中国农业科学 ›› 2014, Vol. 47 ›› Issue (5): 995-1005.doi: 10.3864/j.issn.0578-1752.2014.05.016

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

玉米DDGS中抗生素残留分析

 姚婷1, 秦玉昌2, 吴宁鹏3, 彭丽3, 孙丹丹1, 谷旭1   

  1. 1、中国农业科学院饲料研究所,北京 100081;
    2、农业部食品与营养发展研究所,北京100081;
    3、河南省饲料产品质量监督检验站,郑州450014
  • 收稿日期:2013-05-29 出版日期:2014-03-01 发布日期:2013-10-17
  • 通讯作者: 谷旭,Tel:010-82106069;E-mail:guxu@caas.cn
  • 作者简介:姚婷,E-mail:yaoting0515@126.com
  • 基金资助:

    国家公益性行业(农业)科研专项(201203015)

Analysis of Antibiotic Residues Detection in Corn DDGS

 YAO  Ting-1, QIN  Yu-Chang-2, WU  Ning-Peng-3, PENG  Li-3, SUN  Dan-Dan-1, GU  Xu-1   

  1. 1、Feed Research Institute, Chinese Academy of Agricultural Sciences, Bejing 100081;
    2、Food and Nutrition Development Institute, Ministry of Agriculture, Bejing 100081;
    3、Quality Supervision and Inspection Station of Feed Products of Henan Province,  Zhengzhou 450014
  • Received:2013-05-29 Online:2014-03-01 Published:2013-10-17

摘要: 【目的】玉米发酵生产燃料乙醇的副产物酒糟蛋白饲料(DDGS)常作为蛋白质替代饲料广泛用于动物生产中。随着DDGS产业链的扩大,其生产过程中所带来的抗生素的残留问题应该得到重视,但目前关于DDGS中抗生素的残留研究报道较少。对全国经营和使用环节的玉米DDGS进行抽样调查,对70种抗生素的残留含量进行分析测定,以了解目前中国饲料用DDGS中抗生素的残留情况。【方法】首先利用四级杆飞行时间质谱(quadrupole time-of-flight mass spectrometry,QTOF)建立了大环内酯类、氟喹诺酮类、磺胺类、喹恶啉类、β-内酰胺类、林可胺类、四环素类、酰胺醇类等共70种抗生素药物基于质量数、保留时间和特征碎片离子的数据库。在全国9个省、市、自治区抽取经营和使用环节的玉米DDGS样品74批次,筛查其中可能含有的抗生素药物。样品的前处理:取一定量的均质样品,加入乙腈和水的混合溶液进行提取,经涡旋、震荡、离心后,取上清液过NH2固相萃取柱,收集淋洗液N2气浓缩至一定体积,过0.22 μm滤膜。样品的分析:首先利用QTOF建立的筛查列表对样品DDGS中可能残留的抗生素进行初步筛选,以确定残留抗生素的种类,而后采用液相色谱串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)对发现疑似含有抗生素残留的样品进行定量分析,以确定抗生素的残留量。【结果】欧盟指令2002/657/EC规定了在应用质谱仪对目标化合物进行确证时的准则。一个母离子为2分,一个子离子为2.5分。在本试验条件下所有抗生素的检测结果都能达到这个要求,符合对目标化合物的确证准则。经QTOF定性分析后,抽样的所有DDGS中有一批次样品中含有抗生素残留,种类为甲氧苄啶。样品母离子峰、二级特征离子碎片、出峰时间与标准品完全一致,样品和标准品所有提取碎片离子及离子丰度比一致。利用LC-MS/MS对残留抗生素进行定量分析后,确定其残留量为107.7 ng•g-1。甲氧苄啶在添加浓度为10—1 000 ng•mL-1,平均添加回收率为75.2%—97.9%,线性相关系数大于0.99,日内标准偏差为2.8%—5%,日间标准偏差为8%—10%。【结论】利用QTOF建立DDGS中抗生素类药物的定性筛选方法可作为玉米DDGS中残留抗生素快速筛查方法。存在残留的甲氧苄啶抗生素,可能在玉米发酵生产DDGS前被添加,目的是抑制有害微生物的生长繁殖。抽样分析结果表明,目前中国玉米DDGS中抗生素残留的种类较少、残留量也较低,但DDGS中抗生素残留情况仍不容忽视,相关部门也未给出DDGS发酵过程中可使用的抗生素的种类范围、使用限量以及使用规范。此外,还应该研究直接采用QTOF进行定量分析的方法,以及研究包括维吉尼亚霉素在内的其他抗生素的QTOF分析方法。

关键词: 抗生素 , QTOF , LC-MS/MS , 玉米DDGS

Abstract: 【Objective】 DDGS, which is the byproduct in corn fermentation production, is always used as a protein substitute in animal production for its low starch, high protein, high fat, high digestible fiber and high effective phosphorus. With the expansion of DDGS industry, the residues of antibiotics caused by production process should be taken seriously. However, there is seldom reports about this. In this paper, some DDGS samples from the parts of the operation and use in China were collected, and then 70 kinds of antibiotics were analyzed, in order to know the current situation of antibiotics residues in feed of China. 【Method】 In this study, quadrupole time of flight mass (QTOF) was firstly used to establish a database based on drug mass, retention time and characteristic fragment, which contained macrolides, fluoroquinolones, sulfonamides, quinoxaline, β-lactam, lincosamides, tetracyclines and amphenicols, totally 70 kinds. A total of 74 samples from 9 provinces and autonomous regions were collected to detect the possible antibiotic residues. For pretreatment of the samples, a certain amount of a homogeneous sample was taken and a mixed solution of acetonitrile and water for extraction was added. After vortex, shock and centrifuged, the supernatant was passing over NH2 SPE column. The eluent was collected and concentrated by N2, and filtered by 0.22 μm. For analysis of samples, the antibiotic residues were initially screened to determine the type of residual antibiotics by the established database, and then the liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to determine the amount of the residual antibiotics. 【Result】The EU Directive 2002/657/EC regulations identify mass spectrometer of target compounds in application. A parent ion is 2 minutes while a product ion is 2.5. In this paper, all antibiotic results could meet the requirements, which was in accordance with the rule of corroboration target compounds. After qualitative analysis by QTOF qualitative analysis, only one DDGS sample contains antibiotic residues, which is typeoftrimethoprim. Parent ion peak, characteristics of secondary ion fragments, the peak time in samples was in accordance with the spiked completely, the same of all fragment ions and ion abundance. After quantitative analysis by LC-MS/MS, the residual amount of it was 107.7 ng•g-1. In added concentration range 10-1000 ng•mL-1 of Trimethoprim, the average recoveries were 75.2%-97.9%. The linear correlation coefficient was greater than 0.99, and the standard deviation within a between days was 2.8%-5%, and the standard deviation between days was 8%-10%. 【Conclusion】In this study, the antibiotics qualitative screening method of DDGS in QTOF can be used as a rapid screening method for antibiotic residues in corn DDGS. Typeoftrimethoprim may be added before the corn fermentation production of DDGS, in order to inhibit the growth of harmful microbes. Results indicated that the species of antibiotic residues in corn DDGS was less, and residual volume is low. But the potential risk can not be ignored. The relevant departments have not given the information which DDGS can be used in the fermentation process, and the range of species of antibiotics limited. The method of quantitative analysis QTOF should be studied directly as well as other antibiotics including virginiamycin QTOF analysis methods.

Key words: antibiotic , QTOF , LC-MS/MS , corn DDGS