同位素稀釋質譜法分析豬肉中4種β-受體激動劑
發(fā)布時間：2019-05-18 14:07
【摘要】：食品中獸藥殘留可以直接引發(fā)對人體的急慢性中毒、過敏和變態(tài)反應,并通過環(huán)境和食物鏈的作用對人體造成直接或潛在危害,因此各國在動物飼養(yǎng)過程中都嚴禁使用β-受體激動劑類藥物、鎮(zhèn)定劑類藥物和硝基呋喃類藥物等獸藥,我國農業(yè)部235號公告要求β-受體激動劑等類獸藥在動物源性食品中不得檢出。為了滿足對這些獸藥檢測的高靈敏度要求,并盡量避免檢測結果的假陽性,有必要開發(fā)具有更高檢測靈敏度的分析技術。本實驗通過優(yōu)化豬肉樣品基質中的4種β-受體激動劑的前處理方法和氣相色譜串聯三重四級桿質譜的檢測條件,采用同位素稀釋質譜法,結合固相萃取技術,建立了豬肉中克倫特羅、妥布特羅、溴布特羅、沙丁胺醇4種β-受體激動劑殘留量的氣相色譜-串聯三重四級桿質譜(Gas Chromatography-tandem triple quadruple Mass Spectrometry,GC-MS/MS)同時檢測方法。本論文的主要工作如下:1、考察了豬肉中4種β-受體激動劑的前處理方法和GC-MS/MS檢測方法。絞碎的豬肉樣品中經β-鹽酸葡萄糖醛苷酶/芳基硫酸酯酶酶解,醋酸銨緩沖溶液提取,高氯酸調pH, HLB-MCX固相萃取柱串聯凈化,用N,O-雙三甲基硅基三氟乙酰胺(BSTFA)+1%三甲基氯硅烷(TMCS)衍生,再進行GC-MS/MS測定,外標法定量。結果表明,外標法的線性關系良好,4種β-受體激動劑的線性相關系數r2≥0.9987,在動物組織中添加5.4μg/kg～70.0μg/kg水平的β-受體激動劑,回收率為60.2%～80.5%,相對標準偏差(RSD)為2.0%～10.9%�？梢钥闯�,使用簡單標樣的外標法對豬肉樣品中4種β-受體激動劑進行定量時無法避免前處理過程中β-受體激動劑的損失,以至于得到的回收率比較低,難以實現豬肉中β-受體激動劑的準確痕量分析。2、建立了穩(wěn)定同位素標記的氘代克倫特羅、氘代妥布特羅和氘代溴布特羅3種試劑的同位素豐度和化學純度的液相色譜質譜儀表征方法。先采用外標法結合液相色譜儀對上�；ぱ芯吭褐苽涞姆�(wěn)定同位素標記的克倫特羅-D9、妥布特羅-D9、溴布特羅-D9的化學純度進行表征,三者的標準曲線線性關系良好,相關系數r20.9998,平行配制3個樣品,外標法定量得到的三者的化學純度分別大于98.4%、93.7%和99.1%, RSD均小于0.3%,回收率分別為99.4%～100.3%、99.3%～100.2%和99.3%～100.2%,且重復性良好。然后,采用“質量簇”計算分類方法,利用液質聯用儀測得到的多組同位素豐度數值,對穩(wěn)定性同位素標記克倫特羅-D9、妥布特羅-D9、溴布特羅-D9的同位素豐度進行測定,每個樣品連續(xù)進樣6次,三者的同位素豐度分別為97.8%、98.6%、98.9%。因此,3種氘標記化合物的同位素豐度和化學純度均滿足同位素內標試劑的要求。3、使用穩(wěn)定性同位素標記的克倫特羅-D9、妥布特羅-D9、溴布特羅-D9做內標,建立了豬肉中克倫特羅、妥布特羅、溴布特羅、沙丁胺醇等4種β-受體激動劑殘留量的GC-MS/MS檢測方法。絞碎的動物組織中加入同位素內標克倫特羅-D9、妥布特羅-D9、溴布特羅-D9,經β-鹽酸葡萄糖醛苷酶/芳基硫酸酯酶酶解,醋酸銨緩沖溶液提取,高氯酸調βH,HLB-MCX固相萃取柱串聯凈化,用N,O-雙三甲基硅基三氟乙酰胺(BSTFA)+1%三甲基氯硅烷(TMCS)衍生,再進行GC-MS/MS進行測定,采用穩(wěn)定同位素內標法定量。結果表明,動物組織中添加0.9μg/kg～35.0μg/kg水平的β-受體激動劑,回收率為92.2%～1112.8%,批內相對標準偏差(RSD)為1.0%～12.6%,批間相對標準偏差(RSD)為1.9%～13.2%。4種β-受體激動劑的線性范圍為0.001 mg/L～0.200mg/L,相關系數≥0.9997,方法最低檢測限為0.13μg/kg～0.40μg/kg,最低定量限為0.40μg/kg～1.27μg/kg,該分析方法在回收率、精密度和檢測限方面均能滿足獸藥殘留的分析要求。
[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.
【學位授予單位】：上海應用技術大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.63;TS251.51
[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.
【學位授予單位】：上海應用技術大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.63;TS251.51
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