Pharmaceutical Sciences Faculty Publications

Title

Measuring Seven Endogenous Ketolic Estrogens Simultaneously in Human Urine by High-Performance Liquid Chromatography-Mass Spectrometry

Document Type

Article

Publication Date

10-1-2004

Journal Title

Analytical Chemistry

ISSN

0003-2700

Volume

76

Issue

19

First Page

5829

Last Page

5836

PubMed ID

15456304

PubMed Central® ID

10.1021/ac049405i

Abstract

A rapid, sensitive, and specific high-performance liquid chromatography-electrospray ionization-multistage mass spectrometry (MS) method for measuring endogenous ketolic estrogen metabolites in human urine has been developed. The method requires a single hydrolysis/extraction/derivatization step and only 2.5 mL of urine, yet is able to simultaneously quantify estrone and its 2-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, 16-ketoestradiol, and 2-hydroxyestrone-3-methyl ether metabolites. The combination of a simple hydrazone derivatization step with multistage MS greatly enhances the sensitivity and specificity of the analysis of endogenous estrogen within human urine. Standard curves are linear over a 100-fold concentration range with linear regression correlation coefficients typically greater than 0.99. The lower limit of quantitation for each ketolic estrogen is 0.2 ng/2.5-mL urine sample (10 pg on column), with an accuracy of 93-103% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-13% relative standard derivation (RSD) for samples prepared concurrently and 8-16% RSD for samples prepared in separate batches. This method also allows for the identification of 2-hydroxyestrone-3-methyl ether in urine obtained from both pre- and postmenopausal women. This potentially protective estrogen metabolite has been previously reported only in the urine of pregnant women. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate and specific measurement of estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Keywords

Chromatography, high pressure liquid, estrogens, ether, hydrolysis, hydroxylation, ketones, methylation, postmenopause, premenopause, spectrometry, mass, electrospray ionization

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