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应用UE-SPE-HPLC/FLD法检测养殖业畜禽粪便中雌激素
引用本文:付银杰,凌婉婷,董长勋,刘娟,高彦征,潘玉兰.应用UE-SPE-HPLC/FLD法检测养殖业畜禽粪便中雌激素[J].生态学杂志,2013,24(11):3280-3288.
作者姓名:付银杰  凌婉婷  董长勋  刘娟  高彦征  潘玉兰
作者单位:(;1.南京农业大学土壤有机污染控制与修复研究所, 南京 210095; ;2.南京农业大学理学院, 南京 210095)
摘    要:本研究建立了超声提取(UE)-固相萃取净化(SPE)-高效液相色谱法(HPLC/FLD)测定畜禽粪便中雌激素(雌三醇、17β-雌二醇、炔雌醇和双酚A)的分析方法.粪便样品用乙酸乙酯超声提取30 min,C18固相萃取柱净化,经Inertsil ODS-SP-C18(150 mm×4.6 mm, 5 μm)反相色谱柱分离4种雌激素,高效液相色谱/荧光检测器(HPLC/FLD)检测,流动相采用甲醇/乙腈/水(体积比为20∶30∶50),激发/发射波长为280/310 nm,流速0.8 mL·min-1,柱温40 ℃,进样量20 μL.结果表明: 4种雌激素在1.00~1000.00 μg·L-1范围内线性良好(相关系数均大于0.9995),粪便中雌三醇、双酚A、17β-雌二醇和炔雌醇的检出限分别为3.35、5.01、2.13和1.12 μg·kg-1;粪便样品在雌激素浓度为0.05、0.40、1.00 μg·kg-1的添加水平下,猪粪、牛粪和鸡粪中4种物质的平均回收率分别为75.1%~91.1%、78.4%~117.0%和78.6%~97.8%;各物质的相对标准偏差RSD(n=6)均小于6%.采用此方法检测了南京市部分规模化养殖场的猪粪、牛粪和鸡粪样品,结果显示,利用本方法得到4种雌激素检测平行性好、检出限低,适用于畜禽粪便中雌激素的检测分析.

关 键 词:畜禽粪便  雌激素  超声提取  固相萃取  高效液相色谱仪  荧光检测器

Estrogens determination of livestock dung based on UE-SPE-HPLC/FLD.
FU Yin-jie,LING Wan-ting,DONG Chang-xun,LIU Juan,GAO Yan-zheng,PAN Yu-lan.Estrogens determination of livestock dung based on UE-SPE-HPLC/FLD.[J].Chinese Journal of Ecology,2013,24(11):3280-3288.
Authors:FU Yin-jie  LING Wan-ting  DONG Chang-xun  LIU Juan  GAO Yan-zheng  PAN Yu-lan
Institution:(;1.Institute of Organic Contaminant Control and Soil Remediation, Nanjing Agricultural University, Nanjing 210095, China; ;College of Sciences, Nanjing Agricultural University, Nanjing 210095, China)
Abstract:A method for detecting the estrogens estriol, 17β-estradiol, ethinyl estradiol, and bisphenol A in livestock dung was established by the combination of ultrasonic extraction (UE), solid phase extraction (SPE) purification, and high performance liquid chromatography (HPLC) with fluorescence detector (FLD). The dung samples were extracted with ethyl acetate ultrasonication for 30 min, and purified with C18 solid phase extraction column and related solvents. The test four estrogens in the dung samples were isolated with Inertsil ODS-SP-C18 reversed phase columns (150 mm×4.6 mm, 5 μm), and the isolated estrogens were detected with HPLC/FLD. The mobile phase of HPLC for the detection was methanol/acetonitrile/water (volume ratio of 20:30:50), with a flow rate of 0.8 mL·min-1. The excitation and emission wavelengths of FLD were 280 and 310 nm, respectively, the HPLC column temperature was 40 ℃, and the injection volume was 20 μL. Good linearity (correlation coefficient greater than 0.9995) was observed by the HPLC/FLD detection when the test four estrogens concentrations were in the range of 1.00-1000.00 μg·L-1. The detection limit of estriol, bisphenol A, 17β-estradiol, and ethinyl estradiol was 3.35, 5.01, 2.13, and 1.12 μg·kg-1, respectively. When the added estrogens concentrations of pig, cow, and chicken dung samples were 0.05, 0.40, and, 1.00 μg·kg-1, the average recovery of the four estrogens was 75.1%-91.1%, 78.4%-117.0%, and 78.6%-97.8%, respectively, with the relatively standard deviations (RSD, n=6) all less than 6%. By adopting the established SPE HPLC/FLD method to detect the estrogens in real pig, cow, and chicken dung samples from parts of the large scale livestock raising farms in Nanjing of East China, the detection reproducibility was high, and the detection limit was low, being available and effective for the detection of the estrogens in livestock dung.
Keywords:livestock dung  estrogens  ultrasonic extraction  solid phase extraction  high performance liquid chromatography  fluorescence detector  
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