反相高效液相色谱法测定人肌腱中的胶原蛋白  

Determination of Collagen in Tendon by Reversed-Phase High Performance Liquid Chromatography

作　　者：邹晓莉[1] 黎源倩[1] 曾红燕[1] 周健[1] 秦廷武[2] 莫湘涛[2] 

机构地区：[1]四川大学华西公共卫生学院,四川成都610041 [2]四川大学华西医院,四川成都610041

出　　处：《色谱》2006年第3期263-266,共4页Chinese Journal of Chromatography

基　　金：国家自然科学基金资助项目(No.30570469);四川省青年科技基金资助项目(No.2001-19-0132)

摘　　要：建立了测定人肌腱中胶原蛋白含量的高效液相色谱法。动物肌腱中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用2,4-二硝基氯苯对水解产物衍生化,然后以0.01m o l/L乙酸钠-乙酸缓冲液(pH6.0)-乙腈(体积比为80∶20)为流动相,经反相C18柱分离,紫外检测器于360nm波长处检测来测定羟脯氨酸的含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量来计算胶原蛋白的含量。该方法对羟脯氨酸的检出限为3μg/L,羟脯氨酸为3μg/L^100m g/L时与峰面积的线性关系良好;样品测定的相对标准偏差为1.95%,加标回收率为98.4%~110.8%。对60份人肌腱样品中胶原蛋白的含量进行了测定。所建立的方法灵敏、准确、干扰少,适用于肌腱中胶原蛋白含量的测定。A method for determining collagen in tendon by reversed-phase high performance liquid chromatography （RP-HPLC） was developed. After hydrolysis with hydrochloric acid, the collagen in samples was decomposed into hydroxyproline which hydroxyproline can be derivatized with 2,4-dinitrochlorobenzene for the determination by HPLC （reversed-phase C18 column, 0.01 mol/L NaAc-HAc（ pH 6.0 ） -CH3 CN （80： 20, v/v） as mobile phase, detection at 350 nm ）. The factors influencing hydrolysis, derivatization and HPLC analysis were studied and optimized. Sixty samples were analyzed with the proposed method. The linear range was from 3 μg/L to 100 mg/L and the detection limit was 3 μg/L. The relative standard deviation （RSD） of determination was 1.95%. The recoveries of spiked samples were 98.4% - 110.8%. The results show that the method is sensitive, accurate and suitable for tendon determination.

关 键 词：高效液相色谱法 羟脯氨酸 胶原蛋白 肌腱 

分 类 号：O658[理学—分析化学]