高效液相色谱法分离测定牦牛乳中的8种蛋白质

Separation and Quantification of Major Protein in Yak Milk by High Performance Liquid Chromatographic Method

建立了同时分离和测定牦牛乳中4种酪蛋白和4种乳清蛋白的反相高效液相色谱方法。脱脂牦牛乳经分散剂处理后,采用C4色谱柱(250 mm×4.6 mm,300,5μm i.d.)进行分离,以0.1%的三氟乙酸水溶液和0.1%的三氟乙酸乙腈溶液为流动相,流速为0.8 mL/min,梯度洗脱,二极管阵列检测器(DAD)在220nm波长下检测,外标法定量。结果表明,牦牛乳中8种主要蛋白质在40 min内完全分离,在各自的线性范围内呈良好线性,除α-乳白蛋白外,其余7种蛋白的相关系数均大于0.99。8种蛋白质的回收率为86%～103%,相对标准偏差(RSDs)为1.7%～8.7%;检出限(LODs)为10.7～39.2 mg/L,定量下限(LOQs)为35.7～130.7 mg/L。该方法的准确度和精密度均较高,能够满足实际检测的要求。

A precise,sensitive and low cost method was developed for the determination of four casein（CN）,e.g.αs1-CN,αs2-CN,β-CN and κ-CN,and four whey protein,e.g.α-lactalbumin（α-La）,β-lactoglobulin A（β-Lg A）,β-lactoglobulin B（β-Lg B） and serum albumin（SA） by reversed phase high performance liquid chromatography.Skimmed yak milk was diluted with denaturing solution,and separated on a Jupiter C4 column（250 mm×4.6 mm,300,5 μm i.d.） at 20 ℃.A gradient programme was carried out at a flow rate of 0.8 mL/min using 0.1% trifluoroacetic acid（TFA） water solution and 0.1% TFA acetonitrile solution as eluent.The detection wavelength was set at 220 nm.The results showed that eight protein fractions could be completely separated within 40 min.The calibration curves were linear in the ranges of 0.56-5.6 g/L for αs1-CN,0.14-1.4 g/L for αs2-CN,1.35-13.5 g/L for β-CN,0.35-3.5 g/L for κ-CN,0.17-1.7 g/L for α-La,0.036-0.36 g/L for β-Lg A and β-Lg B,and 0.98-9.8 g/L for SA,with correlation coefficients more than 0.99 except for α-La.The spiked recoveries of eight protein fractions in yak milk sample were in the range of 86%-103% with RSDs of 1.7%-8.7%.The limits of detection（LODs） of eight protein fractions were in the range of 10.7-39.2 mg/L,and the limits of quantitation（LOQs）were 35.7-130.7 mg/L.The developed method was simple and accurate,and could meet the demands for actual application.