同位素内标-高效液相色谱-串联质谱法测定畜禽粪便中6种雌激素

为了研究畜禽粪便中雌激素的污染特征及其环境影响,建立了一种同时提取、分离和检测畜禽粪便中雌酮(E1)、17α-雌二醇(17α-E2)、17β-雌二醇(17β-E2)、雌三醇(E3)、17α-炔雌醇(17α-EE2)和己烯雌酚(DES)共6种雌激素的高选择性和高灵敏性方法——同位素内标-固相萃取-高效液相色谱-串联质谱法。对提取剂、淋洗剂、洗脱剂、色谱分离条件和质谱测定条件等进行了优化。最终的优化方法为:粪便样品加入100 ng同位素内标后,先后用乙腈重复超声提取3次(每次10 mL,每次15 min),离心后合并上清液,旋转蒸发近干再复溶于1 mL甲醇,加7 mL纯水稀释并超声混匀后离心,上清液过0.45μm滤膜后用HLB固相萃取柱净化,经10 mL 15%乙腈水溶液淋洗后,用10 mL的甲醇进行洗脱,氮吹浓缩定容至1 mL,过0.22μm滤膜后测定。用2 mM氟化铵-甲醇为流动相,经TAB色谱柱分离后采用电喷雾离子源和选择反应监测负离子扫描模式进行检测,同位素内标定量。在5、50、200、1000 ng·g^?1加标水平下,6种雌激素的平均回收率为90.2%—103.2%,相对标准偏差为0.09%—9.08%,方法检出限(LOD)和方法定量限(LOQ)分别为0.25—0.95 ng·g^?1和1.00—3.82 ng·g^?1。应用此方法检测了6个猪粪、3个牛粪和3个鸡粪样品中6种雌激素的含量,结果为?1(干质量)。该方法灵敏度高,定量准确且定量范围广,适用于畜禽粪便中6种雌激素的定性定量分析。

Determination of Six Estrogens in Livestock Manures by Isotope Internal Standard-High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry

In order to study the pollution characteristics and their environmental impact of estrogens in livestock,a highly selective and sensitive method of isotope internal standard-solid phase extraction-high performance liquid chromatography-tandem mass spectrometry was developed for extracting,separating,and determining 6 estrogens(estrone(E1),17α-estradiol(17α-E2),17β-estradiol(17β-E2),estriol(E3),17α-ethinylestradiol(17α-EE2),diethylstilbestrol(DES))in livestock manure simultaneously.The extraction,leaching and elution solvents as well as conditions of chromatographic separation and mass spectrometry determination were optimized as follows:after the introduction of 100 ng isotopic internal standards,the estrogens were subsequently ultrasonic extracted by acetonitrile for 3 times(each time 10 mL,15 minutes),the extracts were centrifuged,combined and rotary evaporated to nearly dry,and then 1 mL methanol and 7 mL pure water were added successively for re-dissolution and dilution,after the ultrasonic mix and centrifuged again,the extracts were filtered through a 0.45μm membrane filter and purified by HLB column.The targets were leached with 10 mL acetonitrile aqueous solution(15%),eluted by 10 mL methanol and concentrated to 1 mL under gentle nitrogen for further instrumental analysis after being filtered through a 0.22μm membrane filter.The estrogens were separated on a TAB column with 2 mM NH4F-methanol as mobile phase,then determined under an electrospray ion source and a selected reaction monitoring negative ions scanning mode,which finally quantitated by internal standard method using isotopic standards as correcting internal standards.The average recoveries at 5,50,200 and 1000 ng·g^?1 spiked levels ranged from 90.2%to 103.2%,with relative standard deviations of 0.09%?9.08%.The limits of detection(LODs)and limits of quantitation(LOQs)were 0.25?0.95 ng·g^?1 and 1.00?3.82 ng·g^?1,respectively.6 pig manure,3 cow lung,and 3 chicken manure samples were analyzed by using the developed method,the contents of such 6 estrogens ranged from?1 dry mass.The results showed that the proposed method is sensitive,accurate,and appliable for determination of a wide range of contents,which is suitable for qualitative and quantitative analysis of those typical estrogens in livestock manure.