在线固相萃取—超高效液相色谱—串联质谱法
张品等
摘 要 建立了乳制品中三氯生、三氯卡班、双酚A和壬基酚4种内分泌干扰物的在线固相萃取超高压液相色谱串联质谱（Online SPE LCMS/MS）检测方法。液态乳制品或奶粉样品中加入乙酸缓冲液，目标物经β葡糖醛酸苷肽酶/芳基磺酸酯酶酶解后， 用乙腈提取， 冷冻离心10 min后，取上清液，用水稀释，在线固相萃取串联质谱法测定。样品溶液经Xbridge C8柱富集，BEH C18色谱柱分离，甲醇和水梯度洗脱，三重四极杆质谱电喷雾负离子模式下采集数据，同位素内标法定量。4种目标化合物的线性范围为0.005～5.0 μg/L，相关系数R2>0.99；方法的定量限为0.03～1.0 μg/kg，3个添加水平的平均加标回收率为80.2%～106.7%，
3.2 提取条件的优化
环境污染物或药物进入生物体后在肝脏酶系统的作用下会转化成葡糖糖醛酸结合态。Zhou等[ 18]发现人体尿液中的三氯卡班及其代谢产物主要以结合态形式存在，尽管奶样中是否存在结合态还未有定论[ 19，20]，但为了保证检测结果的准确性，本研究采用β葡糖醛酸苷肽酶/芳基磺酸酯酶进行酶解。
由于4种目标化合物均有一定的亲脂性，因此，酶解后的样品提取过程比较了乙腈和甲醇两种与水互溶的极性有机溶剂。称取适量液奶样品，加标量为5 μg/kg，如2.3节所述进行处理，每种提取溶剂做3个平行。离心后提取液直接测定，从表观上看，乙腈提取液比甲醇提取液更清澈透明。结果表明，乙腈提取液对TCS，TCC，NP和BPA的绝对回收率分别为95%，89%，91%和78%，比甲醇体系略高（分别为90%，85%，92%和69%）。综合表观结果和回收率数据，确定乙腈为提取溶剂。
3.3 背景污染的考察
由于TCS，NP和BPA广泛应用于多个领域，因此这几种污染物在环境中普遍存在，甚至在Milpore的超纯水中检测到了BPA[ 21]。而且，塑料器皿或橡胶制品等都会迁移出大量NP 和BPA，这些均会对分析结果造成干扰。由于离线固相萃取小柱的筛板为聚丙烯等塑料材质，有机溶剂浸润时会持续溶出NP，其浓度大于0.2 μg/L[ 17]；使用二氯甲烷、正己烷等非极性有机溶剂进行液液萃取时，其背景污染的水平视溶剂体积、纯度、批号及浓缩方式有所不同，以10 mL的农残级二氯甲烷为例（挥干后1 mL甲醇定容测定），旋转蒸发至干时NP的背景污染水平约为0.15 μg/L，若使用氮气吹干，背景污染水平则高达1.0 μg/L。
本实验所用实验器具均为玻璃材质，液相色谱管路为聚四氟乙烯，在线固相萃取柱为不锈钢柱，所有溶剂均为LCMS级。过程空白中TCC的背景污染水平低于0.005 μg/L，其余3种物质TCS、NP和BPA均低于0.05 μg/L，且较稳定。该结果明显低于离线固相萃取、液液萃取等方法所致的背景污染。究其原因，本方法前处理方法步骤简单，可引入污染的环节少；溶剂耗费量低，减少了实验试剂可能的污染；本方法的自动化程度高，使得背景污染稳定性高。
3.4 线性范围和定量限
Fig.1 LCMS chromatograms of the target compounds as well as the internal standards in different samples （A： procedural blank sample； B： standard solution， in which the concentration of TCC is 0.05 μg/L， while BPA， NP and TCS are 0.5 μg/L； C： a positive sample， pure milk）[HT5][TS）]
References
1 Veldhoen N， Skirrow R C， Osachoff H， Wigmore H， Clapson D J， Gunderson M P， Van Aggelen G， Helbing C C. Aquat. Toxicol.， 2006， 80 （3）： 217-227
2 Kumar V， Chakraborty A， Kural M R， Roy P.Reprod. Toxicol.， 2009， 27 （2）： 177-185
3 Ahn K C， Zhao B， Chen J， Cherednichenko G， Sanmarti E， Denison S M， Lasley B， Pessah I N， Kültz D， Chang D P Y， Gee S J， Hammock B D. Environ. Health Perspect， 2008， 116 （9）： 1203-1210
4 Christen V， Crettaz P， OberliSchrammli A， Fent K. Chemosphere， 2010， 81 （10）： 1245-1252
5 Kitamura S， Suzuki T， Sanoh S， Kohta R， Jinno N， Sugihara K， Yoshihara S， Fujimoto N ， Watanabe， H， Ohta S. Toxicol. Sci.， 2005， 84（2）： 249-259
6 ZHAO MeiPing， LI YuanZong， ZHANG XinXiang， CHANG WenBao. Chem. J. Chinese Universities， 2003， 24（7）：1204-1206
赵美萍， 李元宗， 张新祥， 常文保. 高等学校化学学报， 2003， 24（7）： 1204-1206
7 Matozzo V， Gagné F， Marin M G， Ricciardi F， Blaise C. Environ. Int.， 2008， 34 （4）： 531-545
8 ZHAO MeiPing， LI YuanZong， CHANG WenBao. Chinese J. Anal Chem.， 2003， 31（1）： 103-109
赵美萍， 李元宗， 常文保. 分析化学， 2003， 31（1）： 103-109
9 Lu Y Y， Chen M L，Sung F C. Environ. Int.， 2007， 33（7）： 903-910
10 Gyllenhammar I， Glynn A， Darnerud P O， Lignell S， van Delft R， Aune M. Environ. Int.， 2012， 43： 21-28
11 Niu Y， Zhang J， Duan H， Wu Y， Shao B. Food Chem.， 2015， 167： 320-325. doi： 10.1016/j.foodchem.2014.06.115. Epub 2014 Jul 5
12 Allmyr M， AdolfssonErici M， McLachlan M S， SandborghEnglund G S. Sci. Total. Environ.， 2006， 372： 87-93
13 DIAO ChunPeng， ZHAO RuSong， SHI JunBo， LIU RenMin. Chinese J. Anal. Chem.， 2009， 37（1）： 131-135
刁春鹏， 赵汝松， 时军波， 柳仁民. 分析化学， 2009， 37（1）： 131-135
14 Barahona F， Turiel E， MartínEsteban A. J. Chromatogr. Sci.， 2011， 49（3）： 243-248
15 Ye X， Bishop A M， Needham L L， Calafat A M. Anal. Chim. Acta， 2008， 622 （1/2）： 150-156
16 GallartAyala H， Moyano E， Galceran M T. J.Chromatogr. A， 2011， 1218（12）： 1603-1610
17 NIU YuMin， ZHANG Jing， ZHANG ShuJun， SHAO Bing. Chinese J. Anal. Chem.， 2012， 40（4）： 534-538
牛宇敏， 张 晶， 张书军， 邵 兵. 分析化学， 2012， 40（4）： 534-538
18 Zhou X， Ye X， Calafat A M. J. Chromatogr. B， 2012， 881882： 27-33
19 Allmyr M， McLachlan M S， SandborghEnglund G， AdolfssonErici M. Anal. Chem.， 2006， 78（18）： 6542-6546
20 Wang H， Zhang J， Gao F， Yang Y， Duan H， Wu Y， Berset J D， Shao B. J. Chromatogr. B， 2011， 879 （21）： 1861-1869
21 CarabiasMartinez R， RodriguezGonzalo E， RevillaRuiz P. J. Chromatogr. A， 2006， 1137（2）： 207-215
22 EFSA （European Food Safety Authority）. EFSA Reevaluates Safety of Bisphenol A and Sets Tolerable Daily Intake. http：//www.efsa.europa.eu/en/press/news/afc070129.htm
23 Nielsen E， stergaard G， Thorup I， Ladefoged O， Jelhnes O， Jelnes J E. The Institute of Food Safety and Toxicology. Danish Veterinary and Food Administration Environmental Project Copenhagen： Danish Environmental Protection Agency， 2000
Determination of 4 Environmental Endocrine
Disruptors Involving Bisphenol A in Dairy Products
by Online Solid Phase Extraction Coupled with
Liquid ChromatographyTandem Mass Spectrometry
ZHANG Pin1，2， ZHANG Jing1，2， CHEN JieJun3， DUAN HeJun2， SHAO Bing*1，2
1（School of Public Health， Capital Medical University， Beijing 100058， China）
2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，
Beijing Centers for Preventive Medical Research， Beijing 100013， China）
3（China National Center for Biotechnology Development， Beijing 100038， China）
Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.
Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry
（Received 24 September 2014； accepted 7 October 2014）
This work was supported by the National Natural Science Foundation of China （No.21177014）
2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，
Beijing Centers for Preventive Medical Research， Beijing 100013， China）
3（China National Center for Biotechnology Development， Beijing 100038， China）
Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.
Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry
（Received 24 September 2014； accepted 7 October 2014）
This work was supported by the National Natural Science Foundation of China （No.21177014）
2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，
Beijing Centers for Preventive Medical Research， Beijing 100013， China）
3（China National Center for Biotechnology Development， Beijing 100038， China）
Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.
Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry
（Received 24 September 2014； accepted 7 October 2014）
This work was supported by the National Natural Science Foundation of China （No.21177014）
摘 要 建立了乳制品中三氯生、三氯卡班、双酚A和壬基酚4种内分泌干扰物的在线固相萃取超高压液相色谱串联质谱（Online SPE LCMS/MS）检测方法。液态乳制品或奶粉样品中加入乙酸缓冲液，目标物经β葡糖醛酸苷肽酶/芳基磺酸酯酶酶解后， 用乙腈提取， 冷冻离心10 min后，取上清液，用水稀释，在线固相萃取串联质谱法测定。样品溶液经Xbridge C8柱富集，BEH C18色谱柱分离，甲醇和水梯度洗脱，三重四极杆质谱电喷雾负离子模式下采集数据，同位素内标法定量。4种目标化合物的线性范围为0.005～5.0 μg/L，相关系数R2>0.99；方法的定量限为0.03～1.0 μg/kg，3个添加水平的平均加标回收率为80.2%～106.7%，
3.2 提取条件的优化
环境污染物或药物进入生物体后在肝脏酶系统的作用下会转化成葡糖糖醛酸结合态。Zhou等[ 18]发现人体尿液中的三氯卡班及其代谢产物主要以结合态形式存在，尽管奶样中是否存在结合态还未有定论[ 19，20]，但为了保证检测结果的准确性，本研究采用β葡糖醛酸苷肽酶/芳基磺酸酯酶进行酶解。
由于4种目标化合物均有一定的亲脂性，因此，酶解后的样品提取过程比较了乙腈和甲醇两种与水互溶的极性有机溶剂。称取适量液奶样品，加标量为5 μg/kg，如2.3节所述进行处理，每种提取溶剂做3个平行。离心后提取液直接测定，从表观上看，乙腈提取液比甲醇提取液更清澈透明。结果表明，乙腈提取液对TCS，TCC，NP和BPA的绝对回收率分别为95%，89%，91%和78%，比甲醇体系略高（分别为90%，85%，92%和69%）。综合表观结果和回收率数据，确定乙腈为提取溶剂。
3.3 背景污染的考察
由于TCS，NP和BPA广泛应用于多个领域，因此这几种污染物在环境中普遍存在，甚至在Milpore的超纯水中检测到了BPA[ 21]。而且，塑料器皿或橡胶制品等都会迁移出大量NP 和BPA，这些均会对分析结果造成干扰。由于离线固相萃取小柱的筛板为聚丙烯等塑料材质，有机溶剂浸润时会持续溶出NP，其浓度大于0.2 μg/L[ 17]；使用二氯甲烷、正己烷等非极性有机溶剂进行液液萃取时，其背景污染的水平视溶剂体积、纯度、批号及浓缩方式有所不同，以10 mL的农残级二氯甲烷为例（挥干后1 mL甲醇定容测定），旋转蒸发至干时NP的背景污染水平约为0.15 μg/L，若使用氮气吹干，背景污染水平则高达1.0 μg/L。
本实验所用实验器具均为玻璃材质，液相色谱管路为聚四氟乙烯，在线固相萃取柱为不锈钢柱，所有溶剂均为LCMS级。过程空白中TCC的背景污染水平低于0.005 μg/L，其余3种物质TCS、NP和BPA均低于0.05 μg/L，且较稳定。该结果明显低于离线固相萃取、液液萃取等方法所致的背景污染。究其原因，本方法前处理方法步骤简单，可引入污染的环节少；溶剂耗费量低，减少了实验试剂可能的污染；本方法的自动化程度高，使得背景污染稳定性高。
3.4 线性范围和定量限
Fig.1 LCMS chromatograms of the target compounds as well as the internal standards in different samples （A： procedural blank sample； B： standard solution， in which the concentration of TCC is 0.05 μg/L， while BPA， NP and TCS are 0.5 μg/L； C： a positive sample， pure milk）[HT5][TS）]
References
1 Veldhoen N， Skirrow R C， Osachoff H， Wigmore H， Clapson D J， Gunderson M P， Van Aggelen G， Helbing C C. Aquat. Toxicol.， 2006， 80 （3）： 217-227
2 Kumar V， Chakraborty A， Kural M R， Roy P.Reprod. Toxicol.， 2009， 27 （2）： 177-185
3 Ahn K C， Zhao B， Chen J， Cherednichenko G， Sanmarti E， Denison S M， Lasley B， Pessah I N， Kültz D， Chang D P Y， Gee S J， Hammock B D. Environ. Health Perspect， 2008， 116 （9）： 1203-1210
4 Christen V， Crettaz P， OberliSchrammli A， Fent K. Chemosphere， 2010， 81 （10）： 1245-1252
5 Kitamura S， Suzuki T， Sanoh S， Kohta R， Jinno N， Sugihara K， Yoshihara S， Fujimoto N ， Watanabe， H， Ohta S. Toxicol. Sci.， 2005， 84（2）： 249-259
6 ZHAO MeiPing， LI YuanZong， ZHANG XinXiang， CHANG WenBao. Chem. J. Chinese Universities， 2003， 24（7）：1204-1206
赵美萍， 李元宗， 张新祥， 常文保. 高等学校化学学报， 2003， 24（7）： 1204-1206
7 Matozzo V， Gagné F， Marin M G， Ricciardi F， Blaise C. Environ. Int.， 2008， 34 （4）： 531-545
8 ZHAO MeiPing， LI YuanZong， CHANG WenBao. Chinese J. Anal Chem.， 2003， 31（1）： 103-109
赵美萍， 李元宗， 常文保. 分析化学， 2003， 31（1）： 103-109
9 Lu Y Y， Chen M L，Sung F C. Environ. Int.， 2007， 33（7）： 903-910
10 Gyllenhammar I， Glynn A， Darnerud P O， Lignell S， van Delft R， Aune M. Environ. Int.， 2012， 43： 21-28
11 Niu Y， Zhang J， Duan H， Wu Y， Shao B. Food Chem.， 2015， 167： 320-325. doi： 10.1016/j.foodchem.2014.06.115. Epub 2014 Jul 5
12 Allmyr M， AdolfssonErici M， McLachlan M S， SandborghEnglund G S. Sci. Total. Environ.， 2006， 372： 87-93
13 DIAO ChunPeng， ZHAO RuSong， SHI JunBo， LIU RenMin. Chinese J. Anal. Chem.， 2009， 37（1）： 131-135
刁春鹏， 赵汝松， 时军波， 柳仁民. 分析化学， 2009， 37（1）： 131-135
14 Barahona F， Turiel E， MartínEsteban A. J. Chromatogr. Sci.， 2011， 49（3）： 243-248
15 Ye X， Bishop A M， Needham L L， Calafat A M. Anal. Chim. Acta， 2008， 622 （1/2）： 150-156
16 GallartAyala H， Moyano E， Galceran M T. J.Chromatogr. A， 2011， 1218（12）： 1603-1610
17 NIU YuMin， ZHANG Jing， ZHANG ShuJun， SHAO Bing. Chinese J. Anal. Chem.， 2012， 40（4）： 534-538
牛宇敏， 张 晶， 张书军， 邵 兵. 分析化学， 2012， 40（4）： 534-538
18 Zhou X， Ye X， Calafat A M. J. Chromatogr. B， 2012， 881882： 27-33
19 Allmyr M， McLachlan M S， SandborghEnglund G， AdolfssonErici M. Anal. Chem.， 2006， 78（18）： 6542-6546
20 Wang H， Zhang J， Gao F， Yang Y， Duan H， Wu Y， Berset J D， Shao B. J. Chromatogr. B， 2011， 879 （21）： 1861-1869
21 CarabiasMartinez R， RodriguezGonzalo E， RevillaRuiz P. J. Chromatogr. A， 2006， 1137（2）： 207-215
22 EFSA （European Food Safety Authority）. EFSA Reevaluates Safety of Bisphenol A and Sets Tolerable Daily Intake. http：//www.efsa.europa.eu/en/press/news/afc070129.htm
23 Nielsen E， stergaard G， Thorup I， Ladefoged O， Jelhnes O， Jelnes J E. The Institute of Food Safety and Toxicology. Danish Veterinary and Food Administration Environmental Project Copenhagen： Danish Environmental Protection Agency， 2000
Determination of 4 Environmental Endocrine
Disruptors Involving Bisphenol A in Dairy Products
by Online Solid Phase Extraction Coupled with
Liquid ChromatographyTandem Mass Spectrometry
ZHANG Pin1，2， ZHANG Jing1，2， CHEN JieJun3， DUAN HeJun2， SHAO Bing*1，2
1（School of Public Health， Capital Medical University， Beijing 100058， China）
2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，
Beijing Centers for Preventive Medical Research， Beijing 100013， China）
3（China National Center for Biotechnology Development， Beijing 100038， China）
Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.
Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry
（Received 24 September 2014； accepted 7 October 2014）
This work was supported by the National Natural Science Foundation of China （No.21177014）
2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，
Beijing Centers for Preventive Medical Research， Beijing 100013， China）
3（China National Center for Biotechnology Development， Beijing 100038， China）
Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.
Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry
（Received 24 September 2014； accepted 7 October 2014）
This work was supported by the National Natural Science Foundation of China （No.21177014）
2（Beijing Key Laboratory of Diagrostic and Traceability Technologies Food Poisoning，
Beijing Centers for Preventive Medical Research， Beijing 100013， China）
3（China National Center for Biotechnology Development， Beijing 100038， China）
Abstract A simple analytical method by means of online solid phase extraction followed liquid chromatographytandem mass spectrometry （SPELCMS/MS） was developed for the simultaneous quantitation of 4 endocrine disruptors （triclosan， triclocarban， bisphenol A and nonylphenol） in dairy products. Infant formula and milk samples were dissolved in acetic acid buffer and hydrolyzed by βglucuronidase/arylsulfatase. Acetonitrile was used as the extract. Then， the mixture was freezecentrifuged for 10 min and the supernatant was diluted with water， and analyzed via online SPELCMS/MS. The sample extracts were concentrated by an Xbridge C8 cartridge and separated on a BEH C18 column with a gradient mobile phase of methanol and water； then analyzed by triple quadrupole mass spectrometry. Mass acquisition was conducted under negative electrospray ionization mode. Quantification was performed by isotopic internal standard calibration. Acceptable linearity （R2>0.99） was achieved over the range of 0.005-5.0 μg/L， with limits of quantification of 0.03-1.0 μg/kg. Average recoveries of four target compounds （spiked at three concentration levels） ranged from 80.2%-106.7%，with relative standard deviation less than 15%. Due to its rapidity， simplicity， and high sensitivity， the method is suitable for the analysis of endocrine disruptors in dairy products. It has been applied in the analysis of raw milk and milk products collected in Beijing. As a result， nonylphenol was found with a high detectable frequency.
Keywords Dairy products； Triclosan； Bisphenol A； Nonylphenol； Online solid phase extraction liquid chromatographytandem mass spectrometry
（Received 24 September 2014； accepted 7 October 2014）
This work was supported by the National Natural Science Foundation of China （No.21177014）
