Establishment Of Detection Method For Halofuginone Residue In Animal Derived Food

Halofuginone is a quinazolinone alkaloid isolated from the plant Changshan which has a broad spectrum to coccidiosis,including sporozoites,first-generation schizonts,and second-generation schizonts.Owing to its outstanding effect,it is widely used in poultry farming as a feed additive for the control of coccidiosis.Not only does fluoxetine have anticoccidial activity,but also inhibits the development of collagen synthesis and tumor cells.The existing national standards method have some limitations to detecting the residue of Halofuginone in animal tissues.such as low sensitivity.And it is only avalible for chickens muscle and liver tissues.So we need to establish a rapid,simple,and easy-to-promote standard method of high performance liquid chromatography for the detection of halofuginone residues in most animal tissues,including muscle,liver,kidney,and sebum of bovine,poultry and swine,to ensure the safety of animals edible tissues and promote the foreign trade exports.According to the "Agricultural Industry Standards Development and Revision(Agricultural Product Quality Safety)Project Guide 2016" document submitted by the National Veterinary Drug Residue Experts Committee Office,the sensitivity of the new method should not be lower than the original standard,and covering more kinds of animal tissues.This project undertook the formulation of "Determination of halofuginone residues in animal foods" in the formulation and revision of agricultural industry standards(quality of agricultural products safety),and optimized the original test methods.To establish a rapid,easy to operate and easy to promote method to meet the needs of Halofuginone residues determination in animal foods.This study has explored and optimized sample pretreatment methods and HPLC conditions,based on a large number of literatures,and established a better method for detecting halofuginone drugs in animal tissues.Based on the principle of accuracy,practicality,rapidity,meeting with the world standards.the similar standard method was formulated,strictly followed the scientific basis,and developed and completed the formulation of standard testing methods,which were handed over to the Anhui Provincial Institute of Veterinary Medicine and Feed Supervision,Jiangsu Province Livestock Products.The quality inspection and testing center and the three inspection agencies of the Henan Provincial Institute of Veterinary Medicine and Feed Inspection conducted methodological verification.Finally,draft standards for drafting and preparation instructions were drafted.1 Determination of haIofuginone residue in animal tissues by high performance liquid chromatography instrument.The purpose of this study was to establish a high performance liquid chromatography method for detected halofuginone concentration which has residued in muscle,sebum,kidney and liver tissues of bovines,poultries and swines.The animal tissues were extracted with ethyl acetate in an alkaline environment,and then continue using 0.125 mol·L-1 ammonium acetate buffer solution for further liquid-liquid extraction.The lower aqueous phase was taken out and the extraction process was repeated again.Then combined the two aqueous phase,and dried the residual ethyl acetate with nitrogen.After purification with HLB column,the eluent was dried with nitrogen and resuspended with mobile phase.The filtrate was filtered by organic filter membrane and then injected by high performance liquid clrromatography instrument.The mobile phase containing acetonitrile-0.006 25 mol·L-1 ammonium acetate buffer(25+75 with acetic acid to adjust the pH to 4.3)and the detection wavelength was 243 nm.According to the results,it have showed that the calculated limit of-detection in this method was 8 μg·kg-1 and the limit of quantitation was 10 μg·kg-1.At halofuginone concentrations between 10 μg·kg-1 to 400 μg·kg-1,The recovery rates were 70.68%～110.91%.the precision of the method was expressed by RSD%of inter-day and intra-day assays were ≤20.62%and ≤16.07%,reapsctively.The results has showed us with a good linear relationship,precision and recovery rate about the method we have established,which was easible,flexible and quickly for detected the content of halofuginone residued in animal tissues.2 Review and verification of《Determination of halofuginone residue in animal derived food by high performance liquid chromatography》method.While formulating the《Determination of halofuginone residue in animal derived food by high performance liquid chromatography》method,in order to meet the requirements of the national standards,after the preparation and completion of the standard test methods,they were handed over to the Anhui Provincial Veterinary Medicine and Feed Inspection Office.Jiangsu Province Animal Products Quality Inspection and Testing Center,and Henan Provincial Institute of Veterinary Medicine and Feed Inspection 3 inspection institutions to conduct methodological review verification,so as to ensure the accuracy and reliability of the test method.The verification test repeats the test method of Chapter Ⅱ.The specificity of the method is good,the detection limit is 8 μg·kg-1,and the limit of quantitation is 10 μ·kg-1.All of these can be verified.The verification test at the Animal and Veterinary Feed Inspection Office in Anhui Province is The recoveries at the concentration levels of 10 μg·kg-1 to 400 μg·kg-1 were 62.1%～116.0%,the intra-day relative standard deviation of this method was ≤19.7%,and the relative standard deviation during the day was≤16.97%;The product quality inspection and test center verification test has obtained recovery rates of 68.5%～118.1%at the concentration levels of 10 μg·kg-1 to 400 μg·kg-1.The relative standard deviation of the method is less than or equal to 19.35%,and the relative standard during the day is The deviation is less than or equal to 14.76%.The recovery rate at the concentration of 10 μg·kg-1 to 400 μdg·kg-1 obtained by the veterinary medicine and feed inspection institute of Henan Province was 76.9%～118.6%.The relative standard deviation of the method within days was ≤12.44%,daytime relative standard deviation≤10.70%.The test results show that the method for determining the content of halofuginoneresidues in animal foods is accurate and reliable and has good repeatability.3《Determination of halofuginone residue in animal derived food by high performance liquid chromatography》Standard text formulation of high-performance liquid chromatography.Based on the laboratory standard test methods,the test was verified by the methodological veriflcation conducted by the Anhui Provincial Institute of Animal and Veterinary Surge Supervision,the Jiangsu Animal Products Quality Inspection and Testing Center,and the Henan Provincial Institute of Veterinary Medicine and Feed Inspection.More than 20 experts in the field received advice and guidance to form a standard manuscript for the "Determination of halofuginoneresidue in animal foods",which specifies the content of halofuginonein animal foods.The sample preparation and high performance liquid chromatography determination method.The method is applicable to the determination of halofuginone content in muscle,sebum,kidney and liver tissues of chickens,swines and cattle.The method has the characteristics of high sensitivity,wide application range,rapid and accurate,and good reproducibility,and is convenient for animal-derived foods.The drug residues of fluoxetine are effectively monitored to ensure the safety of animal-origin foods.