柱前衍生HPLC法测定复方首乌藤合剂多糖中5种单糖的含量

Determination of five monosaccharides in polysaccharides from compound Polygoni Multiflori Caulis mixture by pre-column derivation HPLC method

目的:建立一种同时测定复方首乌藤合剂多糖成分中5种单糖含量的方法。方法:采用水提醇沉法提取复方首乌藤合剂多糖成分。多糖经三氟乙酸水解后,用1-苯基-3-甲基-5-吡唑啉酮(PMP)衍生水解产物,再利用HPLC法分析单糖衍生物,色谱条件如下:依利特Krosmasil-C18柱(250 mm×4.6 mm,5μm),柱温35℃,以乙腈-0.1 mol/L磷酸二氢钠缓冲液(pH=6.8)(18∶82)为流动相进行洗脱,流速1.0 ml/min,检测波长250 nm,进样量10μl。结果:甘露糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖分别在0.628~40.833(r=0.9990)、0.500~30.239(r=0.9999)、0.499~29.970(r=0.9999)、1.060~63.600(r=1.0000)和0.485~29.100μg(r=1.0000)范围内与各自峰面积线性关系良好,平均加样回收率分别为(103.73±2.25)%、(99.41±0.64)%、(103.44±1.75)%、(103.19±1.15)%和(97.64±2.98)%(n=6)。10批样品中甘露糖、半乳糖醛酸、葡萄糖、半乳糖和阿拉伯糖的含量分别为3.35~5.21、0.17~0.37、1.59~2.32、0.46~0.78和0.15~0.27 mg/ml。结论:该方法准确,重复性高,可用于复方首乌藤合剂多糖中单糖的含量测定。

Objective:To establish a method for simultaneous determination of 5 monosaccharides in polysaccharides from compound Polygoni Multiflori Caulis mixture.Methods:The polysaccharides from compound Polygoni Multiflori Caulis mixture were extracted by water extraction and alcohol precipitation,and hydrolyzed into monosaccharides wtih trifluoroacetic acid.The hyrolysate was derived with 3-methyl-1-phenyl-2-pyrazolin-5-one(PMP),and then the PMP derivates of monosaccharides were analyzed by HPLC method.The column was Elite Krosmasil-C18 column(250 mm×4.6 mm,5μm).The column temperature was 35℃and the acetonitrile-0.1%NaH2PO4(pH=6.8)(18∶82)was the mobile phase with a flow rate of 1.0 ml/min.The determination wavelength was 250 nm and injection volume was 10μl.Results:The linear ranges of mannose,galacturonic acid,glucose,galactose and arabinose were 0.628-40.833μg(r=0.9990),0.500-30.239μg(r=0.9999),0.499-29.970μg(r=0.9999),1.060-63.600μg(r=1.0000),0.485-29.100μg(r=1.0000)respectively.The average recovery rates were(103.73±2.25)%,(99.41±0.64)%,(103.44±1.75)%,(103.19±1.15)%,(97.64±2.98)%(n=6)respectively.The contents of mannose,galacturonic acid,glucose,galactose and arabinose in the 10 samples were respectively 3.35-5.21,0.17-0.37,1.59-2.32,0.46-0.78,0.15-0.27 mg/ml.Conclusion:The method was accurate with good reproducibility,which could be used for the determination of monosaccharides in polysaccharides from compound Polygoni Multiflori Caulis mixture.