The Establishment And Application Of Ultra Performance Liquid Chromatography-tandem Mass Spectrometry Methods For Determination Of Eleven Aminoglycosides In Aquatic Products And Fishery Feed

Aminoglycosides?AGs?is a kind of broad-spectrum bactericidal antibiotic,which can promote the growth development of animals and prevent animal diseases,and is widely used in the breeding industry.However,excess AGs drugs have toxic effects such as ototoxicity and nephrotoxicity.Hence,it is very important to accurately detect the residual of AGs in food.At present,the measurement of AGs was mainly field in honey,milk,animal innards and muscles,etc.The research on aquatic products is scarce,the determined species is rare,and sensitivity of the method is low and lack of universality.Therefore the development of an efficient multiresidue detection method of AGs is imperative.Meanwhile,AGs should also be supervised in fishery feed to overall control the drug abuse.The establishment of AGs determination method in fishery feed is of great significance.The detection methods of AGs mainly include microbiological method,immunoassay and instrumental analysis.Among them,LC-MS/MS technique plays an important role due to its strong anti-background interference ability and high sensitivity.In this study,an ultra performance liquid chromatography-tandem mass spectrometry?UPLC-MS/MS?method was developed for the determination of eleven AGs,including paromomycin,spectinomycin,tobramycin,gentamycin,kanamycin,hygromycin B,apramycin,streptomycin,dihydrostreptomycin,amikacin and neomycin in aquatic products and fish feed.This method was also applied for the detection of AGs content in actual aquaculture products and real fishery feed.The main research results was as follows:?1?Establish the detection method of AGs in aquatic products.Through comparing different chromatographic columns,adjusting the composition of mobile phase,optimization of chromatography and mass spectrum parameters,the optimum detection conditions were as follows:1%formic acid aqueous solution?including 2 mM ammonium acetate?-1%formic acid acetonitrile?including 2 mM ammonium acetate?as mobile phase gradient elution,the flow rate of 0.3 mL/min,and 20?L injection volume.Parallel reaction detection mode?PRM?and external standard method was adopted for quantitation.The limit of detection?LOD?,limit of quantitation?LOQ?,linear range,recovery and precision of the method are determined.The LOD?S/N?3?was ranged from 1.0?g/kg to 10.0?g/kg,and the LOQ?S/N?10?was ranged from 2.0?g/kg to 20.0?g/kg.It showed a good linearity relationship in the AGs concentration range of 0.5⁵00?g/L with the correlation coefficient R²>0.991.Besides,the average recoveries presented70.0%¹20.0%with the intra-batch precision less than 20%,and the inter-batch precision less than 10%.?2?The detection method of AGs in fishery feed was established.The samples were extracted by 10%trichloroacetic acid water solution?including 2 mM EDTA?,and C₁₈ solid phase extraction column was used for purification.The LOD?S/N?3?is 10⁵0?g/kg,and the LOQ?S/N?10?is 20¹00?g/kg.It performed good relationship of the eleven AGs in the range of 2.0¹000?g/L?R²>0.994?,and the recovery rate is 70.0%¹15.0%.The intra-batch precision is less than 16%,and the inter-batch precision is less than 10%.?3?The AGs residue in aquatic products and fishery feed was investigated using UPLC-MS/MS methods established above.These aquatic products were obtained from aquaculture base located in Zhuhai,Sihui,Zhaoqing city,Guangdong province,and the fishery feed was sourced from four provinces of Jiangsu,Shanghai,Hainan,Zhejiang.The result showed that only 3 samples of 31 aquatic samples were detected with neomycin and dihydrostreptomycin,and 4 samples of 30 fishery feed samples were detected with gentamicin and streptomycin.And the AGs contents in positive samples were lower than the limit of quantitation,and the remaining samples were not detected.An UPLC-MS/MS method was developed for the determination of eleven AGs in aquatic products and fishery feed in this study through optimizing the liquid chromatography and mass condition,improving the pretreatment method.Compared with present detection methods,UPLC-MS/MS method established in this paper has the characteristics of strong anti-interference ability,and more accurate in qualitative and quantitative analysis.And this method is suitable for simultaneously determination of 11 AGs with high sensitivity in aquatic products and fishery feed.Hence,this study could provide data support and scientific evidence for setting standards of AGs in aquatic products and fishery feed,and provides relevant technical support for the government to supervise the abuse of antibiotics.