Abstract 587: Highly sensitive and accurate method for measuring serum estradiol in postmenopausal women and other population subgroups
Authors: Duke Lumi , Kim Paul H. , Botelho Julianne Cook , Zhou Hui , Ulmer Candice Z. , Vesper Hubert W. 
Published: August 2019

Abstract
Meta-analyses consistently show that postmenopausal women with elevated levels of estradiol have an increased risk for certain cancers, such as breast cancer. At the same time, estradiol therapy is used to treat menopausal symptoms. Data on blood levels of estradiol are highly variable, especially in postmenopausal women, which prevents the formulation of generally recognized normal ranges and the consistent treatment of postmenopausal women. Similarly, no generally recognized reference ranges for estradiol exist for men and children. One reason for this variability in estradiol blood levels is the lack of appropriate analytical methods. To overcome these challenges, new, highly sensitive, specific, and standardized methods for measuring estradiol are needed. This study describes a new analytical method for measuring estradiol in serum from post and premenopausal women, men and children, which is standardized to the CDC Hormone Standardization Program (HoSt). Data produced by this analytical method can be compared with data produced by other methods standardized to CDC HoSt. The analytical method optimizes the extraction of estradiol from serum using two liquid-liquid extractions with pH and polarity adjustments prior to LC-MS/MS analysis. All sample handling and extraction procedures are automated using 96-well plates. Chromatographic separation is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. E2 and its C13 internal standard were analyzed by selected reaction monitoring (SRM) in the negative ion mode with transitions of m/z 271 to 145 and 274 to 148, respectively.
The mean biases of this method to certified reference materials ranged between −1.3% and −1.7% and were statistically not significant. No significant difference to established metrological reference methods was determined. The limit of detection for estradiol using 200 µL of serum is 11.0 pM (2.99 pg/mL). The method is highly precise with a within-run, among-day (determined over 68 days), and total within-laboratory imprecision ranging between 2.9-5.0%, 1.5-1.8%, and 3.3-5.3% CV, respectively [1].
The sensitivity of the method was found to be suitable for determining estradiol levels in postmenopausal women, men, and children, and is being applied to measure estradiol in postmenopausal women in NHANES 2013-2014. Data generated by this method are comparable to those of other analytical methods that are standardized to the CDC HoSt program. This method allows for the development of generally recognized normal ranges of estradiol in postmenopausal women and other population subgroups.
References:
1. Zhou, Hui et al. “Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry” Analytical and bioanalytical chemistry vol. 409,25 (2017): 5943-5954.
Citation Format: Lumi Duke, Paul H. Kim, Julianne Cook Botelho, Hui Zhou, Candice Z. Ulmer, Hubert W. Vesper. Highly sensitive and accurate method for measuring serum estradiol in postmenopausal women and other population subgroups [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 587.