A gas chromatography/mass spectrometry assay to measure estradiol, catecholestradiols, and methoxyestradiols in plasma.

We have developed a gas chromatography/mass spectrometry (GC/MS) assay to measure 17beta-estradiol (E) and its biologically active metabolites 2-hydroxyestradiol (2OHE) and 4-hydroxyestradiol (4OHE), and 2-methoxyestradiol (2MEOE) and 4-methoxyestradiol (4MEOE) in rat plasma. All analytes are well separated and show a linear relationship between concentration (0.25-5 pg/microl) and signal, and coefficients of variation (CVs) are low. Intra-assay CV for the lowest quality control samples (QCs) (0.375 pg/microl) were on average for 17beta-estradiol 20.5%, for 2-hydroxyestradiol 15.6%, for 4-hydroxyestradiol 16.5%, for 2-methoxyestradiol 16.5%, and for 4-methoxyestradiol 11.5%. The inter-assay CVs for the lowest QCs were for 17beta-estradiol 12.1%, for 2-hydroxyestradiol 7.1%, for 4-hydroxyestradiol 15.5%, for 2-methoxyestradiol 16.7%, and for 4-methoxyestradiol 9.7%. The highest sensitivity for this assay was observed for hydroxyestradiols followed by the methoxyestradiols and 17beta-estradiol. In summary, we describe a convenient, sensitive, and specific assay to measure 17beta-estradiol and its biologically active metabolites.