高效液相色谱柱后衍生荧光法测定尿液中硫酸软骨素分解产生的不饱和双糖

目的 探讨高效液相色谱柱后衍生荧光法测定尿液中硫酸软骨素(CS)分解产生的不饱和双糖的效果.方法 分别用离心过滤法和常规沉淀法进行尿液的预处理,处理后的样品加CS裂解酶消化,用高效液相色谱柱后衍生荧光法测定分解产生的不饱和双糖(ADi-0S、△Di-4S、ADi-6S)的量,比较两种预处理方法中CS标准品回收率的差别.结果 高效液相色谱柱后衍生荧光检测系统运转效果良好,该法对于双糖的最低检出量为10 ng.尿样经离心过滤法处理后,CS标准品分解产生的△Di-0S、△Di-4S、△Di-6S的回收率分别为86.93%、87.28%、85.03%;而在常规沉淀法中,3种双糖的回收率仅为75.09%、72.96%、77.70%.结论 高效液相色谱柱后衍生荧光法较为灵敏,可用于测定CS分解产生的不饱和双糖.在尿液样品的预处理方法中,离心过滤法优于常规沉淀法,可应用于尿液中CS的微量测定.

Determination of unsaturated disaccharides produced from chondroitin sulfate in urine using high performance liquid chromatography with fluorometric post column derivatization

Objective To explore the effectiveness of the unsaturated disaccharides derived from chondroitin sulfate(CS)in urine using post column derivation fluorometric method of high performance liquid chromatography (HPLC).Methods Urine samples were prepared with the centrifugal flirtation and the routine precipitation methods separately,then digest the sample with CS lyses,use the post column derivation method of HPLC to fluorometric determine the unsaturated disaecharides(△Di-0S,△Di-4S and △Di-6S),compare the recovery differences of the CS standard in the urine pretreated by the two methods.Results The HPLC system ran well,the lowest detection limit of the method was 10 ng.When the urine was pretreated by the centrifugal flirtation method,the recovery of △Di-0S,△Di-4S and △Di-6S produced from the CS standard were 86.93%,87.28%and 85.03%,respectively,while the recovery of the three disaccharides in routine precipitation method were 75.09%,72.96%and 77.70%merely.Conclusions The HPLC post column derivatization fluorometric method is comparatively sensitive,it can be used to determine unsaturated disaccharides decomposed by CS.In the pretreatment procedure of the urine,the centrifugal flirtation method was superior to the routine precipitation method,it can be used as microdetermination for the unsaturated disaccharides.