Research On Methodology For The Confirmatory Analysis Of Two Macrolides And Their Metabolites And Four Aminoglycosides Residues In Livestock And Poultry Tissues

Currently,macrolide and aminoglycoside antibiotics are widely used in livestock production.However,due to the abuse,misuse,and non-compliance with the withdrawal time of these two types of veterinary drugs in the process of raising livestock and poultry,resulting in excessive residues of veterinary drugs in animal-derived foods,affecting the health of consumers.Therefore,it is of great significance to develop a rapid confirmatory detection technology for veterinary drug residues in animal-derived foods.A novel non-targeted screening method based on in vitro incubation combined with liquid chromatographyquadrupole-time-of-flight mass spectrometry was established to confirm the analysis of Erythromycin and Clarithromycin and their metabolites in chicken liver microsomes in this study.Meanwhile,a developed and improved QuEChERS and SPE sample preparation technique combined with liquid chromatography-tandem mass spectrometry method was used to determine two macrolides and their metabolites and four aminoglycosides residues in livestock and poultry edible tissues.The purpose of this study is to provide a scientific basis for the development of monitoring and testing standards for macrolide and aminoglycoside antibiotic residues in animal-derived foods.The main research contents and results are as follows:(1)The first confirmatory analysis of Erythromycin(ERY)and Clarithromycin(CLA)and their metabolites in chicken liver microsomes was petformed by in vitro incubation combined with liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-Q-TOF-MS).The incubation system(100 μL)contained 65 μL of potassium phosphate buffer(0.1 M,pH 7.4),20 μL of chicken liver microsomes(5 mg/mL),5 μL of a compound(20 μM)and 10μL of NADPH(10 mM).After a 5 min preincubation period at 37 ℃,the reaction was started by introducing 10 μL of NADPH(10 mM).After 60 min of incubation,the reaction was quenched with 100 μL of ice-cold ACN with IS(1 μg/mL;CLA and ERY for ERY and CLA,respectively).The samples were detected by LC-Q-TOF-MS after ultrasonication,vortexing and centrifugation.The result was found that Erythromycin A can produce N-desmethylerythromycin A in chicken liver microsomes,but CLA cannot produce N-desmethylclarithromycin.The characteristic fragment ion information of Erythromycin,Clarithromycin and N-desmethyl-erythromycin A was obtained by LC-Q-TOF-MS,and the mass spectral information of the target compounds was provided to the high-resolution mass spectrometry veterinary drug accurate mass database,which provides a technical support for the development of non-targeted screening methods for the detection of Erythromycin,Clarithromycin and N-desmethyl-erythromycin A residues in animal-derived foods.Compared with low-resolution mass spectrometry,LC-Q-TOF-MS detection technique has stronger qualitative confirmation ability.This study confirmed for the first time that Erythromycin can generate the major metabolite N-desmethyl-erythromycin A in chicken liver microsomes,providing a scientific basis for subsequent studies on the monitoring of macrolides and their metabolite residues in animal-derived foods.(2)An improved QuEChERS combined with ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)confirmatory analytical method was developed for the determination of ERY,CLA and N-D-ERY residues in egg and chicken tissues.The samples were extracted with acetonitrile-water(80:20,v/v),cleaned up with Cleanert MAS-Q cartridge,and analyzed by UHPLC-MS/MS.The mobile phase used in the gradient of elution was composed of 0.1%formic acid in water and 0.1%formic acid in acetonitrile,and ERY,CLA and N-D-ERY were separated on a Waters ACQUITY UPLC BEH C18 column(2.1 mm x 50 mm,1.7 μm)at 0.4 mL/min.In this study,Roxithromycin was used as the internal standard.In the concentration range of 0.2-30 ng/mL,the linear relationship was good,and the correlation coefficients(R2)were all greater than 0.9961.At the three spiked concentration levels of 2,20 and 200 μg/kg,the average recoveries were 87.78%-104.22%,the relative standard deviations(RSDs)did not exceed 6.83%,and the intra-day RSDs and inter-day RSDs were all less than 7.10%,indicating that the method has good repeatability and reproducibility.The limits of detection(LODs)and limits of quantification(LOQs)of ERY,CLA and ND-ERY in eggs and chicken tissues were 0.5 μg/kg and 2 μg/kg.respectively.The method combines the QuEChERS technique with UHPLC-MS/MS to achieve accurate qualitative and quantitative determination of ERY,CLA and N-D-ERY residues in eggs and chicken tissues with only one pretreatment and detection.(3)An improved QuEChERS combined with ultra-high performance liquid chromatography-tandem quadrupole/orbitrap mass spectrometry(UHPLCQ/Orbitrap-MS)confirmatory analytical method was developed for the determination of ERY,CLA and N-D-ERY residues in pork,bovine and sheep muscle.The samples were extracted with acetonitrile-water(80:20,v/v),cleaned up with Cleanert MAS-Q cartridge,and analyzed by UHPLC-Q/Orbitrap-MS.The detection conditions of liquid chromatography and mass spectrometry were optimized to obtain good separation and sensitivity.In this study,Roxithromycin was used as the internal standard.In the concentration range of 0.2-30 ng/mL,the linear relationship was good,and the correlation coefficients(R2)were all greater than 0.9983.At the three spiked concentration levels of 2,20 and 200 μg/kg,the average recoveries were 88.96%-101.76%,the RSD did not exceed 5.95%,and the intraday RSDs and inter-day RSDs were 2.64%-6.65%and 3.08%-6.38%,respectively.The LOD and LOQ were 0.4μg/kg and 2 μg/kg,respectively,and the method can meet the requirements of veterinary drug residue detection.The QuEChERS-UHPLC-Q/Orbitrap-MS method shortens the overall sample pretreatment time and detection time,and allows batch processing of samples,which greatly improves the work efficiency,sensitivity,repeatability and reproducibility.(4)An improved SPE sample preparation technique combined with UHPLC-MS/MS confirmatory analytical method was established for the determination of Gentamicin(GEN),Netilmicin(NET),Sisomicin(SIS)and Kanamycin(KAN)residues in livestock and poultry edible tissues.The samples were extracted with 5%trichloroacetic acid solution(containing 0.4 mM EDTA and 10 mM ammonium acetate),purified by an Oasis PRiME HLB cartridge(6 mL/200 mg),eluted with formic acid-acetonitrile-2 mM ammonium acetate in water(10:5:85,v/v/v),and analyzed by UHPLC-MS/MS.The chromatographic separation was performed at 35℃and 0.3 mL/min on a SiELC Obelisc R column(2.1 mm×50 mm,100(?),5 μm).The mobile phase used in the elution gradient was composed of 1%formic acid in water(containing 2 mM ammonium acetate)and 90%acetonitrile in water.In the concentration range of 10-1000 ng/mL,the linear relationship was good,and the correlation coefficients(R2)were all greater than 0.9972.At the three spiked concentration levels of 20,100 and 200 μg/kg,the average recoveries were 78.90%-105.52%,the intra-day RSDs and inter-day RSDs were all less than 8.31%,indicating that the method has good repeatability and reproducibility.The LODs and LOQs of GEN,NET,SIS and KAN in livestock and poultry edible tissues were 5 μg/kg and 10 μg/kg,respectively.This method combines an improved SPE technique with UHPLCMS/MS to achieve accurate qualitative and quantitative analysis of GEN,NET,SIS and KAN residues in eggs,chicken tissues and porcine,bovine and sheep muscle with only one pretreatment and detection.In conclusion,this study first established a LC-Q-TOF-MS method to confirm the analysis of ERY and CLA and their metabolites in chicken liver microsomes,and it was confirmed that ERY can produce the major metabolite N-D-ERY in chicken liver microsomes.In addition,this study established QuEChERS-UHPLC-MS/MS,QuEChERS-UHPLCQ/Orbitrap-MS and SPE-UHPLC-MS/MS confirmatory analytical methods for accurate qualitative and quantitative determination of two macrolides and their metabolites and four aminoglycosides residues in livestock and poultry edible tissues by optimizing the sample pretreatment techniques and chromatographic and mass spectrometric conditions,and the method performance indicators meet the requirements of veterinary drug residue detection.The above three confirmatory analytical methods are rapid,simple and sensitive,which can be applied to the routine monitoring of macrolides and their metabolites and aminoglycosides residues in livestock and poultry edible tissues.