A validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for measuring the absolute quantities of fifteen endogenous estrogens and their metabolites in human urine.

Available for licensing and commercial development is a patent-pending, validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for measuring the absolute quantities of fifteen endogenous estrogens and their metabolites in human urine. The method is sensitive, specific, accurate, and precise. It requires a single hydrolysis/extraction/derivatization step and only 0.5 mL of urine, yet is capable of simultaneously quantifying estrone, its 2- and 4-methoxy derivatives, and its 2-, 4-, and 16alpha- hydroxy derivatives; estradiol, its 2- and 4-methoxy derivatives, and its 2- and 16alpha-hydroxy derivatives; 2- hydroxyestrone-3-methyl ether; 16-epiestriol; 17-epiestriol; and 16-ketoestradiol in premenopausal and postmenopausal women as well as men. Standard curves are linear over a 103-fold concentration range with the relative standard error of the estimate for the linear regression line ranging from 1.2 to 7.3%, respectively. The lower limit of quantitation for each estrogen is 0.02 ng per 0.5-mL urine sample (only 2 pg placed on column). The percent recovery of a known added amount of estrogen metabolite ranges from 96 to 107%. The overall precision, including the hydrolysis, extraction, and derivatization steps, is 1-5% relative standard deviation for samples prepared concurrently and 1-12% relative standard deviation for samples prepared in separate batches.

U.S. Provisional Application No. 60/688,160 filed 07 Jun 2005 (HHS Reference No. E-207-2005/0-US-01) Inventors: Xia Xu, Timothy Veenstra, Larry Keefer, Regina Ziegler (NCI)

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