鸡组织和蛋中二硝托胺及其代谢产物残留气相色谱—串联质谱确证分析方法的研究

发布时间：2018-10-19 13:10

【摘要】：二硝托胺是一种有效的硝基酰胺类抗球虫药,在禽类生产中应用十分广泛。然而,长期连续不规范用药会使药物以原形药物或代谢产物的形式在禽类组织中累积,造成禽组织中兽药残留。长期食用含有二硝托胺及其代谢产物的动物食品及其制品,兽药会在人体中累积,对人类健康造成威胁。目前,关于二硝托胺及其代谢产物(3-氨基-5-硝基邻甲苯酰胺,3-ANOT)在家禽组织中的残留确证分析方法主要为液相色谱-串联质谱法,而用气相色谱-串联质谱仪器分析方法对禽组织中二硝托胺及其代谢产物残留检测的研究还未见报道。本研究以京海黄鸡为试验素材,利用加速溶剂萃取和固相萃取技术,建立了鸡肌肉、肝脏、肾脏、皮肤+脂肪及鸡蛋中二硝托胺及其代谢产物残留气相色谱-串联质谱确证分析方法(GC-MS/MS),为动物源性食品中二硝托胺及其代谢产物残留检测标准的制定提供科学依据,为残留监控提供新的技术手段。本试验主要研究结果如下:1.首次分别建立和优化了采用加速溶剂萃取仪(ASE350)从鸡组织(肌肉、肝脏、肾脏、皮肤+脂肪)和鸡蛋中同时提取二硝托胺及其代谢产物残留的方法。乙腈作为提取溶剂,样品与硅藻土研磨混匀装填入萃取池,80℃、1500 psi条件下,在线去脂(正己烷作为除脂剂)与二硝托胺及其代谢产物萃取(乙腈作为萃取剂)连续进行。当二硝托胺及其代谢产物添加水平分别为定量限(LOQ)、0.5MRL(最高残留限量)、1MRL、2MRL时,鸡肌肉、肝脏、肾脏、皮肤+脂肪样品中二硝托胺及其代谢产物的平均回收率均在81.96%-94.31%之间,相对标准偏差均在1.72%-5.37%之间;鸡蛋清、蛋黄和全蛋样品中二硝托胺及其代谢产物的平均回收率均在82.74%-87.49%之间,相对标准偏差均在1.73%-4.63%之间。该提取方法节省溶剂、样品基质影响小、萃取效率高、回收率高、方便、快速和全自动。2.首次分别建立和优化了鸡组织(肌肉、肝脏、肾脏、皮肤+脂肪)和鸡蛋中同时检测二硝托胺及其代谢产物残留的气相色谱-串联质谱(GC-MS/MS)确证检测方法。采用EI模式,全扫描(SCAN)定性,Auto SRM结合外标法定量,在空白肌肉、肾脏、皮肤+脂肪、蛋清中添加二硝托胺及其代谢产物LOQ-200.0 μg/kg浓度范围内,定量离子对的色谱峰面积与其浓度呈线性相关,且线性关系良好,决定系数(R2)均高于0.9993;在空白肝脏中添加二硝托胺及其代谢产物LOQ-1600.0 μg/kg浓度范围内,定量离子对的色谱峰面积与其浓度呈线性相关,且线性关系良好,决定系数(R2)高于0.9993;在空白蛋黄和全蛋中添加二硝托胺及其代谢产物LOQ-1800.0 μg/kg浓度范围内,定量离子对的色谱峰面积与其浓度呈线性相关,且线性关系良好,决定系数(R2)均高于0.9995。精密度测定结果:当二硝托胺及其代谢产物添加水平分别为定量限(LOQ)、0.5MRL(最高残留限量)、1MRL、2MRL时,鸡肌肉、肝脏、肾脏、皮肤+脂肪样品中二硝托胺及其代谢产物日内相对标准偏差均在1.87%-5.74%之间,日间相对标准偏差均在3.34%-7.36%之间;鸡蛋清、蛋黄和全蛋样品中二硝托胺及其代谢产物日内相对标准偏差均在2.96%-5.21%之间,日间相对标准偏差均在3.94%-6.34%之间。在优化的试验条件下,二硝托胺及其代谢产物在鸡肌肉、肝脏、肾脏、皮肤+脂肪中的检测限(LOD)在 0.8-2.5 μg/kg(S/N≥3),LOQ 在 2.7-8.0 μg/kg(S/N10);二硝托胺及其代谢产物在鸡蛋清、蛋黄和全蛋中的LOD在0.8-2.8 μg/kg(S/N≥3),LOQ在3.0-10.0μg/kg(S/N≥10)。方法验证参数均满足中国农业部、欧盟和美国FDA兽药残留检测的要求,定量准确、快速、灵敏度高。
[Abstract]:Nitinol is an effective class of anti-coccidial drugs, which is widely used in poultry production. However, long-term continuous non-standardized administration will cause the drug to accumulate in poultry tissue in the form of a prototype drug or metabolite, causing the veterinary drug residue in the avian tissue. The long-term edible animal food containing dinitrogen and its metabolites and its products and veterinary drugs are accumulated in the human body and pose a threat to human health. At present, the residual confirmatory analysis method for the dinitrogen and its metabolites (3-amino-5-nitro o-toluidine, 3-ANOT) in poultry tissues is mainly liquid chromatography-tandem mass spectrometry, The results of GC-tandem mass spectrometer (GC-MS) analysis were not yet reported. In this study, the method of GC-MS/ MS was established by using accelerated solvent extraction and solid phase extraction technique to establish the residual gas chromatography-tandem mass spectrometry (GC-MS/ MS) in chicken muscle, liver, kidney, skin + fat and eggs. The invention provides a scientific basis for establishing a standard for detecting the residual detection standards of the dinitrogen and the metabolites in the animal source food, and provides a new technical means for the residual monitoring. The main results of this study were as follows: 1. A method was established and optimized for the first time using an accelerated solvent extractor (ASE350) from chicken tissue (muscle, liver, kidney, skin + fat) and eggs. Acetonitrile is used as an extraction solvent, and the sample is ground and mixed with diatomite to be filled into an extraction tank, 80 DEG C and 1500 psi, and the on-line de-fat (n-hexane as a degreasing agent) is continuously carried out with the dinitrobutamine and the metabolite thereof (acetonitrile as an extracting agent). The average recoveries were 81. 96%-94. 31% in chicken muscle, liver, kidney, skin + fat sample, and the average recovery was 81. 96%-94. 31%, respectively, when the levels of nitrate and their metabolites were the limit of quantitation (LOQ), 0. 5MRL (maximum residual limit), 1MRL and 2MRL, respectively. The relative standard deviation was between 1. 72% and 5. 37%; the average recovery rate of sodium dinitrate and its metabolites in egg white, yolk and whole egg samples was 82. 74% -87. 49%, and the relative standard deviation was between 1.73% and 4.63%. The extraction method saves the solvent, the influence of the sample matrix is small, the extraction efficiency is high, the recovery rate is high, and the extraction method is convenient, rapid and full-automatic. Gas chromatography-tandem mass spectrometry (GC-MS/ MS) was established and optimized for the first time in chicken tissues (muscle, liver, kidney, skin + fat) and eggs. The chromatographic peak area of quantitative ion pair was linearly related to its concentration in the range of blank muscle, kidney, skin + fat and egg white by using EI mode, full-scan (SCAN) qualitative and auto SRM combined with external standard method. In the blank liver, the peak area of the chromatographic peak of the quantitative ion pair is linearly related with its concentration, and the linear relation is good, and the coefficient of separation (R2) is higher than 0.99993; The peak area of the chromatographic peak of the quantitative ion pair was linearly related to its concentration in the blank yolk and the whole egg and the concentration range of LOQ-1800. 0ug/ kg. The linear relation was good and the thermodynamic coefficient (R2) was higher than 0. 9995. The results of precision determination: The relative standard deviation was between 1. 87% and 5. 74% in chicken muscle, liver, kidney, skin + fat sample, and the relative standard deviation in chicken muscle, liver, kidney, skin + fat samples, respectively, when the levels of nitrate and its metabolites were at the limit of quantitation (LOQ), 0. 5MRL (maximum residual limit), 1MRL and 2MRL, respectively. The relative standard deviation of day relative standard deviation was between 3.34% -7.36%, the relative standard deviation was 2.96% -5.21% in egg white, yolk and whole egg sample, and the relative standard deviation of day was between 3.94% -6.34%. The detection limit (LOD) in chicken muscle, liver, kidney, skin + fat (LOD) in chicken muscle, liver, kidney, skin + fat was 0.8 -2. 5 ug/ kg (S/ N/ 3), the LOQ was 2. 7-8. 0 ug/ kg (S/ N10), the LOD was 0.8 -2. 8 ug/ kg (S/ N/ 3) in egg white, yolk and whole egg. LOQ was 3.0-10.0. mu.g/ kg (S/ N/ 10). Method Validation parameters met the requirements of China's Ministry of Agriculture, the European Union and the United States Agency for Veterinary Drug Residues Detection, with accurate, accurate, rapid and high sensitivity.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.84

【相似文献】