Study On The Determination Method Of Neomycin Residues In Edible Bovine Tissues And Milk By HPLC-MS/MS

Neomycin is a aminoglycoside antibiotic produced by Streptomyces fischeri,which mainly acts on gram-negative bacteria and some gram-positive bacteria.Neomycin has the characteristics of strong antibacterial activity,wide range of action,stable physical and chemical properties,and is not easily absorbed by oral administration.It has ototoxicity and nephrotoxicity,and is easy to cause kidney damage and irreversible hearing damage after long-term use.Veterinary clinic is often based on soluble powder and premix.Human medicine is mainly used for the treatment of bacterial infection,suppurative folliculitis and chronic eczema;veterinary medicine is mainly used for the treatment of animal bacterial gastroenteritis and urinary system infection caused by sensitive bacteria,as well as ear bacterial infection and cow mastitis.The use of neomycin in food animals will inevitably lead to neomycin residues in animal edible tissues.At present,the detection methods of neomycin residues in animal edible tissues,milk and eggs include microbiological method,immunoassay,instrumental analysis and so on,but there is no method for the detection of neomycin residues in bovine edible tissues and milk.The purpose of this study is developing an efficient,convenient,sensitive and specific HPLC-MS/MS method for the determination of neomycin residues in bovine edible tissues and milk,and to provide necessary technical support for the monitoring of neomycin residues in bovine edible tissues and milk,which has important practical significance for ensuring the safety of food consumption and protecting the import and export trade of animal-derived food in China.A HPLC/MS-MS method for determination of neomycin residues in edible bovine tissues and milk was established.Beef muscle,liver,kidney,fat,milk and other edible tissues were extracted with phosphate buffer solution,protein precipitation,purification and elution by PCX cationic SPE column,nitrogen blow-dried,redissolved with 1%sevoflubutanic acid solution,and then detected by HPLC-MS/MS.The samples to be tested were separated and analyzed on a BEH-C8 column with gradient elution using 0.1%formic acid water（containing 2 mM ammonium acetate）and 0.1%formic acid acetonitrile as mobile phases.The elution procedure was as follows:0.1%formic acid acetonitrile 0.0～1.0 min 2%,1.0～1.1 min 8%,1.1～3.0 min 40%,3～3.5 min 90%,3.5～4.5 min 90%,4.5～5 min 2%,5～6 min 2%.Mass spectrometry conditions:electrospray ionization（ESI）,positive ion scanning mode,multiple reaction monitoring（MRM）,external standard quantification.The results showed that when neomycin was added in the range of 20～1000 ng/g,there was a good linear relationship between the concentration of neomycin in tissues and milk and the target peak area,with the correlation coefficients greater than 0.999.The minimum detection limit of this method was 10μg/kg,and the minimum quantification limit was 20 μg/kg.The average recoveries of neomycin were 80.85%～104.19%at 20,250,500 and 1000 μg/kg.The average recoveries of bovine fat at 20、250、500 and 1000 μg/kg were 60.18%～92.86%.The average recoveries of bovine liver at 20,2750,5500 and 11000 μg/kg were 60.52%to 81.20%.The average recoveries of bovine kidney at 20,4500,9000,18000 μg/kg were 75.85%～96.57%.The average recoveries of milk in the range of 20,750,1500 and 3000 μg/kg were 62.22%～86.72%.The intra-batch coefficient of variation（RSD）was 1.29%to 10.43%,and the inter-batch coefficient of variation（RSD）was 3.33%to 8.58%.The HPLC-MS/MS method for the determination of neomycin residues established in this study can meet all the requirements for the determination of edible bovine tissues and milk.