Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking.
Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking.
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DOI：
10.1002/jssc.200500181
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发表时间：
2005-11
影响因子：
3.1
通讯作者：
Yifang Yang;M. Breadmore;W. Thormann
中科院分区：
文献类型：
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作者：
Yifang Yang;M. Breadmore;W. Thormann
报道了用碱性四硼酸缓冲液、电动进样和232 nm紫外吸收检测不饱和双糖透明质酸(di-HA)和硫酸软骨素(di-CS)的毛细管电泳法。分离在未涂覆的熔融石英毛细管中进行，该毛细管具有反转的极性和通过向缓冲液中添加CTAB而产生的反电渗透。考察了CTAB浓度、缓冲液pH、四硼酸根浓度、外加电压等分离参数对两种双糖拆分的影响。基线分离采用25 mM四硼酸盐，pH为10.0，CTAB为0.05 mM。样品中的氯化物和磷酸盐有利于二糖的堆积，其中di-HA形成的峰比di-CS尖锐得多。在25 mM Tris-HCl(pH 7.5)中制备样品，在-10千伏的阴极上电动进样40 S，校正峰面积与双糖浓度在1.0-400.0和0.1-1.0微克/毫升(双壳聚糖为0.2-1.0微克/毫升)范围内呈线性关系，相关系数均为&0.9933。检测次数和校正峰面积的RSD分别为1.13~1.24和1.57~2.13%。应用于乙醇沉淀和软骨素酶解聚制备的人血清样品，ABC法发现内源化合物与di-HA共迁移，且检测时间依赖于样品。血清样品中的di-HA含量可以通过减去空白峰来估计，该空白峰是在没有酶解的情况下获得的。
CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 microg/mL (0.2-1.0 microg/mL for di-CS), with correlation coefficients being >0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.