Development And Application Of Residue Analytical Methods For Five Antiviral Drugs In Honey

With the rapid development of China's beekeeping industry,the production of bee products has taken the lead in the world,and its quality and safety issues have gradually become the focus of increasing attention.In the process of beekeeping,irrational use of drugs and migration of environmental pollutants can easily lead to the accumulation of drug residues in bee products.Veterinary drug residue pollution is one of the most important safety issue of bee products in China.In recent years,the viral diseases in bee colonies have caused huge economic losses to beekeepers.However,the effects of the existing Chinese medicine treatment methods are not significant,and the biological preparations are not yet mature.Therefore,some beekeepers try to use antiviral drugs as a treatment method for viral diseases which resulting in the accumulation of antivirals in honey.At present,there is no introduction of multi-residue detection methods for antiviral drugs in bee products around the world.The purpose of this study is to develop and establish a multi-residue detection method for antiviral drugs in honey and to test honey samples in various regions of China,to determine the exposure level of antiviral drugs in honey,and to further explore the drug reduction of antiviral residues under different processing and storage conditions in honey.This research is divided into two parts.In the first part,a new pretreatment method was developed for distributed collection using PBA(Phenylboronic Acid)solid phase extraction column and combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)for the determination of ribavirin,moroxydine,amantadine,rimantadine and memantine in honey.The quantification limits(LOQ)of ribavirin,moroxydine,amantadine,rimantadine and memantine are 5,0.5,0.5,0.2,0.2 ?g/kg,and the detection limits(LOD)are 2,0.2,0.2,0.1,0.1 ?g/kg,respectively.Internal standard was used for quantification,the recovery range is between 82.46% to 116.34%.The day-to-day precision(RSD <15%)reflects the good reproducibility and stability of this method.This method was successfully applied to the bee products risk monitoring project of the Ministry of Agriculture in China,testing of 563 samples from 7 provinces(Hu Bei,LiaoNing,Ji Lin,Hei Long Jiang,Shan Xi,He Bei,Si Chuan)and the status of antiviral drug residues in China was analyzed.Among all honey tested samples,the positive rate of antiviral was 2.84%,while the detection rates of antiviral drugs in apis cerana honey and apis mellifera honey were 1.38% and 3.35%,respectively.The second part explores the law of residue reduction of antiviral drugs in honey,and is divided into two parts: normal temperature storage experiment and high temperature processing experiment.At different temperatures,the degradation law of the five antiviral drugs are different,ribavirin,amantadine,rimantadine and memantine degrade relatively slow and no half-life was observed during the test period at 4?,25? and 60?.At 80?,the half-lives of ribavirin,amantadine and rimantadine are 9,12,and 10 hours,respectively.Among them,moroxydine degrades the fastest,the half-life of moroxydine was about 165 days and 130 days at 4? and 25?,respectively.Furthermore,the half-life of moroxydine at 60? is about 22 hours while only 2 hours at 80?.This study for the first time explored the law of residue reduction of five antivirals in honey at different temperatures,providing data support for beekeepers to select appropriate antiviral drugs for the treatment of bee cystic larval disease,it also provides technical support for the development of suitable processing and storage conditions for honey that may remain antiviral drugs.