RP-HPLC测定乳清蛋白糖基化产物中的氨基葡萄糖
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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 ODS25μm 4.6 mm×250 mm)utilizing an isocratic elution of acetic acid sodium acetate buffer solution(containing 68%0.025 mol·L-1p H 3.6)and methyl alcohol(containing 32%)as the mobile phase at a flow rated of 1 m L·min-1.Injiction volume and column temperature were set at 10μL and 35℃.The results indicate that the linear ranage was from 0.1 to 100μg·m L-1for glucosamine and correlation coefficient(R2was greater than 0.99).The average recoveries spiked at the three concentration levels of 0.30,0.50,1.00 mg ranaged between88.05%and 110.79%with the relative standard deviations from 1.22%to 5.92%.The limit of detection was 0.02μg·m L-1.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.
Glucosamine in glycosylated whey protein was released hydrochloric acid hydrolysis.The RP-HPLC separation was performed on an Elite C18(Hypersil ODS25μm 4.6 mm×250 mm)utilizing an isocratic elution of acetic acid sodium acetate buffer solution(containing 68%0.025 mol·L-1p H 3.6)and methyl alcohol(containing 32%)as the mobile phase at a flow rated of 1 m L·min-1.Injiction volume and column temperature were set at 10μL and 35℃.The results indicate that the linear ranage was from 0.1 to 100μg·m L-1for glucosamine and correlation coefficient(R2was greater than 0.99).The average recoveries spiked at the three concentration levels of 0.30,0.50,1.00 mg ranaged between88.05%and 110.79%with the relative standard deviations from 1.22%to 5.92%.The limit of detection was 0.02μg·m L-1.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.
引文
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[20]杨岚,李铜铃,黄婷,等.RP-HPLC测定盐酸氨基葡萄糖片的含量[J].华西药学杂志,2005,20(3):259-260.
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[23]刘玥,刘晓兰,周利敏,等.DNS法测定D-氨基葡萄糖含量方法的研究[J].中国调味品,2014(10):89-93.
[2]Kika K,Korlos F,Kiosseoglou V.Improvement,by dry-heating,of the emulsion-stabilizing properties of a whey protein concentrate obtained through carboxymethylcellulose complexation[J].Food Chemistry,2007,104(3):1153-1159.
[3]Jiang S J,Zhao X H.Transglutaminase-induced cross-linking and glucosamine conjugation in soybean protein isolates and its impacts on some functional properties of the products[J].European Food Research and Technology,2010,231(5):679-689.
[4]Jiang S J,Zhao X H.Transglutaminase-induced cross-linking and glucosamine conjugation of casein and some functional properties of the modified product[J].International Dairy Journal,2011,21(4):198-205.
[5]Jiang S J,Zhao X H.Cross-linking and glucosamine conjugation of casein by transglutaminase and the emulsifying property and digestibility in vitro of the modified product[J].International Journal of Food Properties,2011,15(6):1286-1299.
[6]李铮,冯力更,郑喆,等.糖基化反应条件对乳清蛋白——麦芽糖复合物抗原性的影响[J].中国乳品工业,2011,39(1):8-11.
[7]王维,尤瑜敏,周培根.D-氨基葡萄糖测定方法的比较[J].食品研究与开发,2003,24(2):84-87.
[8]Shao Y,Alluri R,Mummert M,et al.A stability-indicating HPLC method for the determination of glucosamine in pharmaceutical formulations[J].Journal of Pharmaceutical&Biomedical Analysis,2004,35(3):625-631.
[9]黄婷,李铜铃,孙健,等.2-羟丙基-β-环糊精对尼扎替丁的包合作用[J].华西药学杂志,2005,20(6):523-524.
[10]Harazono A,Kobayashi T,Kawasaki N,et al.A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals[J].Biologicals,2011,39(3):171-180.
[11]Harvey D J.Harvey,Derivatization of carbohydrates for analysis by chromatography,electrophoresis and mass spectrometry[J].Journal of Chromatography B,2011,879(17-18):1196-1225.
[12]Yan X,Evenocheck H M.Chitosan analysis using acid hydrolysis and HPLC/UV[J].Carbohydrate Polymers,2012,87(2):1774-1778.
[13]Stepan H,Staudacher E.Optimization of monosaccharide determination using anthranilic acid and 1-phenyl-3-methyl-5-pyrazolone for gastropod analysis[J].Analytical Biochemistry,2011,418(1):24-29.
[14]Guan Y,Tian Y,Li Y,et al.Application of a liquid chromatographic/tandem mass spectrometric method to a kinetic study of derivative glucosamine in healthy human urine[J].Journal of Pharmaceutical&Biomedical Analysis,2011,55(1):181-186.
[15]Eikenes M,Fongen M,Roed L,et al.Determination of chitosan in wood and water samples by acidic hydrolysis and liquid chromatography with online fluorescence derivatization[J].Carbohydrate Polymers,2005,61(1):29-38.
[16]曾茂法,李俊,王英瑛,等.D-氨基葡萄糖硫酸钾盐含量测定方法探讨[J].中国现代应用药学,2008,25(3):251-254.
[17]姜红,但晓梦,胡远华,等.HPLC-RID法测定氨基葡萄糖的含量[J].中国药品标准,2013,14(4):259-263.
[18]郑家概,王飞,农云军,等.氨基葡萄糖盐酸盐含量的HPLC柱前衍生法测定[J].分析测试学报,2009,28(9):1081-1083.
[19]王英瑛,李俊,曹苏.氨基葡萄糖3种含量测定方法的比较[J].中国现代应用药学,2009,26(4):307-309.
[20]杨岚,李铜铃,黄婷,等.RP-HPLC测定盐酸氨基葡萄糖片的含量[J].华西药学杂志,2005,20(3):259-260.
[21]杨华良,雷勇胜.HPLC法测定盐酸氨基葡萄糖颗粒中盐酸氨基葡萄糖的含量[J].天津药学,2011,23(6):17-19.
[22]陈金东,李蔚.滴定分析法和分光光度法测定保健食品中D-氨基葡萄糖盐酸盐含量的研究[J].中国卫生检验杂志,2002,12(2):143-144.
[23]刘玥,刘晓兰,周利敏,等.DNS法测定D-氨基葡萄糖含量方法的研究[J].中国调味品,2014(10):89-93.