Estrogens are metabolized to active quinones that modify DNA and may lead to various cancers. To extend the analytical methodology for estrogen-modified purine bases, we report here a simple modification to existing synthetic procedures that use 2-iodoxybenzoic acid (IBX) as the oxidizing agent for the reference material and putative biomarker, 4-hydroxyestrone-1-N3adenine (4-OH-E1-1-N3Ade). The reaction leads to two catechol estrogen quinones, CE1-2,3-Q and CE1-3,4-Q, both of which react via Michael additions to afford 4-OH-E1-1-N3Ade and other DNA adducts. Liquid chromatography separation permits the isolation of high-purity 4-OH-E1-1-N3Ade. With this method, we also prepared single 13C and uniformly 15N (U-15N) labeled 4-OH-E1-1-N3Ade with 8-13C-labeled Ade and U-15N-labeled adenosine 5’-monophosphate (AMP). The approach is also effective for the synthesis of 4-hydroxyestradiol-1-N3adenine, 4-OH-E2-1-N3Ade, and 4-hydroxyestrone(estradiol)-1-N7guanine, 4-OH-E1(E2)-1-N7Gua. The tandem mass spectra (MS2 and MS3) of 4-OH-E1-(unlabeled, 8-13C-, and U-15N-labeled)1-N3Ade and accurate mass measurements for MS2 product ions allow us to assign unambiguously the formula of fragments and delineate the fragmentation pathways. One important reaction is dehydration, which occurs at the ketone oxygen in the C-17 position of estrone. Another is loss of NH3, an ubiquitous process for purines and modified purines, which is affected by the steroid modification. Evidence from MS/MS supports the migration of H-atom(s) from estrone in the loss of NH3. An interesting interaction occurs between the steroid and the Ade in the modified base, promoting loss of CH2NH, a loss that distinguishes modified Ade from unmodified Ade. The synthesis of a stable isotope-labeled 4-OH-E1-1-N3Ade and the understanding of the fragmentation processes will enable studies aimed at the etection of naturally occurring 4-OH-E1-1-N3Ade in biological samples.