Study on standardization method of Astragalus polysaccharide reference substance / 中草药

ABSTRACT

Objective A variety of techniques were adopted to establish a standardization method of Astragalus polysaccharides (APs) reference substance. This reference substance was used for quality control of APs for injection. Methods The total sugar content was quantified by UV spectrophotometry ration. The content of monosaccharide was determined by HPLC at detective wavelength of 250 nm after derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). The high performance size exclusion chromatography method was used for the determination of molecular weight and distribution. TLC was applied to check the free monosaccharide. Then the glycosidic linkage position and configuration were detected by using gas chromatography tandem mass spectrometry and nuclear magnetic resonance spectroscopy (1H-NMR). A number of experimental parameters were applied to control the quality of APs reference substance. Results The APs was composed of rhamnose (Rha), galactose acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Arb), and Glc was the main component and free monosaccharide were not detected in APs. The result of molecular weight test showed that there were four chromatographic peaks in APs, of which the main peak (peak 3) MW was 12 531 and the peak area ratio was 83%. The amount of sugar was 94% by UV test in which the glc was calculated as reference substance. There were eight main types of glycosidic bonds detected by GC-MS. Among them, the 1,4-glucose linkage was the main one. The glycosidic bond confirmed by NMR was α configuration. Conclusion The standardization method of APs reference substance has been established in this study. The identification method and determination of sugar amount are accurate and reliable. In the further research, APs reference substance will be used in quality control of APs products.