Profiling Endogenous Serum Estrogen and Estrogen-Glucuronides by liquid Chromatography-Tandem Mass Spectrometry

Estrogens, namely, 17 beta-estradiol (E-2), are conjugated to glucuronides (G), and this metabolic conversion is part of their tissular-concentration control-mechanism. This inactivation process has been observed, in addition to the liver, in several estrogen-dependent tissues and the resulting polar metabolites are detected in circulation. We developed and validated a highly sensitive and specific mass spectrometry-based method to directly measure estrogen-G serum levels. The method uses deuterated standards but does not involve enzymatic hydrolysis, a major improvement over previous techniques. Estrone (E-1), E-1-sulfate, E-2, the 3-G of E-1, E-2, 2-methoxy-E-1 (2-MeOE1) and 2-methoxy-E-2 (2-MeOE2), and the 17-G of E-2 were measured in serum of 19 premenopausal and 10 postmenopausal healthy women. Two extractions, solid-phase and liquid-liquid, were performed to isolate the estrogens. Estrogens were then quantified by mass spectrometry in the negative MRM ion mode using an API3200 spectrometer with a turbo ionspray source. The method selectively measured estrogen glucuronides with sensitivity >= 5 pg/mL, accuracy 90-111%, and reproducibility (CV = 1.4-13.3%). The method is applicable between 5 and 1000 pg/mL. For the ovarian follicular phase, the major metabolite found was E-1-3G, with E-2-3G and 2-MeOE1-3G found in lesser amounts (54, 10.4, and 7.8 pg/ml, respectively) These concentrations are 2.6- to 3-fold greater than found for luteal-phase estrogens. The concentrations of E-2-17G and 2-MeOE2-3G were usually less than the limit of quantification. In serum of postmenopausal women, E-1-3G was the most abundant estrogen found (30.9 pg/mL). Our method profiles estrogens and estrogen-glucuronides and may represent a new tool to identify biomarkers in hormone-dependent diseases.