摘要
利用酸水解释放出的糖基化产物中氨基葡萄糖,采用Elite C18色谱柱分离,以68%的0.025 mol·L^(-1) pH 3.6乙酸-乙酸钠缓冲溶液和32%甲醇作为流动相等度洗脱,流速为1 m L·min^(-1),进样体积10μL,柱温35℃。定量分析结果表明,氨基葡萄糖0.1~100μg·m L^(-1)与氨基葡萄糖峰面积线性关系良好(R2=0.9998);以0.3、0.5、1.0 mg 3个添加水平作回收试验,氨基葡萄糖平均回收率为88.05%~110.79%,相对标准偏差为2.45%~5.92%;氨基葡萄糖检出限为0.02μg·m L^(-1)。糖基化乳清蛋白中氨基葡萄糖含量为2.03 mg·g^(-1)。RP-HPLC测定法用于乳清蛋白-氨基葡萄糖糖基化产物制备条件优化,方法简单、重复性好、灵敏度高,可用于蛋白质-氨基葡萄糖糖基化产物中氨基葡萄糖含量分析测定。
Glucosamine in glycosylated whey protein was released hydrochloric acid hydrolysis. The RP-HPLC separation was performed on an Elite C18 （Hypersil ODS25 μm 4.6 mm ×250 mm） utilizing an isocratic elution of acetic acid sodium acetate buffer solution （containing 68% 0.025 mol. L1 pH 3.6） and methyl alcohol （containing 32%） as the mobile phase at a flow rated of 1 mL. min-1. Injiction volume and column temperature were set at 10 IJL and 35 ℃. The results indicate that the linear ranage was from 0.1 to 100 μg·mL^-1 for glucosamine and correlation coefficient （R2 was greater than 0.99）. The average recoveries spiked at the three concentration levels of 0.30, 0.50, 1.00 mg ranaged between 88.05% and 110.79% with the relative standard deviations from 1.22% to 5.92%. The limit of detection was 0.02 pg. mLL Content of glucosamine in glycosylated whey protein was 2.03 mg·g^-1. Therefore, this method had the characteristics of simple operation, high reproducibility and sensitivity, It could be widely applied to determine glucosamine in glycosylated protein.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2017年第5期58-64,共7页
Journal of Northeast Agricultural University
基金
国家自然科学基金(31201453)
关键词
反相高效液相色谱
乳清蛋白
氨基葡萄糖
reversed-phase high-performance liquid chromatography （RP-HPLC）
whey protein
glucosamine
