Pharmaceutical Sciences Faculty Publications

Quantitative Measurement of Endogenous Estrogens and Estrogen Metabolites in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry

Document Type

Article

Publication Date

10-15-2007

Journal Title

Analytical Chemistry

ISSN

0003-2700

Volume

79

Issue

20

First Page

7813

Last Page

7821

DOI

10.1021/ac070494j

PubMed ID

17848096

Abstract

Endogenous estrogen plays a key role in the development of human breast cancer, yet the contribution of specific estrogen metabolites and patterns of estrogen metabolism remains unclear. To determine their individual and joint roles in breast carcinogenesis, it is necessary to be able to measure quantitatively each estrogen metabolite in epidemiologic and clinical biospecimens. In this report, we detail a sensitive, specific, accurate, and precise high-performance liquid chromatography-tandem mass spectrometry method utilizing selected reaction monitoring for measuring the absolute quantities of free (unconjugated) and total (conjugated + unconjugated) endogenous estrogens and estrogen metabolites in human serum from premenopausal and postmenopausal women. The method requires a simple sample preparation and only 0.5 mL of serum, yet is capable of quantifying simultaneously 15 estrogens and estrogen metabolites (EM): estrone and its 2-, 4-, and 16alpha-hydroxy and 2- and 4-methoxy derivatives; 2-hydroxyestrone-3-methyl ether; 17beta-estradiol and its 2-hydroxy and 2- and 4-methoxy derivatives; and estriol, 17-epiestriol, 16-ketoestradiol, and 16-epiestriol. The lower limit of quantitation for each EM was 0.4 pg on-column, equivalent to 8 pg/mL (26.5-29.6 fmol/mL) in the original serum sample. Calibration curves were linear over a 10(3)-fold concentration range. For a stripped serum sample containing 8 pg/mL of each EM, accuracy (percent recovery of a known added amount) ranged from 91 to 113%. Intrabatch precision (including hydrolysis, extraction, and derivatization steps) ranged from 7 to 30% relative standard deviation (RSD), and interbatch precision ranged from 8 to 29% RSD. Since distinct roles have been proposed for many of these estrogen metabolites, an accurate, precise, sensitive, and specific method for measuring their levels in circulation should suggest new approaches to breast cancer prevention, screening, and treatment.

Keywords

Chromatography, high pressure liquid, estrogens, hydrolysis, ions, spectrometry, mass, electrospray ionization

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