An improved carbohydrate derivatization method employing 1-phenyl-3-methyl-5-pyrazolone(PMP) was developed in this study.Liquid ammonia was used instead of sodium hydroxide aqueous solution to provide the base medium ...An improved carbohydrate derivatization method employing 1-phenyl-3-methyl-5-pyrazolone(PMP) was developed in this study.Liquid ammonia was used instead of sodium hydroxide aqueous solution to provide the base medium for the derivatization reaction.Derivatization reaction and HPLC separation conditions were optimized,including reaction time,reaction temperature and PMP consumption.The derivatives of five reducing monosaccharides were well separated by HPLC. Because we employed ammonia instead of sodium hydro-xide,the excessive volatile ammonia can be removed by vacumm drying,these derivatives could be directly analyzed by MALDI-TOF-MS without desalting procedure,possessing potential application for monosaccharide and oligosaccharide separation and subsequent MS analysis.This method not only increased the MS detection sensitivity,but also decreased sample wastes during purification. We successfully applied this method to mo-nosaccharide composition analysis of polysaccharides SPPA-1,SPPB-1 and SPPC-1 purified from Spirulina(Platensis).展开更多
目的:测定复方南板蓝根颗粒中多糖的含量及其单糖组成。方法:采用硫酸-苯酚法测定多糖含量;测定条件:5%苯酚溶液1 m L,硫酸5 m L,检测波长490 nm。设计正交试验对该制剂中多糖全降解条件进行优化;以1-苯基-3-甲基-5-吡唑啉酮(PMP)为衍...目的:测定复方南板蓝根颗粒中多糖的含量及其单糖组成。方法:采用硫酸-苯酚法测定多糖含量;测定条件:5%苯酚溶液1 m L,硫酸5 m L,检测波长490 nm。设计正交试验对该制剂中多糖全降解条件进行优化;以1-苯基-3-甲基-5-吡唑啉酮(PMP)为衍生试剂,采用毛细管区带电泳法,分析其单糖组成;电泳条件:未涂层弹性石英毛细管柱(52 cm×50μm,有效长度43 cm),运行缓冲液40 mmol·L^(-1)硼砂缓冲液(pH 10.1),检测波长245 nm,运行电压12 k V,进样量8 cm×10 s。结果:测得该制剂中多糖含量为1.708%~1.828%;多糖全降解的最优条件为硫酸浓度0.75 mol·L^(-1),温度100℃,时间5 h;其单糖组成为木糖、阿拉伯糖、葡萄糖、鼠李糖、甘露糖、半乳糖、葡萄糖醛酸和半乳糖醛酸。对所建立的毛细管区带电泳法进行方法学验证,结果显示其精密度RSD≤3.8%,重复性RSD≤5.5%,并且稳定性满足要求,回收率结果显示:半乳糖和半乳糖醛酸的平均回收率分别为99.7%~106.9%和94.8%~102.5%,其RSD分别为2.4%~4.9%和1.7%~4.6%。结论:本文为复方南板蓝根颗粒中多糖的测定及其质量标准的完善提供参考。展开更多
文摘An improved carbohydrate derivatization method employing 1-phenyl-3-methyl-5-pyrazolone(PMP) was developed in this study.Liquid ammonia was used instead of sodium hydroxide aqueous solution to provide the base medium for the derivatization reaction.Derivatization reaction and HPLC separation conditions were optimized,including reaction time,reaction temperature and PMP consumption.The derivatives of five reducing monosaccharides were well separated by HPLC. Because we employed ammonia instead of sodium hydro-xide,the excessive volatile ammonia can be removed by vacumm drying,these derivatives could be directly analyzed by MALDI-TOF-MS without desalting procedure,possessing potential application for monosaccharide and oligosaccharide separation and subsequent MS analysis.This method not only increased the MS detection sensitivity,but also decreased sample wastes during purification. We successfully applied this method to mo-nosaccharide composition analysis of polysaccharides SPPA-1,SPPB-1 and SPPC-1 purified from Spirulina(Platensis).
