液相色谱-串联质谱法测定猪肝中痕量己烯雌酚、己烷雌酚和双烯雌酚

建立了液相色谱-串联质谱法测定猪肝中痕量己烯雌酚、己烷雌酚和双烯雌酚。以氟化铵为流动相添加剂，目标物响应值平均提高约10倍，向样品中加入1%（V/V）乙酸乙腈超声提取，提取液经分散固相萃取净化、水稀释后，采用Waters Acquity UPLC HSS T3色谱柱，以0.1 mmol/L氟化铵和乙腈为流动相，梯度洗脱分离，电喷雾电离源负离子模式和多反应监测模式检测，外标法定量。在0.4、0.8、4.0 μg/kg添加水平下，3种目标物的加标回收率为89.5%～109.4%，相对标准偏差（RSD）为2.2%～7.8%，方法定量限为0.4 μg/kg。本方法前处理过程无需浓缩，操作简便、灵敏度高、重现性好、环保节约，适用于猪肝中痕量己烯雌酚、己烷雌酚和双烯雌酚的定性与定量分析。

Determination of Trace Diethylstilbestrol,Hexestrol and Dienestrol in Pork Liver by LC-MS/MS

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of trace levels of diethylstilbestrol, hexestrol and dienestrol in pork liver. The sensitivity was achieved by using ammonium fluoride as an eluent additive, which significantly enhanced the analytical signals on average about 10 times. The analytes were extracted with acetonitrile containing 1%（V/V） acetic acid by ultrasonic extraction. Then, the extracts were purified by dispersive solid phase extraction with C18 and then diluted with water for LC-MS/MS analysis. Chromatographic analysis was conducted on a Waters Acquity UPLC HSS T3 column using acetonitrile-0.1 mmol/L ammonium fluoride as mobile phase with gradient elution. The analytes were detected by negative electrospray ionization-tandem mass spectrometry under multiple reaction monitoring mode. The enzymolysis method was investigated using β-glucuronidase/sulfatase enzyme from Helix pomatia for 12 h at 37 ℃. The results showed the cis-trans-tautomerism of diethylstilbestrol and diethylstilbestrol-d8 during enzymolysis, and the signals of the two compounds were reduced significantly by 60%. Only the same isotope internal standard calibration was used to obtain the accurate results with enzymolysis method.The quantification was achieved using external standard method without enzymolysis. Because the samples turned out to be sufficiently dilute, the matrix effects were soft (enhancement of 9.6%-13.6%) and negligible. The recoveries at fortification levels of 0.4, 0.8, 4.0 μg/kg in pork liver ranged from 89.5% to 109.4% with the relative standard deviations of 2.2%-7.8%. The limits of quantification (S/N>10) were 0.4 μg/kg for all analytes. The method does not require concentration for the whole sample processing, and need not employ isotope internal standard or matrix-matched calibration for quantification. It is also highly simple, reliable and sensitive in detecting the trace diethylstilbestrol, hexestrol and dienestrol in pork liver.