Determination Methods Study For The Three Estrogens In Feed

Hormone residues in foodstuffs of animal origin have become one of the hotissues of the current food safety. The feed is the main source of the exogenoushormones intake for food-borne animal.These hormones can cause the mutation ofanimal physiological characteristics. In the metabolic processes of animal life, hormoneresidues were found in animal muscle and blood. Finally, the hormone residues in foodof animal origin may affect human health. Therefore, the safety of feed quality is foodsafety. In order to better monitor the levels of hormones in feed, the solid phaseextraction-high performance liquid chromatography and liquid chromatographytandem mass spectrometry were developed to determinate17β-estradiol, estradiolbenzoate and estradiol valerate.The research results are list below.（1） Solid phase extraction-high performance liquid chromatography wasemployed to determinate17β-estradiol, estradiol benzoate and estradiol valerate infeed. The effects for the mobile phase composition, gradient elution conditions,column type, column temperature, injection volume and mobile phase flow rate on theretention time and peak area of the three estrogens have been studied. Combined withthe determination of samples, the optimal chromatographic conditions were obtained.The characteristic UV-Vis absorption spectroscopy of three compounds was alsoconfirmed. The detection wavelength of three compounds was230nm（2） The effect of the pre-treatment comditions on the extraction efficiency wasdetailed researched. The results show that solid phase extraction has high extractionefficiency for extracting tested substances, with the extraction efficiency up to85%.With25.0mL acetonitrile and30.0min extraction, the extraction efficiency reachesthe maximum value. Four kinds of solid phase extraction column packed non-polarity materials were used to sample solution purification. The comparisons between thesefour columns showed that Heaion C18was the best, with highest recovery rate.（3） Liquid chromatography tandem mass spectrometry （LC-MS/MS） as aquantitation and confirmation method was employed to determinate17β-estradiol,estradiol benzoate and estradiol valerate in feed. The optimal MS parameters wereobtained.17β-estradiol and estradiol valerate were detected with negative ion mode.Estradiol benzoate was detected with positive ion mode. Their precursor ions were271.37、355.43、377.40, and their qualitative and quantitative ions were145.03and183.13,253.29and101.03,135.08and104.98. Qualitative and quantitative method ofthree compounds was established.17β-estradiol13^C₂isotope internal standardeliminated sample matrix effect.（4） The methodologies of solid phase extraction-high performance liquidchromatography and liquid chromatography tandem mass spectrometry （LC-MS/MS）were investigated. With solid phase extraction-high performance liquidchromatography, the linear range of17β-estradiol, estradiol benzoate and estradiolvalerate were0.20¹20.00μg/mL,0.20¹50.00μg/mL,0.20¹30.00μg/mL; theirlinear relationships were greater than0.9999; their limits of detection were0.95¹.00mg/kg,0.50⁰.75mg/kg,0.80¹.00mg/kg; their recoveries were63.40%¹05.55%,68.00%¹03.23%,68.55%¹07.95%. With liquid chromatography tandem massspectrometry （LC-MS/MS）, the linear range of17β-estradiol, estradiol benzoate andestradiol valerate were10.00²00.00ng/mL,5.00²00.00ng/mL,10.00²00.00ng/mL;their linear relationships were greater than0.9900; their limits of detectionwere0.200⁰.060mg/kg,0.200⁰.025mg/kg,0.220⁰.050mg/kg, their recoverieswere64.67%¹05.55%,68.33%⁹4.69%,68.11%⁹5.66%.