高效液相色谱法测定土垅大白蚁菌圃多糖的单糖组成

Determination of monosaccharide composition in polysaccharide of ant nest of Macrotermes annadalei by HPLC

目的:测定土垅大白蚁菌圃多糖中单糖的组成。方法:采用水提醇沉法提取土垅大白蚁菌圃多糖,三氟乙酸水解后,经衍生化试剂1-苯基-3-甲基-吡唑啉酮(PMP)衍生化,高效液相色谱法(HPLC)分析。色谱条件:色谱柱C18(4.6 mm×250 mm,5μm),柱温25℃;流动相为0.05 mol.L-1磷酸二氢钾溶液(pH 6.8)-乙腈,流速1.0 mL.min-1,梯度模式:时间梯度为0→5→10→30→35 min,相应的磷酸盐缓冲溶液梯度为83%→82%→81%→80%→83%;检测波长250 nm;进样体积10μL。结果:土垅大白蚁菌圃多糖中含甘露糖、鼠李糖、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖的摩尔比3.55∶1.00∶1.19∶3.54∶2.54∶4.41∶1.90。结论:该方法具有简单、快速、分离效率高等特点,可用于测定土垅大白蚁菌圃多糖中单糖的组成。

Objective： To determine monosaccharide composition in polysaccharide of ant nest of Macrotermes art-nadalei by high performance liquid chromatography. Methods： The polysaccharides of ant nest of Macrotermes an-nadalei were extracted by water extraction and alcohol precipitation method, and then hydrolyzed by trifluoroacetic acid. The hydrolysate was reacted with 1-phenyl-3-methyl-5-pyrazolone （PMP）to produce the PMP deriva- tives that were analyzed by HPLC. The chromatographic conditions were as follows：chromatographic column Cls （4.6 mm × 250 mm, 5 μm） , column temperature 25 ℃ ; the mobile phase consisted of 0. 05 mol · L^ - 1 potassium di-hydrogen phosphate solution （ pH 6. 8 ） and methyl cyanide, the flow rate was 1.0 mL · min^ - 1 with gradient mode of time gradient 0→5→10→30→35 min,the corresponding gradient of PBS phosphate buffer solution was 83%→82% →81%→80%→83 % ; the detection wavelength was 250 nm with the sample volume of 10 μL. Results： The molar ratio of mannose, rhamnose, galacturonicaied, glucose, galactose and xylose arabonose was 3.55： 1.00： 1.19： 3.54： 2. 54： 4.41：1.90 in polysaccharide of ant nest of Macrotermes annadalei. Conclusion： This method is sim-ple, rapid, with high separating efficiency, and can be used to determine monosaccharide composition in polysaccha-ride of ant nest of Macrotermes annadalei.