柱前衍生HPLC法分析网地藻多糖的单糖组成

Analysis of monosaccharide compositions in Dictyota dichotoma polysaccharides by precolumn derivatization HPLC

目的:建立测定网地藻多糖的单糖组成方法。方法:采用超声辅助提取、乙醇沉淀、Sevage法脱蛋白制备网地藻多糖,采用硫酸水解后,水解产物以1-苯基-3-甲基-5-吡唑酮(PMP)柱前衍生化后采用反相高效液相色谱法于248 nm处测定单糖组成。色谱柱为Diamonsil C18(250mm×4.6 mm,5μm);流动相为0.02 mol/L磷酸缓冲盐(A)-乙腈(B),洗脱梯度(0 min,0%B;10min,18%B;25 min,25%B;30 min,30%B)。结果:网地藻多糖含有甘露糖、核糖、鼠李糖、葡萄糖、半乳糖和木糖,其摩尔比为0.24:0.04:0.03:1:0.19:0.1。结论:该方法准确可靠、分离效果较好,可用于网地藻多糖的单糖组成测定。

Objective： To determine the monosaccharide compositions in polysaccharide from Dictyota dichotoma polysaccharides. Methods： The polysaccharide which was extracted by ultrasonic-assisted technology, ethanol precipitation and deproteinization with Sevage method was hydrolyzed with sulfuric acid （H2SO4）. The hydrolysate was derivatized with 1-phenyl-3-methyl-5-pyrazolone （PMP） and then the PMP derivates of monosaccharides were determined by RP-HPLC at 248 nm. The HPLC separation was achieved on a Diamonsil C18 （250mm×4.6mm,5μm） column with a gradient elution process （0 min, 0%B; 10 min, 18% B; 25 min, 25% B; 30 min, 30% B） using 0.02 mol/L phosphate buffer salt （A）-acetonitrile（B） as mobile phase. Results： Polysaccharide from Dictyota dichotoma was composed of mannose, ribose, rhamnose, glucose, galactose and xylose and their molar ratio was about 0.24：0.04：0.03：1：0.19：0.1. Conclusion： This method was accurate and sensitive, and it can be used for determination of polysaccharide compositions in Dictyota dichotoma.