Establishment And Application Of LC-MS/MS Method For The Determination Of Tylosin Residues In Animal Derived Food

Tylosin is a macrolide antibiotic for livestock and poultry.It is widely used in clinical practice to prevent and treat diseases caused by mycoplasma,gram-positive bacteria and certain gram-negative bacteria in livestock and poultry,which can effectively reduce breeding costs and improve economic efficiency.However,improper use can cause residues in animal tissues,and if animal tissues with excessive residues are consumed,they can cause gastrointestinal discomfort and damage to the cochlear nerve in humans.The current standards for the determination of tylosin residues in animal food in China cover mainly meat,liver,milk and honey,which cannot cover all the matrices required in GB31650-2019,and the detection limits and recovery rates cannot meet the relevant testing technical requirements.In order to meet the needs of food safety supervision and enforcement and testing of animal origin,a liquid chromatography-tandem mass spectrometry method was developed for the determination of tylosin residues in animal origin foods.The method was applied to investigate the status of tylosin residues in foods of animal origin in Henan Province for the first time,to provide a scientific basis for assessing the status of tylosin residues in the province,and to provide technical support for the comprehensive monitoring of tylosin residues,the study is as follows:1、Establishment of a liquid chromatography tandem mass spectrometry method for tylosin residue markers(tylosin A)in food of animal originA liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was developed by investigating the pretreatment conditions and optimizing the instrument conditions.The sample was extracted by acetonitrile,cleaned up by a PCX solid phase extraction column,and quantified by internal standard method using 0.1%formic acid water and acetonitrile as mobile phases.The linearity of the method was good in the concentration range of 1 μg/L to 100 μg/L with the correlation coefficient r greater than 0.99.The limits of detection were 2 μg/kg and the limits of quantification were 5 μg/kg in muscle,liver,kidney,skin+fat and eggs from four different animals: pigs,cattle,chickens and turkeys,and 2 μg/L and 5 μg/L in milk.The average recoveries of tylosin A ranged from 95.1% to 109.8% at concentrations ranging from 5 μg/kg to 600 μg/kg,with intra-and inter-batch coefficients of variation ranging from 1.83% to 13.2%.This method covers all matrices required by GB31650-2019,and has high sensitivity,accuracy and precision,and is suitable for the determination of tylosin A residue in muscle,liver,kidney,skin + fat,milk and egg tissues of four different animals: pig,cattle,chicken and turkey.2、Assessment of the residue status of tylosin in commercially available samples in Henan Province using the established method for tylosin A residuesA total of 485 samples,including 112 chicken,42 beef,121 pork,54 eggs,44 milk,65 chicken liver and 50 pig liver,were collected from supermarkets,farmers’ markets and slaughterhouses in various cities of Henan Province.A total of 13 positive samples were detected using the tylosin residue marker(tylosin A)assay developed in this paper,with a total detection rate of 2.7% and total detection amounts ranging from 3.6 μg/kg to 30.3 μg/kg,all of which did not exceed the MRLs,indicating that the risk of tylosin residues in chicken,beef,pork,chicken liver,pig liver,eggs and milk in various cities of Henan Province is low.It also verified that the established liquid chromatography tandem mass spectrometry detection method could meet the actual sample detection requirements.