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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,5μm)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20μL.结果表明:4种雌激素在1.00~1000.00μg·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1.12μg·kg-1;粪便样品在雌激素浓度为0.05、0.40、1.00μg·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.
In this study, the determination of estrogen (estriol, 17β-estradiol, ethinylestradiol and bis (2-ethylhexyl) estradiol) in livestock and poultry manure by ultrasonication (SPE-HPLC- Phenol A). The stool samples were extracted with ethyl acetate for 30 min and cleaned up with a C18 solid phase extraction column. The four estrogens were separated on a reversed-phase Inertsil ODS-SP-C18 column (150 mm × 4.6 mm, 5 μm) (HPLC / FLD). The mobile phase consisted of methanol / acetonitrile / water (volume ratio 20:30:50), the excitation / emission wavelength was 280/310 nm and the flow rate was 0.8 mL · min -1, the column temperature was 40 ℃, and the injection volume was 20 μL.The results showed that the linearity of the four estrogens in the range of 1.00-1000.00 μg · L-1 (correlation coefficient> 0.9995), estriol, bisphenol A , And the detection limits of 17β-estradiol and ethinyl estradiol were 3.35, 5.01, 2.13 and 1.12 μg · kg-1, respectively. The stool samples were tested at estrogen concentrations of 0.05, 0.40 and 1.00 μg · kg-1, The average recoveries of four substances in pig manure, cow manure and chicken manure were 75.1% -91.1%, 78.4% -117.0% and 78.6% -97.8%, respectively. The relative standard deviations RSD (n = 6) 6%. South was detected using this method Pig, cow and chicken manure sample portion of city-scale farms, the results show that the use of this method to obtain four kinds of parallel detection of estrogen is good, low detection limit, detection and analysis of livestock manure applied to estrogen.