建立了液相色谱/串联质谱（LC-MS/MS）同时测定牛奶中雌酮、17α-雌二醇、17β-雌二醇和雌三醇4种雌性激素残留的方法。牛奶样品用乙腈振荡提取，经过NaCl脱水和低温冷冻除脂，然后用C18固相萃取柱净化，最后分析物采用Hydrosphere C18色谱柱分离，以乙腈和水为流动相进行梯度洗脱，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（流动相、柱流速和柱温）和质谱参数以提高检测灵敏度。以基质匹配外标法定量，方法线性相关系数r>0.999；雌酮、17α-雌二醇、17β-雌二醇和雌三醇定量限均为0.2 μg/kg；各雌性激素的浓度添加水平均为0.2 μg/kg、0.4 μg/kg、1.0 μg/kg时，雌性激素加标回收率范围在69.7~108.0%之间，相对标准偏差在4.3~10.5%之间。结果表明，该方法准确、可靠，适用于牛奶中雌性激素残留的检测。

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of four estrogens residues (estrone, 17 β-estradiol, 17α-estradiol, estriol) in milk samples. The milk samples were extracted by oscillation with acetonitrile, dehydrated with NaCl and defatted under freezing condition, and then purified by solid-phase extraction using C18 cartridges. The analytes were separated using Hydrosphere C18 chromatographic column with gradient elution of acetonitrile/water. Finally, the eluents were qualitatively and quantitatively determined by mass spectrometer with electrospray ionization in negative multiple reaction monitoring mode. The parameters of the liquid chromatography (mobile phase, flow rate, temperature of column) and mass spectrometry were also optimized to enhance detection sensitivity. Using matrix matched external standard method, good linearity in response could be obtained with correlation coefficients larger than 0.999. All quantification limits of estrone, 17β-estradiol, 17α-estradiol and estriol were 0.2 μg/kg. At the spike levels of 0.2 μg/kg, 0.4 μg/kg and 1.0 μg/kg for each estrogen, the recoveries of estrogens were within the range of 69.7%~108.0%, and the relative standard deviation were from 4.3% to 10.5%. The above results showed that the method was accurate and reliable, and suitable for the determination of estrogens residues in milk samples.

国家质检总局局科技计划项目（2010IK137）；广东省省部产学研结合项目（2010B090400342）；“双打”质检公益项目子课题（2012104003-4）