Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC–MS/MS
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We show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs 13C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®.
KeywordsEstrogens Phenyl-hexyl stationary phase LC–MS/MS Dansylation 13C-labeled internal standards
We thank Ms. Shastazia White for her excellent technical assistance. This work was supported in part by the National Institutes of Health (grant number AG031535 to LP and AG031421 to KPT) and the Robert A. Welch Foundation (endowment BK-0031 to LP).
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