Fu Yin-Jie, Ling Wan-Ting, Dong Chang-Xun, Liu Juan, Gao Yan-Zheng, Pan Yu-Lan
Organic Contaminant Control and Soil Remediation, Nanjing Agricultural University, Nanjing 210095, China.
College of Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Ying Yong Sheng Tai Xue Bao. 2013 Nov;24(11):3280-8.
A method for detecting the estrogens estriol, 17beta-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 x 4.6 mm, 5 microm), 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 x min(-1). The excitation and emission wavelengths of FLD were 280 and 310 nm, respectively, the HPLC column temperature was 40 degrees C, and the injection volume was 20 microL. 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 microg x L(-1). The detection limit of estriol, bisphenol A, 17beta-estradiol, and ethinyl estradiol was 3.35, 5.01, 2.13, and 1.12 microg x kg(-1), respectively. When the added estrogens concentrations of pig, cow, and chicken dung samples were 0.05, 0.40, and, 1.00 microg x 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.
通过超声提取（UE）、固相萃取（SPE）净化和配备荧光检测器（FLD）的高效液相色谱（HPLC）相结合的方法，建立了一种检测家畜粪便中雌三醇、17β-雌二醇、乙炔雌二醇和双酚A的方法。粪便样品用乙酸乙酯超声提取30分钟，并用C18固相萃取柱及相关溶剂进行净化。粪便样品中的四种受试雌激素用Inertsil ODS-SP-C18反相柱（150 mm×4.6 mm，5μm）进行分离，分离后的雌激素用HPLC/FLD进行检测。用于检测的HPLC流动相为甲醇/乙腈/水（体积比为20:30:50），流速为0.8 mL·min⁻¹。FLD的激发波长和发射波长分别为280和310 nm，HPLC柱温为40℃，进样量为20μL。当四种受试雌激素浓度在1.00 - 1000.00μg·L⁻¹范围内时，HPLC/FLD检测呈现出良好的线性关系（相关系数大于0.9995）。雌三醇、双酚A、17β-雌二醇和乙炔雌二醇的检测限分别为3.35、5.01、2.13和1.12μg·kg⁻¹。当猪、牛和鸡粪便样品中添加的雌激素浓度分别为0.05、0.40和1.00μg·kg⁻¹时，四种雌激素的平均回收率分别为75.1% - 91.1%、78.4% - 117.0%和78.6% - 97.8%，相对标准偏差（RSD，n = 6）均小于6%。采用所建立的SPE-HPLC/FLD方法对华东地区南京部分大型养殖场实际猪、牛和鸡粪便样品中的雌激素进行检测，检测重现性高，检测限低，适用于家畜粪便中雌激素的检测且有效。
