title: Stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine.
abstract: A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography-mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97-99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1-3% for samples prepared concurrently and 2-11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.
authors.affiliation: Epidemiology and Biostatistics Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892-7246, USA.
authors.forename: Xia
authors.initials: X
authors.lastname: Xu
authors.forename: Regina G
authors.initials: RG
authors.lastname: Ziegler
authors.forename: David J
authors.initials: DJ
authors.lastname: Waterhouse
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: Larry K
authors.initials: LK
authors.lastname: Keefer
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D006868:Hydrolysis; D006894:Hydroxyestrones / Q000652:urine*; D007554:Isotopes; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006894:Hydroxyestrones; D007554:Isotopes; C026418:4-hydroxyestrone; C024980:2-hydroxyestrone