Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC-MS/MS

Anal Bioanal Chem. 2013 Apr;405(10):3399-406. doi: 10.1007/s00216-013-6710-y. Epub 2013 Feb 1.


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 (13)C-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®.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Estradiol / administration & dosage
  • Estradiol / chemistry*
  • Estradiol / isolation & purification
  • Estrogens / administration & dosage
  • Estrogens / chemistry*
  • Estrogens / isolation & purification
  • Estrone / administration & dosage
  • Estrone / chemistry*
  • Estrone / isolation & purification
  • Humans
  • Isomerism
  • Male
  • Rats
  • Rats, Sprague-Dawley
  • Tandem Mass Spectrometry / methods*


  • Estrogens
  • Estrone
  • Estradiol