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.