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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.