Studies On The Analytical Method Of Major Proteins And 17 Kinds Of β-Agonists In Milk

Nutrient-rich milk is indispensable to daily life drinks. So, quality and safe of the milk has always been highly concerned. The problem of quality mainly comes from two aspects:one is non-protein nitrogen illegal adding; the other is caused by veterinary drug residues for dairy consumption of veterinary drugs in dietary or medicinal for therapy. Therefore, it is significative to establish methods to determine the content of proteins and veterinary drugs residues in milk.A RP-HPLC method for the quantification of the five major bovine milk proteins (κ-CN, as2-CN, as1-CN,β-CN, Whey) is described. Separation and quantification were achieved by using a reversed-phase analytical column (Agilent Zorbax 300SB-C8,250×4.6 I.D.,5μm) and column temperature was at 45℃. The gradient elution solvents of 0.1% TFA in water and 0.1% TFA in acetonitrile at a flow rate of 0.8 mL/min were used. Photodiode array detector was monitor at 214 nm. A linear relationship(R>0.999) between the concentrations of proteins and peak areas was observed over the concentration range. Method was validated including testing linearing, repeatability, reproducibility and accuracy. Recoveries of six target proteins spiked in milk were from 74.8% to 132.5%.9 kinds of milks of different brands were determined by this method, the results showed that this method is accurate and reliable. The difference of the concentration and relative ration ofκ-CN,αs2-CN, as1-CN,β-CN and whey were found.A liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on a solid phase extraction (SPE) procedure using mixed cation-exchange reversed-phase sorbents, for the simultaneous extraction/preconcentration, separation and detection of 17β2-agonists andβ-blockers in milk was studied and optimized.3% trichloroacetic acid was used as protein precipitation solution, and then centrifuged to lipids under 4℃, 5000rpm, take the upper clear liquid. Extraction and purification were achieved by solid phase extraction cartridges (Waters Oasis MCX), and the elution was dried by nitrogen, then dissolved with mobile phase, and filtered. Separation was achieved by using a reversed-phase analytical column (Waters XBridge TM Shield RP18,50 mm×2.1mm,2.5μm) and the gradient elution solvents of 0.1% formic acid-methanol, and monitored with ESI quadrupole mass spectrometry.Under optimal SPE, chromatographic and mass spectrometry conditions, recoveries of 17 compounds ranged from 61.6% to 134.3% and the RSDs were less than 25%. LOD and LOQ of the method ranged from 0.024 to 3.960μg·kg-1 and from 0.354 to 13.200μg·kg-1, respectively. The established method is simple, rapid, sensitive and specific, and is appropriate for the identification and quantification mutiresidues ofβ-agonists in milk.