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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (2): 191-196    DOI: 10.3785/j.issn.1008-9209.2012.11.612
论文     
超高效液相色谱大体积流通池荧光法检测奶及奶制品中的黄曲霉毒素M1
王军淋1， 蔡增轩2， 任一平1,2*
1.浙江工业大学化学工程与材料学院，杭州 310014；2.浙江省疾病预防控制中心，杭州 310054
Determination of aflatoxin M1 in milk by ultraperformance liquid chromatography and fluorimetric detection combined with large volume flow cell. Journal of Zhejiang University
WANG Junlin1, CAI Zengxuan2, REN Yiping1, 2*
1. College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310014, China; 2. Zhejiang Provincial Center for Disease Prevention and Control, Hangzhou 310054, China
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摘要： 采用超高效液相色谱系统，并配有大体积流通池的荧光检测器快速检测奶及奶制品中的黄曲霉毒素M1.在样品中按m(牛奶)∶V(乙腈)=1∶2.5的比例加入乙腈，采用涡旋及超声辅助液液萃取法对样品中的黄曲霉毒素M1进行提取，经离心后，取上清液用磷酸盐缓冲液稀释，过黄曲霉毒素M1免疫亲和柱进行净化及浓缩；采用V(甲醇)∶V(乙腈)=50∶50及纯水作为流动相，经UPLC BEH C18 柱 (100 mm×2.1 mm, 1.7 μm)分离.结果表明：该方法的定量限为0.03 μg/kg，低于现行食品安全国家标准对食品中黄曲霉毒素M1的最低限量标准，符合检测要求；同时，在0.06~1.2 μg/kg范围内具有良好的线性，其线性相关系数大于0.999；3个加标浓度的回收率在81.95%~94.20%之间，效果较好；采用大体积流通池检测的灵敏度较普通流通池提高了近3倍；经用于奶及其制品中黄曲霉毒素M1的检测结果与现行标准方法一致；另外，在前处理中，用乙腈对样品中的黄曲霉毒素M1进行提取，可以有效地沉淀样品中的蛋白质，从而得到澄清的提取液，在过免疫亲和柱净化时只需利用溶液自身重力作用即能过柱.综上，此定量方法具有样品前处理简单、检测速度快和灵敏度高等优点，适用于对牛奶中黄曲霉毒素M1的检测.
Abstract: Aflatoxins (AFT), whose basic structure is composed of difuran and coumarin, have 17 kinds of derivatives including B1, B2, G1, G2, M1, M2, etc. AFT M1 was firstly separated from milk. AFT M1 and AFT M2 were the derivatives of AFT B1 and AFT B2 through the animal metabolism. Especially, AFT B1 and AFT M1 have been defined as a category A and 2B carcinogen by the International Agency for Research on Cancer (IARC) from World Health Organization (WHO) in 1993, respectively. Moreover, AFT B1 and AFT M1 are regarded as strong carcinogens, the carcinogenic mechanism of which is achieved via affecting the pericellular membrane, inhibiting the synthesis of RNA and interfering the inductive style of specific enzymes. In December 2011, the General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ) announced the selective examination results of 200 kinds of liquid milk products. The aflatoxin M1 in parts of milk products were over ranging the maximum residue limits (MRLs) of M1 in milk and milk products, of which the maximum superscalar were 140% exceeded. Moreover, the contents of aflatoxin M1 were found exceeded in milk powder again in July 2012. The method used now was to first heat the milk and milk products in water bath, then samples were cleanup and concentrated by immunoaffinity column after filtered or centrifuged. In this method, no clear sample solutions were obtained, when passing through the immunoaffinity column, sometimes the immunoaffinity column would be blocked, and the recovery would not in expectation. So a better method was needed for determinating the aflatoxin M1 in milk and milk products. The present study developed an improved analytical method for the fast determination of aflatoxin M1 in milk and milk products by ultraperformance liquid chromatography (UPLC) combined with large volume flow cell fluorescence detection (FLD). The milk sample was extracted by acetonitrile, and the ratio of the milk sample to acetonitrile was 1 to 2.5 in mass to volume. Then, the sample was extracted by vortex and ultrasound assisted liquidliquid extraction (ULLE), and was cleanedup and concentrated by aflatoxin M1 immunoaffinity column. The analyte was separated by UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), and was eluted with acetonitrilemethanol (50∶50) and pure water. The results showed that the limit of quantitation (LOQ) of aflatoxin M1 was 0.03 μg/kg, which was lower than the national criteria on determination of the minimum level of aflatoxin M1 in milk and milk products. Meanwhile, high correlation coefficient (R2>0.999) was obtained within linear range from 0.06 to 1.2 μg/kg, and reasonable recoveries (81.95%94.20%) were in different spike level. In addition, acetonitrile could effectively precipitate protein in milk during the pretreatment to obtain clear extraction which could rapidly pass through immunoaffinity column only by gravity. When using the large volume flow cell, the sensitivity was increased, which was three times than the standard flow cell. The results obtained from this method were similar to the classical method. In conclusion, this quantitative method has many advantages including simple pretreatment, rapid determination and high sensitivity, which can be applied to the determination and quantification of aflatoxin M1 in milk sample.
出版日期: 2013-03-20
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王军淋1
蔡增轩2
任一平1
2*

引用本文:

王军淋1， 蔡增轩2， 任一平1,2*. 超高效液相色谱大体积流通池荧光法检测奶及奶制品中的黄曲霉毒素M1[J]. 浙江大学学报(农业与生命科学版), 2013, 39(2): 191-196.

WANG Junlin1, CAI Zengxuan2, REN Yiping1, 2*. Determination of aflatoxin M1 in milk by ultraperformance liquid chromatography and fluorimetric detection combined with large volume flow cell. Journal of Zhejiang University. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(2): 191-196.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.11.612    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I2/191

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