利用核磁共振(^(31)P-NMR)测定b型流感嗜血杆菌荚膜多糖中磷含量的新方法研究

Determination of the Phosphorus Content of Haemophilus influenza Type b Polysaccharides Through Nuclear Magnetic Resonance(^(31)P-NMR) Compared with Traditional Method

目的:利用^(31)P-NMR核磁共振定量法(QNMR)测定b型流感嗜血杆菌(Hib)荚膜多糖中的磷含量,建立QNMR的质控方法。方法:采用Bruker AVANCEⅡ400核磁共振谱仪,以六甲基磷酰三胺为内标,采集一维定量^(31)PNMR谱对不同浓度的磷酸二氢钾进行定量分析,采用传统方法验证该新方法的精密度及准确度,并对2种方法进行比较研究。结果:QNMR定量结果相对标准偏差(RSD)均小于1.7%,相对误差为2.1%～3%,不同浓度样品回收率为97.0%～98.1%;传统的化学法定量结果 RSD为1.2%～3.2%,相对误差为5%～7%,不同浓度样品回收率为93.0%～94.9%;2种方法检测的3批Hib荚膜多糖中磷含量无明显差别,均符合《中国药典》三部(2010版)规定的68～90 mg/g。结论:^(31)P-NMR定量方法操作简单,耗时短,重现性好,精密度和准确度高,且不破坏多糖成分,可应用于细菌荚膜多糖质量控制的磷定量。

Objective:The quantitative nuclear magnetic resonance(QNMR)method was employed to detect the phosphorus content in Haemophilus influenzae type b(Hib)polysaccharide.Methods:Potassium phosphate monobasic at various concentrations were tested by one-dimensional quantitative31P-NMR spectrum with the internal standard hexamethylphosphoramide by a Bruker AVANCEⅡ400spectrometer.This new method was comfirmed by traditional chemical analysis with precision and accuracy.Results:The relative standard deviations(RSD)of results of QNMR were below1.7%,while the relative errors were2.1%~3%.The recovery rates of samples at various concentrations were97.0%~98.1%.However,the RSD value of traditional chemical analysis were1.2%~3.2%,while the relative errors were5%~7%,and its recovery rates were93.0%~94.9%.Conclusion:The quantitative31PNMR method is easily operated and time-saving.According to its high precision,reproducibility and accuracy,this method may be used in quality control of bacterial polysaccharide,withstabilization of polysaccharide.