柱前衍生化HPLC法分析沙参多糖中单糖组成

目的建立北沙参多糖中单糖的组成和含量的检测方法。方法以三氟乙酸水解沙参多糖,水解产物加入衍生化试剂1-苯基-3-甲基-5-吡唑酮(PMP)生成衍生化产物,采用RP-HPLC法,色谱柱:C18色谱柱(250 mm×4.6 mm,5μm),流动相:体积分数为19%的乙腈溶液-醋酸铵缓冲液(醋酸铵-冰醋酸,pH5.5,体积比为100∶1),等度洗脱,流速:1.0 mL.min-1,检测器:紫外检测器,检测波长:250 nm,柱温:35℃。结果沙参多糖由D-甘露糖、D-鼠李糖、半乳糖醛酸、D-葡萄糖、D-半乳糖、L-(+)-阿拉伯糖等8种单糖(有2种未鉴定出的单糖)组成。D-甘露糖、D-鼠李糖、半乳糖醛酸、D-葡萄糖、D-半乳糖、L-(+)-阿拉伯糖的线性分别为4~80μmol.L-1(r=0.999 0)、12~240μmol.L-1(r=0.999 0)、5.6~1.12×103μmol.L-1(r=0.999 4)、1.54×103~30.80×103μmol.L-1(r=0.999 0)、80.0~1.6×103μmol.L-1(r=0.999 0)、64.00~1.28×103μmo.lL-1(r=0.9992);平均加样回收率分别为94.0%、88.5%、93.9%、95.9%、89.2%、90.8%。含量分别为0.22%、0.61%、8.55%、72.88%、4.79%、2.71%。结论该方法可以准确的测定出北沙参多糖中含有的各种单糖和含量。

Analysis of monosaccharide composition in Radix Glehniace by precolumn derivatization high rerformance liquid chromatography

Objective To develop a method for the determination the contents of monosaccharide compositions in Radix Glehniace polysaccharide (GLP). Methods GLP sample was hydrolyzed with trifluoroacetic acid. 1-phenyl-3methyl-5pyrazolone (PMP) was added as derivative agent. The hydrolyzed monosaccharides were converted into derivatives. The analytical column was a C18（250 × 4.6 mm, 5 µm）. The mobile phase was 19%（w）acetonitrle– ammoniumacetact (ammoniumacetact –glacial acetic acid pH5.5); isocratic elution process was performed; the flow rate was 1.0 mL /min and the column temperature was 35℃. The ultraviolet detector was used and the detection wavelength was 250 nm. Results Concentrations of eight monosaccharides, including mannose, rhamnose, galacturonic-acid, glucose, galactose, arabinose, and two unidentifiable monosaccharides, were determined in GLP sample. The contents of mannose rhamnose, galacturonic-acid, glucose, galactose and arabinose were good linearity over the range of 0.004– 0.080 mmol&;#1468;L^-1（ r=0.990 0）,0.012- 0.240 mmol&;#1468;L^-1 （r=0.990 0）,0.056-1.120 mmol&;#1468;L^-1（ r=0.990 0）,1.54 – 30.80 mmol&;#1468;L^-1（ r=0.990 0）,0.08– 1.60 mmol&;#1468;L^-1 （r=0.990 0） and 0.064 – 1.280 mmol&;#1468;L^-1（ r=0.990 0）, respectively; the average recoveries were 94.0%,88.5%,93.9%,95.9%,89.2% and 90.8%, respectively. The contents were 0.22％, 0.61％, 8.55％, 72.88％, 4.79％ and 2.71％, respectively. Conclusion The method reported in this paper is simple, quick and accurate. It can be used for analysis of monosaccharide hydrolyzed from GLP.