同位素稀释-超高效液相色谱-串联质谱法同时测定精油中的7种雌性激素

Simultaneous determination of 7 female sex hormones in essential oil by high performance liquid chromatography-tandem mass spectrometry with isotope dilution

建立了采用同位素稀释-超高效液相色谱-串联质谱同时快速测定精油中7种雌性激素（雌三醇、雌二醇、雌酮、炔雌醇、己二烯雌酚、己烷雌酚、己烯雌酚）的方法。样品中雌性激素用乙酸乙酯-正己烷（2∶98,v/v）溶液提取后,经硅胶固相萃取小柱净化,通过ACQUITY UPLCTMBEH SHELD RP18色谱柱（100mm×2.1mm,1.7μm）、以水-乙腈作流动相梯度洗脱对7种雌性激素进行分离,采用串联质谱在负离子扫描方式下通过多反应监测（MRM）模式进行定性定量分析。以雌三醇-D3、雌二醇-D3、己烯雌酚-D6为内标,有效减少了样品基质的影响。该方法对精油中7种雌激素的检出限（LOD）为0.3-7μg/kg,定量限（LOQ）为1-20μg/kg。待测物与内标物定量离子的峰面积比值与待测物的质量浓度在20-500μg/L范围内呈良好的线性关系,相关系数（r^2）均大于0.997;在20-500μg/kg范围内3个水平的加标平均回收率为88.5%-114.8%,日内精密度（以相对标准偏差计）（n=6）为4.8%-18.9%。应用该方法对浙江杭州地区不同超市或美容院随机采集的12份精油样品进行测定的结果显示,有1份样品含有雌二醇和雌酮,其余11份样品均未检出雌性激素。

A reliable ultra-high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） method for the simultaneous determination of 7 female sex hormones（estriol,estradiol,estrone,ethinyloestradiol,dienestrol,hexestrol,diethylstilbestrol） in essential oil was developed.The sample was extracted by ethylacetate-normal hexane solution（2∶98,v/v） and the extract was purified by a silica solid phase extraction-based clean-up column.Then,the analytes were separated on an ACQUITY UPLC BEH SHELD RP18 column（100 mm×2.1 mm,1.7 μm） in gradient elution with the mobile phases of water and acetonitrile.The separated compounds were detected with a Waters Xevo TQ MS tandem quadrupole mass spectrometer operated in negative electro-spray ionization using multiple reaction monitoring mode.Estriol-D3,estradiol-D3 and diethylstilbestrol-D6 were used as the internal standards to reduce the matrix effects.The limits of detection and quantitation for the 7 female sex hormones in essential oil were 0.3-7 μg/kg and 1-20 μg/kg,respectively.Good linear relationships and high correlation coefficients（r^2≥0.997） were obtained in the mass concentration range of 20-500 μg/L.The average recoveries were 88.5%-114.8% and the intra-assay relative standard deviations were 4.8%-18.9% at the spiked levels of 20-500 μg/kg.Finally,a total of 12 samples randomly collected from different supermarkets in Zhejiang Province were screened for the 7 female sex hormones by the proposed method.The results showed that only one sample contained estradiol and estrone.