Research On Determination Of Macrolide Antibiotics And Their Metabolites In Animal Derived Foods

Objective:(1)The determination of 17 macrolide antibiotics(Josamycin,Troleandomycin,Tylosin,Medemycin,Kitasamycin,Clarithromycin,Roxithromycin,Dirithromycin,Azithromycin,Tilmicosin,Erythromycin A,Spiramycin,Abamectin,ivermectin,Doramectin,Eprinomectin,acetylspiramycin)in bovine milk was performed using QuECHERS followed by a liquid chromatography-tandem mass spectrometry method(LC-MS/MS).(2)The determination of 4 macrolide antibiotics metabolites(Erythromycylamine,Neospiramycin,Anhydroerythromycin A,Erythromycin A ene alcohol ether)in milk power were performed using QuECHERS followed by a liquid chromatography-tandem mass spectrometry method(LC-MS/MS).(3)The determination of 21 macrolide antibiotics and their metabolites(Josamycin,Troleandomycin,Tylosin,Medemycin,Kitasamycin,Clarithromycin,Roxithromycin,Dirithromycin,Azithromycin,Tilmicosin,Erythromycin A,Spiramycin I,Abamectin,ivermectin,Doramectin,Eprinomectin,acetylspiramycin,Erythromycylamine,Neospiramycin I,Anhydroerythromycin A,Erythromycin A ene alcohol ether)in bovine milk and mike powers were performed using QuECHERS followed by a liquid chromatography-quadrupole-time of flight mass spectrometry method(LC-Q-TOF/MS).Methods:(1)The macrolide antibiotics in samples were extracted with acetonitrile.The bovine milk was directly purified by QuECHERS pretreatment method.MgSO4 and sodium acetate anhydrous were added into bovine milk sample for precipitating protein and salting out stratification.Then,MgSO4 and PestiCarb were used for dehydration and removing impurity.The upper layer was evaporated to dryness under a stream of nitrogen and the residue obtained was redissolved in methanol.17 macrolide antibiotics were separated by Eclipse Plus C18 chromatographic column.Electro spray ionization was used to analyze these macrolide antibiotics by using positive ion mode.Multiple reaction monitoring(MRM)was used to monitor ions.Matrix-matched calibration curve was used to quantitative calculation.(2)The metabolite of macrolide antibiotics in samples were extracted with water and acetonitrile.The milk power was directly purified by QuECHERS pretreatment method.MgSO4 and sodium acetate anhydrous were added into milk sample for precipitating protein and salting out stratification.Then,MgSO4 and PestiCarb were used for dehydration and removing impurity.The upper layer was evaporated to dryness under a stream of nitrogen and the residue obtained was redissolved in methanol.The 4 macrolide antibiotics metabolites were separated by Eclipse Plus C18 chromatographic column.Electro spray ionization was used to analyze these macrolide antibiotics by using positive ion mode.Multiple reaction monitoring(MRM)was used to monitor ions.Matrix-matched calibration curve was used to quantitative calculation.(3)The macrolide antibiotics and their metabolites in milk power were extracted with water and acetonitrile,and the macrolide antibiotics and their metabolites in bovine milk were extracted with acetonitrile.The samples were directly purified by QuECHERS pretreatment method.MgSO4 and sodium acetate anhydrous were added into milk sample for precipitating protein and salting out stratification.Then,MgSO4 and PestiCarb were used for dehydration and removing impurity.The upper layer was evaporated to dryness under a stream of nitrogen and the residue obtained was redissolved in methanol.The 21 macrolide antibiotics and their metabolites were separated by Eclipse Plus C18 chromatographic column and detected with positive mass spectrometry ionization mode.The extracted ion chromatogram charts of parent ions were obtained according to accurated mass-to –charge ratio,and according to the chart to establish an matrix-matched calibration curve quantitative method.According to optimizing of collision induced dissociation(CID)energy.Results:(1)17 macrolide antibiotics showed good linear relationship in the range of 2-200 ng/mL,and dirithromycin showed good linear relationship in the range of 5-500 ng/mL.The linear correlation coefficient were all greater than 0.99.At three levels of LOQ,2LOQ,and 4LOQ,the average recoveys were 61.24%-119.38% excluding dirithromycin and tylosin.The intra-day precisions were 1.43%-7.01%,and the inter-day precision were 3.29%-13.28%.The limits of detection(LOD)of 17 macrolide antibiotics were 0.6-1.3?g/kg,and the limits of quantitative(LOQ)were 1.1-3.2?g/kg.(2)4 metabolites of macrolide antibiotics showed good linear relationship in the range of 5-500ng/mL,the linear correlation coefficient were all greater than 0.999.At three levels of LOQ,2LOQ,and 4LOQ,the average recoveys were 71.23%-86.15%,Intra-day precision were 1.97%-5.27%,and the inter-day precision were 3.17%-8.27%.The limits of detection(LOD)of 4 metabolites of macrolide antibiotics were 0.6-1.3?g/kg,and the limits of quantitative(LOQ)were 3.1-4.6 ?g/kg.(3)21 macrolide antibiotics and their metabolites showed good linear relationship in the range of 50-5000 ng/mL,the linear correlation coefficient were all greater than 0.999.At three levels of LOQ,2LOQ,and 4LOQ the average recoveys were 51.17%-121.11%,Intra-day precision were 2.37%-6.98%,and inter-day precision were 3.82%-9.21%.The limits of detection(LOD)of 21 macrolide antibiotics and their metabolites were 6.57-12.36?g/kg,and the limits of quantitative(LOQ)were 30.27-50.45?g/kg.Conclusion(s):(1)The analytical method of 21 macrolide antibiotics in animal derived foods using Quick,Easy,Cheap,Effective,Rugged,and Safe extraction(QuEChERS)and high performance liquid chromatography tandem mass spectrometry was simple,rapid,sensitive and specific.The analytical method was suitable to quantitate macrolide antibiotics and their metabolites in bovine milk and milk power.(2)The screening method of 21 macrolide antibiotics in animal derived foods using Quick,Easy,Cheap,Effective,Rugged,and Safe extraction(QuEChERS)and a liquid chromatography-quadrupole-time of flight mass spectrometry method were high resolution and could effectively avoid false positive result,and suitable to confirmation for macrolide antibiotics and their metabolites in bovine milk and milk powe,and provide technical guarantee for food safety monitoring.