Study On The Casein Identification And Detection Technology Of Residual Veterinary Drugs In Buffalo Milk

Buffalo（Bubalus bubalis）is an important livestock species adapted to the southern climate and valuable for economic exploitation.Buffalo milk is known as the"king of milk",because of its rich nutrition and high added value of the product.The buffalo milk industry has become an important composition of the dairy industry in the south of China.However,veterinary drug abuse during buffalo farming has led to drug residues in buffalo milk,posing a threat to human health.On the other hand,adulteration of buffalo milk sources has became a common issues in the society.Therefore,the detection of drug residues and milk components in buffalo milk is a critical aspect to ensure food safety of animal origin.There is an urgent need to develop sensitive and effective analytical methods for the veterinary drug residues detection and casein identification in buffalo milk.Based on this purpose,this study was conducted to investigate and apply veterinary drug residues detection and casein identification by Liquid Chromography tandem mass spectrometry（LC-MS/MS）approach.This study provides new tools for food safety testing and traceability analysis of buffalo milk products,provides a theoretical basis for establishing industrial inspection standards,and contributes to the positive development of the dairy buffalo industry.The main contents of this study were as following:（i）Study of LC-MS/MS method for the detection of multiple veteranary drug residues in buffalo milk.Select reaction monitor（SRM）method for 8 common veterinary drugs,including metronidazole,salbutamol,atropine,rimethoprim,hydrocortisone,kitasamycin,roxithromycin and tylosin by LC-MS/MS.Methodological optimization was carried out for linearity,detection limit,accuracy,precision,recovery,and other indexs specified in the bioanalytical method guidelines of Food and Drug Administration（FDA）.Meanwhile,the sample processing methods,chromatographic conditions,and mass spectrometric methods were compared.The results showed that the precision and accuracy of each analyte ranged from 1.81%～12.35%and 1.25%～14.57%,respectively.Good linearities were obtained with the correlation coefficient（r²）were ranged from 0.9995 to 0.9999,and the detection limits ranged from 0.1 to 10μg·L^-1.The average extraction recoveries ranged from 93.59%to 114.57%and the average matrix effect ranged from 87.12%to 103.76%.Different sample pretreatment methods and data collection methods were established,and all eight analytes exhibited stability in buffalo milk.The technical method has low detection limits,high sensitivity and good reproducibility,and can provide accurate qualitative and quantitative analysis of 8 drugs residues within 20 min.Moreover,the technical system was successfully used to detect drug residues in raw milk and dairy products.This study laid the foundation for the establishment of industrial inspection standards for drug residues detection in buffalo milk.（ii）Study on casein（CN）isolation and peptide fingerprinting of buffalo milk.The casein monomers of buffalo milk were separated and identified by high performance liquid chromatography（HPLC）and mass spectrometry,and the casein peptide fingerprinting of buffalo and cow were comparative analyzed.The results showed that casein could be effectively extracted by chemical precipitation with acetic acid-sodium acetate buffer at p H 4.8.HPLC was performed using acetonitrile（0.1%TFA）and water（0.1%TFA）as mobile phases and a C18 ODS-AP column（300A 5μm 4.6×250 mm）,which could effectively separate buffalo milkα_S1,α_S2,β,κ-CN.After digestion of the casein,16 common peptides of casein were detected between buffalo milk and cow milk and 17 characteristic peptides of buffalo milk casein were detected using matrix-assisted laser desorpt-ion time-of-flight mass spectrometry（MALDI-TOF-MS）.Further identification of casein characteristic peptides by LC-MS/MS allowed for 8 characteristic peptides from buffalo milk casein.These characteristic peptides could be used as peptide markers for the casein traceability of buffalo milk.The method could effectively separate multiple casein monomers and establish an approach for rapid identification and quality inspection of buffalo milk.The characteristic peptides can provide a highly sensitive method for trace analysis of buffalo milk,common milk and its mixtures with mass spectrometry.In summary,a method was successfully established that can be used for the simultaneous detection of multiple veterinary drug residues in buffalo milk.An isolation system of casein for buffalo milk was established.Characteristic peptides of casein in buffalo milk were screened for adulteration analysis of buffalo milk and cow milk.