Analysis Of 4 β-agonists In Pork By Isotope Dilution Mass Spectrometry

Veterinary drug residue in food can cause acute or chronic poisoning, allergy and abnormal reaction to human body, and cause direct or potential harm to human body through the role of the environment and food chain. Veterinary drugs such as β-agonists, tranquilizers and nitrofuran drugs are prohibited use in the process of animal feeding in many countries,ministry of agriculture announcements in our country stipulates many drugs such as(3-agonists should not be detected in animal food. Therefore, in order to meet the high sensitivity of these veterinary drug testing requirements, and try to avoid the false positive test results, it is necessary to develop a high detection sensitivity analysis techniques.In this study, by optimizing the pretreatment of four β-agonists in the matrix of pork samples and the detection conditions of the gas chromatography-tandem triple quadruple mass spectrum(GC-MS/MS), A method for multi-residue analysis of β-agonist, clenbuterol,brombuterol, tulobuterol, salbutamol in pork has been established by using isotope dilution combined with gas chromatography-tandem mass spectrometry and based on solid phase extraction method.The main work of this paper is as follows:1. A pretreatment method and GC-MS / MS detection method were established for fourβ-agonists in pork. The homogenized samples were enzymatically hydrolysed and extracted by acetic acid buffer, adjusted pH by perchloric acid, and cleaned-up on HLB and MCX cartridge, and derivatized with N,O-bis (trimethylsilyl) trifluoro acetamide (BSTFA)+1%trimethylchlorosilane (TMCS), detected with GC-MS/MS, and quantified by external standard method. The results show that a linear relationship of the external standard method is good, the linear correlation coefficient were all greater than 0.9987. The recoveries were 60.2%～80.5%,and the relative standard deviations were 2.0%～10.9% in blank samples spike with the four β-agonists at 5.4pg/kg～70.0μg/kg. So, the result presents that the use of external standard method to quantify four β-agonists in pork samples can not avoid the loss ofβ-agonists during pretreatment, so that the recovery rate is relatively low, it is difficult to achieve trace analysis of β-agonists in pork.2. The isotopic abundances and chemical purities of deuterium clenbuterol, tulobuterol and brombuterol prepared by Shanghai Research Institute of Chemical Industry were established by liquid chromatography-mass spectrometry. Firstly, the chemical purities of stable isotope labeled clenbuterol-D9, tulobuterol-D9 and brombuterol-D9 were determined by external standard method combined with liquid chromategraphy. The results show that a linear relationship of the external standard method is good, the linear correlation coefficient were all greater than 0.9998, the chemical purities of the three compounds were 98.4%,93.7% and 99.1%,and the RSDs were less than 0.3% and the recoveries were 99.4%～100.3% and 99.3%～100.2%,respectively,the RSDs were all less than 0.4%. The isotopic abundances of clenbuterol-D9, tulobuterol-D9 and brombuterol-D9 were determined by LC-MS and calculated by "quality cluster"method, in which the abundances of clenbuterol-D9,tulobuterol-D9 and brombuterol-D9 were 97.8 atom%, 98.6 atom%, 98.9 atom%, respectively.Therefore, the isotopic abundance and chemical purity of the three materials can satisfy the requirements of isotope internal standard reagents.3. A method for multi-residue analysis of β-agonist, clenbuterol, brombuterol, tulobuterol,salbutamol in pork has been established combined with gas chromatography-tandem mass spectrometry by using the stable isotope labeled clenbuterol-D9, tulobuterol-D9 and brombuterol-D9 which were made by Shanghai Research Institute of Chemical Industry . The homogenized samples were spiked with isotope-labeled internal standards clenbuterol-D9,brombuterol-D9, tulobuterol-D9, enzymatically hydrolysed and extracted by acetic acid buffer,adjusted pH by perchloric acid, and cleaned-up on HLB and MCX cartridge, and derivatized with N,O-bis (trimethylsilyl) trifluoro acetamide (BSTFA)+1% trimethylchlorosilane (TMCS),detected with GC-MS/MS. The experimental results showed that spiking with the 4 β-agonists at 0.9μg/kg～35μg/kg in blank samples the recoveries were 92.0%～116.0% , and intra-day and inter-day relative standard deviations were 1.0%～12.6% and 1.9%～13.2%. The linear ranges of 4β-agonists were from 0.001 mg/L ～0.2 mg/L,the coefficient of correlation was not less than 0.9997,detection limits ranged from 0.13～0.4μg/kg,and the quantitation limits ranged from 0.40μg/kg～1.27μg/kg. This analysis method has satisfactory separation, the recovery, detection limit and precision can satisfy the requirement of the veterinary drug residue analysis.