Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 780, Issue 2, Pages 315-330Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S1570-0232(02)00539-1
Keywords
stable isotope dilution LC-MS; hormonal carcinogenesis; hydroxyestrones; p-toluenesulfonhydrazide
Funding
- NCI NIH HHS [N01-CO-56000] Funding Source: Medline
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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. Published by Elsevier Science B.V.
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