食品中兽药残留高通量筛查与检测平台的建立及膳食暴露评估研究
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摘要
近年来，国内外食品安全事故层出不穷，食品安全已经成为全社会广泛关注的重要的公共安全问题。动物养殖过程中出现了使用替代品种代替重点监管兽药和不同类别、功效的兽药同时饲喂的新现象，动物源性食品中出现了多类别兽药、多残留的新问题，给健康带来极大的隐患。本研究以食品中兽药残留为研究对象，利用分析技术和膳食暴露评估技术，从技术层面对食品安全问题进行了研究。为分析和评价由于兽药残留引起的食品安全问题提供了新的思路。
本课题研究内容主要由以下部分组成：
第一部分，主要是分析了我国食品安全现状与产生原因、新形势下食品安全问题面临的挑战以及食品安全相关概念。对食品兽药残留和关键检测技术的相关文献进行了综述。介绍了本课题的意义和研究方法。
第二部分，建立了基于高效液相色谱-高分辨飞行时间质谱联用技术对猪肉中β-兴奋剂、镇静剂、磺胺三大类73种兽药残留的高通量快速筛查平台。样品经提取，分散固相法萃取净化，用液相色谱-飞行时间质谱法测定，同位素内标定量。该方法前处理简单，选择性高，灵敏度好，适宜筛查猪肉中可能存在的多类药物残留。
第三部分，建立了禽畜肉中抗病毒类药物的定量检测平台，包括禽肉中金刚烷胺残留量的测定及禽畜肉中利巴韦林残留量的测定。样品分别采用提取或酶解提取后，经固相萃取净化，用液相色谱串联四极杆线性离子阱质谱法测定，采用同位素内标法定量。检测平台中的两个方法均经过方法学验证，峰面积与浓度线性相关系数r>0.999，回收率均在80%以上。可作为禽畜肉中抗病毒药物的定量检测方法。
第四部分，建立了猪肉和肝脏组织中同时测定20种β-兴奋剂残留的高通量检测平台。动物组织样本酶解提取后，经固相萃取柱萃取净化，用液相色谱串联三重四级杆质谱测定，采用同位素内标或外标法定量测定。方法学验证显示结果准确，适宜作为猪肉类食品中β-兴奋剂的高通量定量检测方法。
用本方法对猪肉和猪肝中莱克多巴胺进行检测，并对结果进行统计分析，采用国际通用的风险评估模式，从危害识别、危害特征描述、暴露评估和风险特征描述等方面对猪肉食品中莱克多巴胺残留进行了膳食暴露评估。结果为，猪肉类食品中莱克多巴胺残留对人体健康风险较低。
第五部分，建立了禽肉中氯霉素残留的快速检测方法。动物组织样本经乙腈提取后，用正己烷除脂净化，浓缩后用液相色谱串联三重四级杆质谱测定，采用同位素内标法定量测定。经方法学验证，结果准确可靠，适宜作为禽肉中氯霉素残留的定量测定方法。
应用本方法对禽肉中氯霉素残留进行检测，并对结果进行统计分析，采用国际通用的风险评估模式，从危害识别、危害特征描述、暴露评估和风险特征描述等方面对氯霉素残留进行了膳食暴露评估。结果为，禽肉中氯霉素残留产生致癌性风险较低，发生氯霉素临床不良反应的可能性较低。
In recent years, food safety accidents have exploded in China and abroad. Food safety has become a major concern of the whole society. All these accidents expose that great hazard is threatening people's health, such as strictly regulated veterinary drugs substituted by new alternative drug, drugs of different categories with different effects feeded at the same time, multi-residues of veterinary drugs appeared in the animal derived food. The objective of this paper is to study the food safety problem by analyzing veterinary residues in food and dietary exposure assessment, providing a new sight for analysis and evaluation of food safety issues caused by veterinary residues.
In the first chapter, it mainly describes the current situation and main sources of food safety problem in China, and discusses the challenges for food safety under the new situation
.Several concepts related to food safety are also introduced. The principal methods for detection of veterinary residues in food are reviewed. The purposes and methods of this study are also introduced in this chapter.
In the second chapter, a high-throughput screening method for73veterinary residues in pork was developed, containing β-agonists, sedatives and sulfonamides. The method was established by high performance liquid chromatography hybrid-quadruple time-of-flight mass spectrometry (HPLC-Q-TOF-MS). These samples were extracted and purified by dispersed solid-phase extraction, then analyzed by HPLC-Q-TOF-MS and quantitated with isotope internal standard. The method was proved to be rapid and simple, with high selectivity and sensitivity. The result showed that the method could be used for both targeted and untargeted veterinary residues in pork.
In the third chapter, a platform for detecting antiviral drugs in livestock and poultry meats was established, including the determination of amantadine in poultry meats and the determination of ribavirin in poultry and livestock meats. The samples were extracted by mixed solvent with or without enzymolysis respectively, purified by solid phase extraction, then determined by linear ion trap with liquid chromatography tandem quadrupole mass spectrometry (QTrap), and quantified by using isotope internal standard method. These two determination methods were both validated. The linear correlation coefficient of peak area and concentration was above0.999, all of the average recoveries were above80%. The platform was proved to be used as a routine method for antiviral drugs determination in poultry and pork meats.
In the fourth chapter, a high-throughput LC-MS/MS method for simultaneous detection of20β-agnoists in poultry and pork meat was developed. The samples were extracted by mixed solvent with enzymolysis, purified by SPE, determined by LC-MS/MS, and quantified by using isotope internal standard method or external standard method. The validation indicated that that the method was suitable for the determination of β-agnoist in poultry and pork.
The dietary exposure assessment of ractopamine residue in pork meats and livers was studied by using the established LC-MS/MS method as well as statistical analysis. The risk assessment included hazard identification, hazard characterization, exposure assessment, risk assessment and risk characterization. The assessment result showed that ractopamine residue in pork food was at a low risk for human health.
In the fifth chapter, a rapid LC-MS/MS method for detection of chloramphenicol (CAP) in poultry meat was developed. The samples were extracted by acetonitrile (ACN), purified by hexane, determined by LC-MS/MS with a concentrated analyte, and quantified by using isotope internal standard method. The validation of the method showed that the method was suitable for the determination of CAP in poultry meat.
The dietary exposure assessment of CAP residue in poultry is studied. The risk assessment included hazard identification, hazard characterization, exposure assessment, risk assessment and risk characterization. The assessment result suggested that CAP residues in poultry meat was at low risk of carcinogenicity for human, and the adverse clinical reaction caused by CAP would occur less likely.
本课题研究内容主要由以下部分组成：
第一部分，主要是分析了我国食品安全现状与产生原因、新形势下食品安全问题面临的挑战以及食品安全相关概念。对食品兽药残留和关键检测技术的相关文献进行了综述。介绍了本课题的意义和研究方法。
第二部分，建立了基于高效液相色谱-高分辨飞行时间质谱联用技术对猪肉中β-兴奋剂、镇静剂、磺胺三大类73种兽药残留的高通量快速筛查平台。样品经提取，分散固相法萃取净化，用液相色谱-飞行时间质谱法测定，同位素内标定量。该方法前处理简单，选择性高，灵敏度好，适宜筛查猪肉中可能存在的多类药物残留。
第三部分，建立了禽畜肉中抗病毒类药物的定量检测平台，包括禽肉中金刚烷胺残留量的测定及禽畜肉中利巴韦林残留量的测定。样品分别采用提取或酶解提取后，经固相萃取净化，用液相色谱串联四极杆线性离子阱质谱法测定，采用同位素内标法定量。检测平台中的两个方法均经过方法学验证，峰面积与浓度线性相关系数r>0.999，回收率均在80%以上。可作为禽畜肉中抗病毒药物的定量检测方法。
第四部分，建立了猪肉和肝脏组织中同时测定20种β-兴奋剂残留的高通量检测平台。动物组织样本酶解提取后，经固相萃取柱萃取净化，用液相色谱串联三重四级杆质谱测定，采用同位素内标或外标法定量测定。方法学验证显示结果准确，适宜作为猪肉类食品中β-兴奋剂的高通量定量检测方法。
用本方法对猪肉和猪肝中莱克多巴胺进行检测，并对结果进行统计分析，采用国际通用的风险评估模式，从危害识别、危害特征描述、暴露评估和风险特征描述等方面对猪肉食品中莱克多巴胺残留进行了膳食暴露评估。结果为，猪肉类食品中莱克多巴胺残留对人体健康风险较低。
第五部分，建立了禽肉中氯霉素残留的快速检测方法。动物组织样本经乙腈提取后，用正己烷除脂净化，浓缩后用液相色谱串联三重四级杆质谱测定，采用同位素内标法定量测定。经方法学验证，结果准确可靠，适宜作为禽肉中氯霉素残留的定量测定方法。
应用本方法对禽肉中氯霉素残留进行检测，并对结果进行统计分析，采用国际通用的风险评估模式，从危害识别、危害特征描述、暴露评估和风险特征描述等方面对氯霉素残留进行了膳食暴露评估。结果为，禽肉中氯霉素残留产生致癌性风险较低，发生氯霉素临床不良反应的可能性较低。
In recent years, food safety accidents have exploded in China and abroad. Food safety has become a major concern of the whole society. All these accidents expose that great hazard is threatening people's health, such as strictly regulated veterinary drugs substituted by new alternative drug, drugs of different categories with different effects feeded at the same time, multi-residues of veterinary drugs appeared in the animal derived food. The objective of this paper is to study the food safety problem by analyzing veterinary residues in food and dietary exposure assessment, providing a new sight for analysis and evaluation of food safety issues caused by veterinary residues.
In the first chapter, it mainly describes the current situation and main sources of food safety problem in China, and discusses the challenges for food safety under the new situation
.Several concepts related to food safety are also introduced. The principal methods for detection of veterinary residues in food are reviewed. The purposes and methods of this study are also introduced in this chapter.
In the second chapter, a high-throughput screening method for73veterinary residues in pork was developed, containing β-agonists, sedatives and sulfonamides. The method was established by high performance liquid chromatography hybrid-quadruple time-of-flight mass spectrometry (HPLC-Q-TOF-MS). These samples were extracted and purified by dispersed solid-phase extraction, then analyzed by HPLC-Q-TOF-MS and quantitated with isotope internal standard. The method was proved to be rapid and simple, with high selectivity and sensitivity. The result showed that the method could be used for both targeted and untargeted veterinary residues in pork.
In the third chapter, a platform for detecting antiviral drugs in livestock and poultry meats was established, including the determination of amantadine in poultry meats and the determination of ribavirin in poultry and livestock meats. The samples were extracted by mixed solvent with or without enzymolysis respectively, purified by solid phase extraction, then determined by linear ion trap with liquid chromatography tandem quadrupole mass spectrometry (QTrap), and quantified by using isotope internal standard method. These two determination methods were both validated. The linear correlation coefficient of peak area and concentration was above0.999, all of the average recoveries were above80%. The platform was proved to be used as a routine method for antiviral drugs determination in poultry and pork meats.
In the fourth chapter, a high-throughput LC-MS/MS method for simultaneous detection of20β-agnoists in poultry and pork meat was developed. The samples were extracted by mixed solvent with enzymolysis, purified by SPE, determined by LC-MS/MS, and quantified by using isotope internal standard method or external standard method. The validation indicated that that the method was suitable for the determination of β-agnoist in poultry and pork.
The dietary exposure assessment of ractopamine residue in pork meats and livers was studied by using the established LC-MS/MS method as well as statistical analysis. The risk assessment included hazard identification, hazard characterization, exposure assessment, risk assessment and risk characterization. The assessment result showed that ractopamine residue in pork food was at a low risk for human health.
In the fifth chapter, a rapid LC-MS/MS method for detection of chloramphenicol (CAP) in poultry meat was developed. The samples were extracted by acetonitrile (ACN), purified by hexane, determined by LC-MS/MS with a concentrated analyte, and quantified by using isotope internal standard method. The validation of the method showed that the method was suitable for the determination of CAP in poultry meat.
The dietary exposure assessment of CAP residue in poultry is studied. The risk assessment included hazard identification, hazard characterization, exposure assessment, risk assessment and risk characterization. The assessment result suggested that CAP residues in poultry meat was at low risk of carcinogenicity for human, and the adverse clinical reaction caused by CAP would occur less likely.
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