素代谢物 (EM) 的水平与其他乳腺癌风险因素相关联的流行病学研究，我们优化了使用超高效液相色谱-高分辨率质谱 (UHPLC) 进行 EM 定量的方法-HRMS）。采用了两步法；第一步包括方法开发和方法性能评估。第二步包括应用这种方法来量化绝经后妇女的雌激素，并确定观察到的模式是否与现有文献和雌激素代谢的先验知识一致。首先，使用 1-甲基咪唑-2-磺酰氯 (MIS) 衍生研究参与者尿液中的内源性雌激素和雌激素代谢物。由于 C18 反相色谱柱无法分离所有结构相关的 EM，我们使用了 C18-五氟苯基 (PFP​​) 色谱柱。母体雌激素和 EM 在此 C18-PFP 色谱柱上使用 30 分钟梯度以不同的保留时间进行基线分离。该方法用于量化在一项涉及男性以及绝经前和围绝经期女性的初步试点研究中收集的尿样中的母体雌激素和 13 种 EM，以评估尿样中的一系列 EM 水平，并能够与之前的文献进行比较用于测定评估。检测限范围为 1 - 20 pg/mL，具体取决于 EM。我们评估了基质效应和干扰以及批次内和批次间的重现性，包括水解、提取、衍生化和使用活性炭剥离的人尿作为基质的 LC-MS 分析。然后将方法应用于测量来自 169 名参加流行病学研究的绝经后妇女的尿液样本中的雌激素，以检查乳腺癌风险、肠道微生物群和尿 EM 之间的关系。我们队列的结果与之前关于绝经后妇女尿 EM 的报告相当，并且能够对该方法进行彻底评估。