Stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine.
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.
Xu
Xia
X
Epidemiology and Biostatistics Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892-7246, USA.
Ziegler
Regina G
RG
Waterhouse
David J
DJ
Saavedra
Joseph E
JE
Keefer
Larry K
LK
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*
D006894:Hydroxyestrones; D007554:Isotopes; C026418:4-hydroxyestrone; C024980:2-hydroxyestrone