Development Of The Certified Reference Material Of Enrofloxacin Residue In Whole Liquid Egg

Enrofloxacin,as a third-generation fluoroquinolone,has a wide range of applications in human and veterinary medicine and can be used to treat a variety of diseases including urinary,respiratory and gastrointestinal infections.However,the illegal use of Enroflxacin in livestock and poultry breeding will increase the resistance of pathogenic microorganisms,side effect to human health and risk to public health and safety.Therefore,in the 235 and other announcements promulgated by the Ministry of Agriculture of the People's Republic of China,the use of Enrofloxacin and its limits were clearly defined,with particular emphasis on the ban on laying hens and the related testing standards to ensure the quality and safety of agricultural products.Enrofloxacin is an important monitoring parameter in the veterinary drug residue monitoring program,which has been included in the routine monitoring and special monitoring programs for aquatic products,livestock and poultry in the national and local scope of the Ministry of Agriculture.However,in recent years,the risk monitoring results of livestock and poultry products have shown that In poultry and other large livestock and poultry products in China,Enrofloxacin and other fluoroquinolones continued to be detected to some extent.Therefore,Enrofloxacin has also become the focus of the current quality monitoring of eggs and other livestock and poultry products in China.In order to ensure the accuracy and reliability of the monitoring results,effectively support government supervision work,and ensure the reliability,comparability,and accuracy of measurement results,it is urgent to develop relevant purity and matrix reference materials.In view of the research on Enrofloxacin matrix reference materials in whole liquid egg,the development of the purity reference material of Enrofloxacin was first carried out to complete the traceability chain.Experiments were conducted to quantify the candidate by infrared spectroscopy and mass spectrometry.Then the purity of the candidate was determined by two different methods-mass balance method and quantitative nuclear magnetic method.Homogeneity and stability were monitored and investigated.At the same time,the uncertainty in the whole process was evaluated.The purity value of the obtained Enrofloxacin purity reference material was accurate,the homogeneity and stability can meet the requirements of technical specification.The purity of Enrofloxacin CRM was 99.7%with the uncertainty value 0.4(k=2),and it has been successfully declared as a national secondary standard reference material(GBW(E)090817).Secondly,a high accuracy method is established for the determination of matrix reference materials.Using Enrofloxacin-D5 as an internal standard,based on the dispersive solid-phase extraction pretreatment technique combined with high-sensitivity triple quadrupole mass spectrometry,a liquid chromatography-isotope dilution mass spectrometry method was established for the analysis of Enrofloxacin residues in liquid eggs.The detection limits and quantification limits of the method were 0.5?g/kg and 1.5?g/kg,respectively.The linearity was good within 0.5?g/kg?100?g/kg,and the recoveries ranged from 95%to 105%with the addition level of 2?g/kg?20?g/kg,which can meet the requirement for the development of matrix reference materials.Thirdly,the development of Enrofloxacin certified matrix reference material has been carried out.The layers were continuously administered orally for 5 days.Eggs were collected from the first day of administration to the 9th day after the end of the administration,the rule of elimination of Enrofloxacin residue was analyzed.By mixing,homogenizing,encapsulating,iradiating,sterilizing,encapsulating and cryopreservation,the egg matrix reference material candidates were obtained.The established isotopic dilution mass spectrometry method was used in the determination of characteristic value in reference materials by several laboratories,and the key techniques such as homogeneity and stability of egg matrix reference materials were systematically and comprehensively studied.Finally,the characteristic value and extended uncertainty of the matrix reference material of Enrofloxacin drug residue in the whole egg liquor were obtained,which were 30.6?g/kg and 2.8?g/kg,and the long-term stability test showed that the matrix reference material was stable for 9 months at-80 ?.The application of this research will guarantee the accuracy,comparability and tracezbility for the monitoring results of egg product's quality and safety.