同位素稀释质谱法分析猪肉中4种β-受体激动剂

发布时间：2019-05-18 14:07

【摘要】：食品中兽药残留可以直接引发对人体的急慢性中毒、过敏和变态反应,并通过环境和食物链的作用对人体造成直接或潜在危害,因此各国在动物饲养过程中都严禁使用β-受体激动剂类药物、镇定剂类药物和硝基呋喃类药物等兽药,我国农业部235号公告要求β-受体激动剂等类兽药在动物源性食品中不得检出。为了满足对这些兽药检测的高灵敏度要求,并尽量避免检测结果的假阳性,有必要开发具有更高检测灵敏度的分析技术。本实验通过优化猪肉样品基质中的4种β-受体激动剂的前处理方法和气相色谱串联三重四级杆质谱的检测条件,采用同位素稀释质谱法,结合固相萃取技术,建立了猪肉中克伦特罗、妥布特罗、溴布特罗、沙丁胺醇4种β-受体激动剂残留量的气相色谱-串联三重四级杆质谱(Gas Chromatography-tandem triple quadruple Mass Spectrometry,GC-MS/MS)同时检测方法。本论文的主要工作如下:1、考察了猪肉中4种β-受体激动剂的前处理方法和GC-MS/MS检测方法。绞碎的猪肉样品中经β-盐酸葡萄糖醛苷酶/芳基硫酸酯酶酶解,醋酸铵缓冲溶液提取,高氯酸调pH, HLB-MCX固相萃取柱串联净化,用N,O-双三甲基硅基三氟乙酰胺(BSTFA)+1%三甲基氯硅烷(TMCS)衍生,再进行GC-MS/MS测定,外标法定量。结果表明,外标法的线性关系良好,4种β-受体激动剂的线性相关系数r2≥0.9987,在动物组织中添加5.4μg/kg～70.0μg/kg水平的β-受体激动剂,回收率为60.2%～80.5%,相对标准偏差(RSD)为2.0%～10.9%。可以看出,使用简单标样的外标法对猪肉样品中4种β-受体激动剂进行定量时无法避免前处理过程中β-受体激动剂的损失,以至于得到的回收率比较低,难以实现猪肉中β-受体激动剂的准确痕量分析。2、建立了稳定同位素标记的氘代克伦特罗、氘代妥布特罗和氘代溴布特罗3种试剂的同位素丰度和化学纯度的液相色谱质谱仪表征方法。先采用外标法结合液相色谱仪对上海化工研究院制备的稳定同位素标记的克伦特罗-D9、妥布特罗-D9、溴布特罗-D9的化学纯度进行表征,三者的标准曲线线性关系良好,相关系数r20.9998,平行配制3个样品,外标法定量得到的三者的化学纯度分别大于98.4%、93.7%和99.1%, RSD均小于0.3%,回收率分别为99.4%～100.3%、99.3%～100.2%和99.3%～100.2%,且重复性良好。然后,采用“质量簇”计算分类方法,利用液质联用仪测得到的多组同位素丰度数值,对稳定性同位素标记克伦特罗-D9、妥布特罗-D9、溴布特罗-D9的同位素丰度进行测定,每个样品连续进样6次,三者的同位素丰度分别为97.8%、98.6%、98.9%。因此,3种氘标记化合物的同位素丰度和化学纯度均满足同位素内标试剂的要求。3、使用稳定性同位素标记的克伦特罗-D9、妥布特罗-D9、溴布特罗-D9做内标,建立了猪肉中克伦特罗、妥布特罗、溴布特罗、沙丁胺醇等4种β-受体激动剂残留量的GC-MS/MS检测方法。绞碎的动物组织中加入同位素内标克伦特罗-D9、妥布特罗-D9、溴布特罗-D9,经β-盐酸葡萄糖醛苷酶/芳基硫酸酯酶酶解,醋酸铵缓冲溶液提取,高氯酸调βH,HLB-MCX固相萃取柱串联净化,用N,O-双三甲基硅基三氟乙酰胺(BSTFA)+1%三甲基氯硅烷(TMCS)衍生,再进行GC-MS/MS进行测定,采用稳定同位素内标法定量。结果表明,动物组织中添加0.9μg/kg～35.0μg/kg水平的β-受体激动剂,回收率为92.2%～1112.8%,批内相对标准偏差(RSD)为1.0%～12.6%,批间相对标准偏差(RSD)为1.9%～13.2%。4种β-受体激动剂的线性范围为0.001 mg/L～0.200mg/L,相关系数≥0.9997,方法最低检测限为0.13μg/kg～0.40μg/kg,最低定量限为0.40μg/kg～1.27μg/kg,该分析方法在回收率、精密度和检测限方面均能满足兽药残留的分析要求。
[Abstract]:The veterinary drug residue in the food can directly cause acute and chronic poisoning, allergy and allergy to the human body, can cause direct or potential harm to the human body through the action of the environment and the food chain, In China's Ministry of Agriculture, China's Ministry of Agriculture, China's Ministry of Agriculture, No.235 of China's Ministry of Agriculture, the animal-derived food should not be detected in animal-derived food. In order to meet the high sensitivity requirements for the detection of these veterinary drugs, and to avoid false positive of the detection results, it is necessary to develop an analytical technique with higher detection sensitivity. In this experiment, by optimizing the pre-treatment method and the detection condition of the four kinds of hormone-receptor agonists in the pork sample matrix, using the isotope dilution mass spectrometry, combined with the solid-phase extraction technology, Clenbuterol in the pork is established, The gas chromatography-tandem triple quadrupole mass spectrometry (GC-MS/ MS) of 4 kinds of opioid-receptor agonist residues in bromobbuterol and salbutamol was also tested. The main work of this thesis is as follows:1. The pretreatment method and the method of GC-MS/ MS for four kinds of hormone-receptor agonists in pork are investigated. The minced pork sample is obtained by the following steps of: carrying out enzymolysis on the gluconic acid glucose aldolase/ aryl sulfate esterase in the minced pork sample, extracting with a buffer solution of acetic acid, adjusting the pH value of the perchloric acid, and carrying out serial purification on the HLB-MCX solid-phase extraction column, and derivatization with N, O-bis (trimethylsilyl) trifluoroethamine (BSTFA) and 1% trimethylchlorosilane (TMCS); The determination of GC-MS/ MS and the quantification of external standard method were carried out. The results showed that the linear relationship of the external standard method was good, the linear correlation coefficient of 4 kinds of antigen-receptor agonists was r2 = 0.9987, and the rate of recovery was 60.2% ~ 80.5% and the relative standard deviation (RSD) was 2.0% ~ 10.9%. it can be seen that the loss of the antigen-receptor agonist in the pre-treatment process cannot be avoided when a simple standard standard external standard method is used for the quantification of the four types of antigen-receptor agonists in the pork sample, so that the recovery rate obtained is relatively low, The accurate and trace analysis of the oxygen-receptor agonists in pork was difficult to achieve. The chemical purity of the stable isotope-labeled Clenbuterol-D9, trastudino-D9 and brot-D9 prepared by the Shanghai Chemical Research Institute was characterized by using an external standard method and a liquid chromatograph, the linear relationship of the standard curves of the three is good, and the correlation coefficient r20.9998, The chemical purity of the three samples is more than 98.4%, 93.7% and 99.1%, RSD is less than 0.3%, the recovery rate is 99.4% ~ 100.3%, 99.3% ~ 100.2% and 99.3% ~ 100.2%, and the repeatability is good. then, using the "mass cluster" calculation and classification method, the isotope abundance values of the stability isotope labeled Clenbuterol-D9, the trastudino-D9 and the bributan-D9 are determined by using the multi-group isotope abundance values obtained by the liquid quality combination instrument, and each sample is injected for six times continuously, The isotopic abundance of the three was 97.8%, 98.6% and 98.9%, respectively. Therefore, the isotopic abundance and the chemical purity of the three kinds of marker compounds meet the requirements of the isotope internal standard reagent.3. Using the stability isotope labeled Clenbuterol-D9, Trota-D9, and the bromobutan-D9 as the internal standard, and establishing the clenbuterol in the pork, The method of GC-MS/ MS for the determination of 4 types of opioid-receptor agonist residues, such as bributamol, salbutamol, and the like. the minced animal tissue is added with the isotope internal standard Clenbuterol-D9, the butetroo-D9 and the bromobutan-D9, and is obtained by the following steps of: carrying out enzymolysis on the gluconic acid glucose aldolase/ arylsulfatase, the extraction of the acetate buffer solution of the acetic acid, the pH of the perchloric acid and the H, and the HLB-MCX solid-phase extraction column to be purified in series, and using N, O-bis (trimethylsilyl) trifluoride (BSTFA) + 1% trimethylchlorosilane (TMCS) was derived, and then GC-MS/ MS was used to determine the amount of stable isotope internal standard. The results showed that the recovery rate was 92.2% ~ 1112.8%, the relative standard deviation (RSD) in the batch was 1.0% ~ 12.6%, the relative standard deviation (RSD) was 1.9% ~ 13.2% in the animal tissue, and the relative standard deviation (RSD) was from 1.9% to 13.2%. The linear range of the four kinds of antigen-receptor agonists was 0.001 mg/ L ~ 0.200 mg/ L, and the correlation coefficient was 0.9997. The minimum detection limit of the method is 0.13 & mu; g/ kg-0.40 & mu; g/ kg, the minimum limit is 0.40 & mu; g/ kg-1.27 & mu; g/ kg, and the analytical method can meet the analysis requirement of the veterinary drug residue in the aspects of recovery rate, precision and detection limit.
【学位授予单位】：上海应用技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.63;TS251.51

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