Bonfatti Valentina, Grigoletto Luca, Cecchinato Alessio, Gallo Luigi, Carnier Paolo
Department of Animal Science, University of Padova, Viale dell'Università 16, 35020 Legnaro, Padova, Italy.
J Chromatogr A. 2008 Jun 27;1195(1-2):101-6. doi: 10.1016/j.chroma.2008.04.075. Epub 2008 May 7.
A new RP-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins is described. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5 microm, 300A, 150 x 4.6 I.D.) was used. All the most common casein (CN) and whey protein genetic variants, including beta-CN(I) were detected and separated simultaneously in less then 40 min, with the exception of alpha(S1)-CN(B) and CN(C) variants. Purified protein genetic variants were employed in calibration and showed different absorbances at 214 nm. The procedure was developed using 40 raw individual milk samples of cows belonging to four different breeds and certified skim milk powder BCR-063R. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship (R(2)>0.99) between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.92 to 4.32% within analytical day and from 0.85 to 9.52% across analytical days. The recoveries, calculated using mixtures of samples previously quantified, ranged from 98.1 to 103.7%.
本文描述了一种用于分离和定量牛乳蛋白最常见遗传变体的新型反相高效液相色谱（RP-HPLC）方法。使用了反相分析柱C8（Zorbax 300SB-C8 RP，3.5微米，300A，150×4.6内径）。除α(S1)-酪蛋白(B)和酪蛋白(C)变体外，所有最常见的酪蛋白（CN）和乳清蛋白遗传变体，包括β-酪蛋白(I)，均在40分钟内同时检测和分离。纯化的蛋白遗传变体用于校准，并在214nm处显示出不同的吸光度。该方法使用来自四个不同品种奶牛的40份生鲜个体乳样和认证脱脂奶粉BCR-063R开发。方法验证包括测试线性、重复性、再现性和准确性。在浓度范围内观察到蛋白质浓度与峰面积之间呈线性关系（R(2)>0.99），检测限较低。保留时间和峰面积的重复性和再现性均令人满意。峰面积的相对标准偏差（RSD）在分析日内为0.92%至4.32%，在不同分析日之间为0.85%至9.52%。使用先前定量的样品混合物计算的回收率为98.1%至103.7%。
