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A simple,fast,robust,sensitive and specific method for the confirmation and quantification of four kinds of estrogens,including 17β-estradiol(E2),estriol(E3),4-hydroxyestradiol(4-OHE2) and 2-methoxyestradiol(2-MeOE2) in fortified and incurred fish and shrimp flesh samples was developed. The method used a simple extraction of sample with acetonitrile,dispersive solid-phase extraction(dispersive-SPE) cleanup,and high performance liquid chromatography(HPLC) coupled with a fluorescence detector for confirmation and quantitation.