| Azithromycin,an antibiotic of the erythromycin group with a 15-membered azalactone ringhas been approved for the treatment of respiratory,skin,otitis media,and sexually transmitted bacterial infections.At present,azithromycin has been classified as banned drugs for the production of livestock bythe Ministry of Agriculture,because it will produce drug resistance problems,affecting animal disease control,food safety and human health.Although azithromycin is banned by our country,but driven by economic benefits of farming,antibiotics are still being abused in livestock,has become a new hidden threat to safety of livestock products.However,there is no standard for the detection of AZM in any matrix at home and abroad.Although domestic and foreign scholarshave carried out the research of AZM detection technology in some matrix,but the detection technology of AZM in feed,eggs and other matrix has not been reported.Therefore,in this experiment,we take theformula feed and animal edible tissue on as the object ofthe researchto establish an accurate and sensitive AZM liquid chromatography tandem mass spectrometry detection technology of animal feed and edible tissues.Then thisdetection method was used to monitor the AZM residues in formula feed and animal edible tissuesof Sichuan.And the technology canprovide the basic data and reliable means for establishing standards ofdetect AZM in feed and animal edible tissue,increasing supervision of AZM,speeding up the realization of non-anti-breeding,so that ensuring food safety.The main research contents and results are as follows:Exp.l Study on the detection of AZM in Pig and chicken formulafeed by LC-MS/MS and risk monitoring A method was developed for the determination of azithromycin in formula feed by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The results are presented as follows:(1)The sample was extracted with acetonitrile under ultrasonic assistance.Oasis MCX solid-phase extraction column were used for purification,and the chromatographic separation was performed on a Hypersil BDS C18 column(2.4 mm,100×2.1 mm)with isocratic elution using 0.05 mol/L ammonium acetate-methanol(1:9)as mobile phase.The detection of azithromycin was performed with positive ion under multiple reaction monitoring(MRM)mode.The internal standard isotope dilution method was used for quantification.(2)A good linearity(r2=0.9991)was obtained for azithromycin in the range of 1-250 ng/mL.The limit of detection(LOD,S/N>3)of azithromycin for laying hens formula feed and fattening pigs formula feed were 2.2 μg/kg and 2.8 μg/kg,respectively.The limit of quantitation(LOQ,S/N≥10)of azithromycin for laying hens formula feed and fattening pigs formula feed were 8.4 μg/kg and 8.4μg/kg,respectively.Average recoveries ranged from 84.86%-102.30%for laying hens formula feed,at spike levels of 0.5,1,5,10 mg/kg.The intra-RSD for azithromycin were 0.88%-4.01%and inter-RSD were 3.24%-6.32%.Average recoveries ranged from 87.0%-106.6%for fattening pigs formula feed,at spike levels of 0.5,1,5,10 mg/kg.The intra-RSD for azithromycin were 0.58%-5.4%and inter-RSD were 3.1%-5.4%.The method is accurate and sensitive,and the method confirmed the absence of the matrix effects.The method can be applied in the determination of azithromycin in the formula feed.(3)Thisl method was applied to the determination of AZM in 36 fattening pigs formula feed and 18 laying hens formula feed in Sichuan.The results showed that AZM were not detected in 36 fatteningpigsfomula feed and 18 laying hens formula feed.It is inferred that the possibility of illegal use of AZM through the lfeed path is small,but does not rule out a small range of mixing.Exp.2 Study on the detection of AZM in pig and chicken edible tissue by LC-MS/MS and risk monitoring A method was developed for the determination of azithromycin in pork,pork liver,chicken,and egg by liquid chromatography-tandem mass spectrometry.The results are presented as follows:(1)The samples were extracted with ether under ultrasonic assistance.Oasis MCX solid-phase extraction column were used for purification,and the chromatographic separation was performed on a Hypersil BDS C18 column(2.4 mm,100×2.1 mm)with isocratic elution using 0.05 mol/L ammonium acetate-methanol(1:9)as mobile phase.The detection of azithromycin was performed with positive ion under multiple reaction monitoring(MRM)mode.The internal standard isotope dilution method was used for quantification.(2)A good linearity(r2 greater than 0.9991)was obtained for azithromycin in pork,pork liver,chicken and egg at the range of 1-150 μg/mL and 1-200μg/L,respectively.The limit of detection(LOD,S/N>3)of azithromycin for pork,pork liver,chicken,andegg were 0.24μg/kg.0.21 μg/kg、0.20μg/kg and 0.28μg/kg,respectively.The limit of quantitation(LOQ,S/N≥10)of azithromycin for pork,pork liver,chicken,andegg were 0.80μg/kg.0.69μg/kg、0.53μg/kg and 0.84μg/kg,respectively.Average recoveries ranged from 97.47%-105.90%、92.75%-104.73%and 104.03%-113.09%for pork,pork liver,chicken,at spike levels of 0.1mg/kg、0.2mg/kg、0.3mg/kg.The intra-RSD for azithromycin were 1.81%-3.99%、2.24%-4.05%、1.62%-3.20 and inter-RSD were 0.90%-4.38%、3.60%-4.80%、2.54%-2.60%,respectively.Average recoveries ranged from 100.32%-109.01%for egg,at spike levels of 0.075mg/kg、0.15mg/kg、0.225mg/kg.The intra-RSD for azithromycin were 0.56%-2.26%and inter-RSD were 3.56%-4.49%.The method is accurate and sensitive,and the method confirmed the absence of the matrix effects.The method can be applied in the determination of azithromycin in the pork,pork liver,chicken,and egg.Meanwhile it was used to analyze residual AZM in products of livestock and poultry in Sichuan.(3)AZM was not observed in all tested samples of pork,but the positive rate of AZM residues in pig liver,chicken and egg were 3.45%、7.69%、1.96%,respectively,and the maximum residue were up to 12.64 μg/kg、27.85 μg/kg、139.70 μg/kg,respectively.The results indicate the presence of AZM in livestock and poultry breeding,but the possibility route through the feed is slim,so the other possible way may be intramuscular injection and drinking or feeding in a small range.AZM metabolizing in animals can produce a variety of metabolites,some of which have pharmacological activity.No AZM prototypes detecting does not indicate that AZM is not used in the breeding process.Therefore,it is necessary to increase the number and coverage of samples,expand the sampling route,determine the metabolites of AZM,and through the detection of AZM and its metabolites to monitor the use of AZM,so as to more comprehensive evaluation of the safety of feed.In summary,the results of the study showed as follows:A method was developed for the determination of azithromycin in formula feed and pork,pork liver,chicken,and egg by LC-MS/MS.The method is accurate and sensitive,and the method confirmed the absence of the matrix effects.This method was applied to the determination of AZM in fattening pigs formula feed(36),laying hens formula feed(18)and pork(87),pork liver(37),chicken(52),and egg(59)in Sichuan.The results showed that AZM were not detected in 36 fattening pigs formula feed and 18 laying hens formula feed.AZM was not observed in all tested samples of,but the positive rate of AZM residues in pig liver,chicken and egg were 3.45%、7.69%、1.96%,respectively,and the maximum residue were up to 12.64 μg/kg、27.85μg/kg、139.70 μg/kg,respectively.The results indicate the presence of AZM in livestock and poultry breeding. |
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