Abstract:A method was developed for the simultaneous determination of five kinds of estrogens (dienestrol, diethylstilbetrol, hexestrol, 17α-estradiol and 17β-estradiol) in feed by ultra performance liquid chromatography-tandem mass spectrometry coupled with solid phase extraction. Estrogens were extracted by ethyl ether from the feed sample. The extract was deproteinized with trichloroacetic acid (TCA), defatted with n-Hexane, and then purified by Max phase extraction column. Five kinds of estrogens were derived by Athena C18-WP chromatographic column with gradient elution of acetonitrile/0.25‰ ammonia water. Finally, identification and quantification were performed using multiple reaction monitoring (MRM) with one precursor ion, and two product ions for each analyte and electrospray ionization in negative mode. The parameters of the liquid chromatography and mass spectrometry were also optimized to enhance detection sensitivity. The quantity of estrongens were determined by internal standard method with 17β-estradiol-D3 and diethylstilbestrol-D8 as reference. The results showed that the linear ranged from 0 to 10 μg/L for five estrogens with the correlation coefficients (r) were no less than 0.9994. The limits of quantitation (LOQ) for all analytics were 1.0 μg/kg. At the spike levels of 1.0 μg/kg, 2.0 μg/kg and 5.0 μg/kg for each estrogen, the recoveries of estrogens were within the range of 70.20%~114.37%, and the relative standard deviation was no more than 9.08% (n=6). The results demonstrated that the developed method can simultaneously determine the five estrogens in feed with advantages of simple pretreatment and high accuracy.
