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A liquid chromatography–mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites

Abstract

The ability to measure estrogen metabolites (EMs) quantitatively is important for investigating their individual roles in cancer screening, treatment and prevention, as well as in a host of other hormone-related disorders. In this protocol we describe a method that is capable of quantitating 15 distinct EMs in urine. Endogenous EMs are quantitatively measured using a liquid chromatography–tandem mass spectrometry method in which the spectrometer operates in a selected reaction monitoring mode. This method is capable of quantifying estrone and its 2-, and 4- and 16α-hydroxy and its 2-, 4-methoxy derivatives, and 2-hydroxyestrone-3-methyl ether; 17β-estradiol and its 2-hydroxy, and 2- and 4-methoxy derivates, and estriol, 16-epiestriol, 17-epiestriol, and 16-ketoestradiol. The method requires only 0.5 ml of urine and approximately 60 urine samples can be quantitatively analyzed per week.

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Figure 1: Endogenous estrogen metabolism in humans.
Figure 2: Summary of method for the analysis of 15 endogenous estrogens and their metabolites in urines by HPLC–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS2).
Figure 3: HPLC–electrospray ionization–tandem mass spectrometry selected reaction monitoring (SRM) chromatographic profiles of dansylated estrogens and estrogen metabolites (EMs) corresponding to (a) a 0.5-ml pre-menopausal urine sample and (b) a 0.5-ml post-menopausal urine sample.

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Acknowledgements

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health (NIH), under Contract NO1-CO-12400 and by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the United States Government.

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Correspondence to Timothy D Veenstra.

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Xu, X., Keefer, L., Ziegler, R. et al. A liquid chromatography–mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites. Nat Protoc 2, 1350–1355 (2007). https://doi.org/10.1038/nprot.2007.176

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