An Improved PMP (1-Phenyl-3-Methyl-5-Pyrazolone) Precolumn Derivatization Method Of Carbohydrate For HPLC And MALDI-TOF Analysis

The important roles of carbohydrates in many biological processes have become more and more recognized, thus, there are considerable interests in carbohydrate researchers, of which high-performance liquid chromatography(HPLC) is an essential analytical tool. Precolum derivatization is usually employed to achieve HPLC analysis coupling with UV or fluorescence detection, precolumn derivatization methods have several advantages, such as high detection sensitivity, short time consumption, small quatities of sample requirement, and so on. PMP(l-Phenyl-3-Methyl-5-Pyrazolone) is one of the most commonly used derivatization agents, in a typical derivazation reaction procedure, sodium hydroxide solution was introduced as alkaline medium to initiate reaction, afterward the neutralization with acid was a necessary step, forming too much salt, which interferes subsequent MALDI-MS analysis although HPLC separation run well.In this paper, ammonia was employed instead of sodium hydroxide, derivazation reaction was systematic optimized, including reaction time, reaction temperature and PMP consumption, high yield was achieved. The derivatives were identified by MALDI-MS. In addition, the stability of derivatives and the fidelity of derivatization method were evaluated. The results indicated that this method was quite suitable for routine analyses of carbohydrate. So far this method has been successfully applied to HPLC analysis of simple monosaccharides. A baseline separation of five monosaccharides was obtained under optimized isolation conditions. Then a routine identification and quantification method has been established. By this method, the real carbohydrate sample was well analyzed. Furthermore, the PMP method was also successfully used for oligosaccharides and the glycans released from glycoprotein.An improved PMP precolumn derivazation of carbohydrate procedure was developed. Because we employed ammonia instead of sodium hydroxide, the excessive volatile ammonia can be removed by vacumm drying, no salt formed. Thederivatives can be analyzed by MS without any cleanup. This method is more convenient and more compatible to subsequent separation and analysis of carbohydrate.