固相萃取-超高效液相色谱-三重四极杆质谱法同时测定饲料中5种雌激素

Simultaneous Determination of Five Estrogens in Feed by Ultra Performance Liquid Chromatography-tandem Mass Spectrometry Coupled with Solid Phase Extraction

建立了固相萃取-超高效液相色谱-三重四极杆质谱法(SPE-UPLC-MS/MS)同时测定饲料中双烯雌酚、己烯雌酚、己烷雌酚、17α-雌二醇和17β-雌二醇5种激素的检测方法。饲料样品采用乙醚振荡提取、三氯乙酸除蛋白和正己烷除脂,提取液通过Max固相萃取柱净化,经过Athena C18-WP色谱柱(2.1×150 mm,5μm)分离,以0.25‰氨水和乙腈进行梯度洗脱,负离子电喷雾电离多反应监测模式进行定性和定量分析。同时优化液相色谱条件和质谱参数,以提高灵敏度,并以17β-雌二醇-D3和己烯雌酚-D8为内标进行定量。结果显示,5种激素在0～10μg/L范围内线性相关系(r)≥0.9994;5种雌激素的定量限均为1.0μg/kg;各雌激素的加标浓度为1.0μg/kg、2.0μg/kg、5.0μg/kg时,其加标回收率均在70.20%～114.37%之间,相对标准偏差为0.15%～9.08%(n=6)。结果表明,该方法前处理简单、准确可靠,适用于饲料中5种雌激素的检测。

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.