Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by high performance liquid chromatography-electrospray ionization tandem mass spectrometry with phenazine derivatization

Article abstract of DOI:10.1016/j.jsbmb.2010.02.016

A sensitive and selective assay method for labile estrogen o-quinones, estrone (E₁)-2,3-quinone (Q), E₁-3,4-Q, estradiol (E₂)-2,3-Q and E₂-3,4-Q, based on the use of phenazine (Phz) derivatization with o-phenylenediamine and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were purified by solid phase extraction and analyzed by HPLC-ESI-MS/MS. The protonated molecule was observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode). In multiple reaction monitoring, the transition from [M+H]⁺ to m/z 231 was chosen for quantification. Calibration curves for the o-quinones were obtained using standard catechol estrogens after sodium metaperiodate treatment and Phz derivatization. Using this method, these four estrogen o-quinones were analyzed with the limit of quantification of 5ng/ml in acetonitrile (MeCN)-blank matrix (1:4, v/v), respectively, on a basis of the weight of catechol estrogens. Assay accuracy and precision for four estrogen o-quinones were 89.6-113.0% and 3.1-12.6% (5, 125 and 2000ng/ml in MeCN-blank matrix). Applications of this method enabled to determine the catalytic activities on hydroxylation and subsequent oxidation of E₁ and E₂ of Mushroom tyrosinase and rat liver microsomal fraction. It was confirmed by this method that tyrosinase exhibited 2- and 4-hydroxylation and further oxidation activities for catechols in the ring-A of estrogens. Whereas rat liver microsomal fraction possessed only 2- and 4-hydroxylation activities, and further oxidation activity for catechol estrogens was low.