Investigation On Residue Analysis Method Of Macrolides Antibiotics And Metabolites In Milk

Objective（1）To establish a method for the determination of 25 macrolides in milk combined optimized Qu ECh ERS（Quick、Easy、Cheap、Effective、Rugged、Safe）method as a sample pretreatment method with ultra-performance lipid chromatography-quadrupole/electrostatic field orbitrap massspectrometry（UHPLC-HESI-Q-Orbitrap-MS）.At the same time,the fragmetion pathway of 25kinds of target were analyzed,in order to detect the trace drug residues quickly and accurately.（2）An analytical method by UHPLC-HESI-Q-Orbitrap-MS was established to detect six macrolide metabolites（Anhydro erythromycin A,erythromycin enol ether,erythromycyl amine,erythromycin A oxime,N-demethyl roxithromycin and N-desmethyl clarithromycin）.（3）A highly sensitive detection and analysis method for the determination of 31 macrolides in milk samples by freezing centrifugation combined with liquid chromatography tandem mass spectrometry had been established to ensure that trace amounts of 31macrolide targets can be detected at a trace concentration and accurately quantificated to ensure food safety.Methods（1）In this experiment,10 m L of a 0.1%（v/v）formic acid acetonitrile solution was added to milk,and 25 macrolide compounds in the sample were extracted.Sodium chloride was added to facilitate protein precipitation in the milk.Anhydrous magnesium sulfate was added for dehydration and delamination,and the adsorbent materials such as N-propylethylenedi amine（PSA）、C18、and sodium acetate were added to purify the sample,and then centrifuged at low temperature.After standing,nitrogen blowing was used.1m L of methanol was reconstituted,filtered through 0.22μm nylon membrane.The 25 macrolides were separated by XBridge-C18 chromatographic column.Electrospray positive and negative ions were monitored simultaneously,and scanning was performed in full-scan（full-ms）and secondary mass spectrometry（dd-ms2）mode.Matrix-matched methods are used for quantification.（2）The six metabolites of macrolides were extracted from milk by 10m L acetonitrile solution.Sodium chloride and anhydrous magnesium sulfate were added to precipitate,dehydrate and separate the proteins in the milk samples.The samples were purified by Qu ECh ERS,SPE and freeze centrifugation procedure respectively.Methanol was redissolved after nitrogen blowing,then filtered by 0.22μm nylon membrane,and separated by XBridge-C18 chromatographic column.The positive and negative ions were detected simultaneously by electrospray full-ms and dd-ms² scanning,matrix matching method quantitative.（3）Six macrolide metabolite and 25 common macrolides were extracted from milk by 0.1%（v/v）formic acid acetonitrile solution.The protein was precipitated and stratified by Na CL and Mg SO₄.Methanol was dissolved after nitrogen blowing,and filtered by 0.22μm nylon membrane,and filtered through a 0.22μm nylon membrane,and selected by XBridge-C18 chromatography column.Multiple reaction mode（MRM）was used for monitoring.Matrix quantitative analysis by matching method.Results（1）The determination of 25 macrolides in milk by UHPLC-HESI-Q-Orbitrap-MS was good in the range of 1～200μg/L（r>0.99）.The recovery can reach80.2%～119.1%.The intra-day precision was 1.2%～14.8%;the inter-day precision was 1.1%～12.9%;the limit of detection was 0.1～0.5μg/kg,and the limit of quantification was 0.3～2.3μg/kg.（2）The determination of 6 compounds in milk by UHPLC-HESI-Q-Orbitrap-MS have a good linearity in a concentration range of 1～200μg/L（r>0.99）,the recovery was up to 84.4%～86.1%,the intra-day precision and the inter-day precision were 1.9%～5.7%and1.1%～9.9%;the limit of detection was 0.1～0.2μg/kg,and the limit of quantification was 0.3～0.7μg/kg.（3）Determination of six macrolide metabolites and 25 macrolides in milk analysed by liquid chromatography-tandem triple quadrupole mass spectrometry have a good linearity in the concentration range of 1～200μg/L（r>0.99）.The recovery can reach 75.6%～117.6%,and the intra-day precision was 1.2%～9.5%,the intra-day precision was 1.1%～9.2%;the detection limit was 0.1～0.5μg/kg,and the limit of quantification was0.3～2.2μg/kg.Conclusions（1）In this study,UHPLC-HESI-Q-Orbitrap-MS was used to monitor positive and negative ions of 25 macrolides in milk.RSM method was used to optimize the best sample purify conditions.After the sample preparation of optimized Qu ECh ERS method,these macrolides had a recovery greater than 70%.The method has short cycle,high resolution,good stability and high accuracy,and was suitable for high-throughput screening and detection of milk samples on the market.（2）This experiment investigated the effects of three different pretreatment methods on the degree of purification,matrix effect,and recovery of six macrolide metabolites in milk samples.UHPLC-Q-Orbitrap mass spectrometry was applied to detect six metabolite residues in milk and screened multiple samples in the market.The trace amount of macrolide antibiotic residues can be detected,which verifies the accurateness and practicability of the method.（3）A liquid chromatography tandem triple quadrupole mass spectrometry method was established for the determination of 31 macrolides compounds.The 31 macrolides compounds were extracted and separated by freeze centrifugation from milk,and then the results were accurate and quantitative.For these macrolides antibiotics and metabolites,the recovery was more than 75%.The method’s sensitivity was high and was suitable for the macrolides residue detection in milk.It was applied to the screening of milk samples in the market.