柱前衍生化高效液相色谱法分析9种多糖中的单糖组成

摘要

Objective To establish a precolumnderivatization procedure with 1-phenyl-3-methyl-5-pyrazolone ( PMP )-high performance liquid chromatography ( HPLC ) method for the determination of the monosaccharide composition of nine polysaccharides.Methods The nine polysaccharides were hydrolyzed, derivatized by PMP and analyzed by HPLC with the Inertsil ODS-SP C18 column(260mm ×4.6mm,5μm).The mobile phase was the 20%triethylamine-buffer solution with KH2PO4(pH=6.9),and the flow rate was 1.0 ml/min.The UV absorbance was detected at a wavelength of 254 nm,and the injection volume was 10μl with the column temperature 30℃.Results The method was successful in the separation of the mixture monosac-charide standards.The calibration line of each monosaccharide had a good relationship.And the precision, stability and reproducibility were also good.The nine polysaccharides were composed of different monosac-charide and the contents had some difference.Conclusion The method was accurate and stable for the deter-mination of the monosaccharide composition and content of nine polysaccharides.%目的：建立1-苯基-3-甲基-5-吡唑啉酮（ PMP）柱前衍生化-高效液相色谱法（ HPLC）对甘草多糖等9种多糖的单糖组成成分进行分析。方法9种多糖样品水解后，利用PMP试剂进行柱前衍生化并进行HPLC分析，采用Inertsil ODS-SP C18色谱柱（260mm ×4．6mm，5μm），流动相为20％乙腈与KH2PO4-三乙胺缓冲溶液（pH＝6．9），流速1．0ml／min，检测波长254 nm，进样体积10μl，柱温30℃。结果建立的方法对多种混合单糖标准品具有良好的分离效果，各单糖在相应的线性范围内线性关系良好，且方法的精密度、稳定性、重现性良好。9种多糖各由不同的单糖组成，且含量存在一定的差异。结论该方法准确稳定，可以满足9种多糖的单糖组成及含量分析。