Developme Nt Of An Indirect Competitive Enzyme Linked Immunoassay For The Analysis Of Amantadine And 3-Methyl-Quinoxaline-2-Carboxylic Acid And Chloramphe Nicol In Chicken Muscle

Nowadays,more and more people concerned about the chicken muscle safety.veterinary drug residues is one of the key factors that affec t the chicken muscle safety,especially the antibiotics and antiviral drug residues in chicken mucle.The fast and sensitive detection methods for veterinary drugs have been developed quickly in recent years.amantadine（AMA）and Chloramphenicol（CAP）and olaquindox（O LA）have been illegally added to animal feed,injectables and internal medicines as antiviral drugs or antibiotics,which are widely used in the prevention and treatment of animal diseases.However,it was reported that AMA,CAP and OLA can easily cause drug-resistant strains,and both show toxicity to human and animal,including hematological toxicity,embryo toxicity and immunotoxicity,which threatens the health of consumers.Therefore,it is very important to strengthen the monitoring of AMA,CAP and OLA,and it is necessary to control the residues in animal derived food of these three drugs.For now,the main detection methods of AMA,C AP and 3-Methyl-quinoxaline-2-carboxylic acid（MQCA）are the instrument methods,including liquid chromatography（LC）,high performance liquid chromatography-mass spectrometry（HPLC-MS/MS）.Although these technologies are accurate and relable,they also have shortcomings,such as tedious operation,high cost,proficient experts and low throughout.As a rapid,se nsitive,and high throughout analytical method,immunoassay can be used conjunction with the instrument techniques to meet the fast screening needs of large quantities of samples.In this study,three rapid,accurate and stable indirect competitive ELISA（ic-ELISA）for the detection of amantadine,MQCA and chloramphenicol residues in chicken muscle were developed.The following are the main content:（1）An ic-ELISA for the detection of amantadine was established.The ic-ELISA showed an IC₅₀ value of 0.69μg/L for amantadine,respectively.The linear ranges were ranged from 0.07μg/L to 6.15μg/L with a limit of detection of 0.21μg/L for amantadine,respectively.There is no cross reaction with other analogues.The kit stored at 37℃ can be kept for 6 days,at 4℃for more than half a year.The average recoveries of amantadine of spiked samples ranged from 101.69%to 108.71%with a coefficient of variation of less than 15%.Good correlations between the results of ic-ELISA and HPLC-MS/MS were obtained.Therefore,this ic-ELISA provided a valid detection method for amantadine in real samples.（2）An ic-ELISA for the detection of MQCA was established.The ic-ELISA showed an IC₅₀ value of 1.88μg/L for MQCA,respectively.The linear ranges were ranged from 0.49μg/L to 7.27μg/L with a limit of detection of 0.32μg/L for MQCA,respectively.There is no cross reaction with other analogues.The kit stored at 37℃ can be kept for 6 days,at 4℃ for more than half a year.The average recoveries of MQCA of spiked samples ranged from90.44%to 107.16%with a coefficient of variation of less than 15%.Good correlations between the results of ic-ELISA and HPLC-MS/MS were obtained.Therefore,this ic-ELISA provided a valid detection method for MQCA in real samples.（3）An ic-ELISA for the detection of CAP was established.The ic-ELISA showed an IC₅₀ value of 0.12μg/L for MQCA,respectively.The linear ranges were ranged from 0.02μg/L to 0.70μg/L with a limit of detection of 0.019μg/L for CAP,respectively.There is no cross reaction with other analogues.The kit stored at 37℃ can be kept for 6 days,at 4℃ for more than half a year.The average recoveries of CAP of spiked samples ranged from 74.02%to 113.48%with a coefficient of variation of less than 15%.Good correlations between the results of ic-ELISA and HPLC-MS/MS were obtained.Therefore,this ic-ELISA provided a valid detection method for C AP in real samples.（4）The sample pretreatment methods of chloramphenicol and amantadine were combined.The extraction efficiency of different extractants and shaking time was compared.Finally,acetic acid-acetonitrile and shaking time of 2 min were chosen for the extraction drugs.The method was simple and provided a new idea for multi-residue detection of veterinary drugs.