Abstract 2206: A new sensitive, accurate liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure circulating estrogens, androgens, and progesterone in postmenopausal and premenopausal women
Authors: Duke Lumi , Kim Paul H. , Botelho Julianne Cook , Ziegler Regina G. , Vesper Hubert 
Published: August 2018

Abstract
Endogenous steroid hormones play a critical role in the etiology of breast cancer. However, the interrelationships among estrogens, androgens, and progesterone in determining risk are not well understood. Relative strengths of associations, independent and joint effects, differential effects for tumor subtypes, and effect modification by lifestyle and genetic profile remain unclear. A major reason is the limited accuracy, specificity, and sensitivity of many steroid hormone assays, especially at the low circulating hormone concentrations characteristic of postmenopausal women.
CDC developed a new sensitive, accurate isotope dilution LC-MS/MS method capable of measuring eight important steroid hormones in 200 µL of female serum. The method uses sequential liquid-liquid extractions for isolation of estradiol (E2), estrone (E1), estrone sulfate (E1S), testosterone (TT), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), progesterone (P), and 17-hydroxyprogesterone (17-OHP) without derivatization or hydrolysis. Chromatographic separation of the steroids is carried out using a phenyl-hexyl HPLC column and a gradient of methanol and methanol:water. Scheduled selected reaction monitoring by electrospray ionization in the positive and negative ion modes is applied to quantify the analytes. Two mass transitions, each specific to the fragmentation of the analyte, are monitored for each analyte: quantitation ion (QI) and confirmation ion (CI). The QI to CI ratios are used as criteria to identify possible interferences.
The method is able to measure each steroid in nearly all postmenopausal women as well as premenopausal women. Limits of detection in serum-based materials are: E2 1.72 pg/mL, E1 0.13 ng/dL, E1S 2.04 pg/mL, TT 0.57 ng/dL, A 0.82 ng/dL, DHEAS 0.22 µg/dL, P 0.86 ng/dL, and 17-OHP 0.41 ng/dL. Precision, expressed as coefficients of variation and evaluated at three levels for each analyte, ranged from 1.2 to 9.9%. Average bias, a measure of accuracy, ranged from -0.3 to 8.8%. The method is certified through the CDC Hormone Standardization Program for estradiol and testosterone and meets the requirements of the College of American Pathologists Proficiency Testing Program for androgens and progesterone.
This new, highly specific and sensitive method demonstrates high accuracy and precision while requiring small amounts of specimens. It can assay up to 140 samples at once. Thus, the method is suitable for large epidemiologic studies investigating the hormonal etiology of breast, endometrial, and ovarian cancer.
Citation Format: Lumi Duke, Paul H. Kim, Julianne Cook Botelho, Regina G. Ziegler, Hubert Vesper. A new sensitive, accurate liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure circulating estrogens, androgens, and progesterone in postmenopausal and premenopausal women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2206.