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目的建立核磁共振波谱(nuclear magnetic resonance,NMR)法测定b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)荚膜多糖中核糖含量的方法。方法利用NMR技术中的氢核磁共振波谱(1H-NMR)的定量功能,对不同浓度的D-核糖进行定量分析,验证该方法的精密度及准确度,并与传统化学方法进行比较。结果 NMR法定量结果相对标准偏差(RSD)均小于1.0%,相对误差除最低浓度20 mmol/L为7.4%外,其余浓度均在1%~3%之间,不同浓度的样品回收率在97.2%~107%之间;化学法定量结果 RSD在0.6%~7.9%之间,相对误差在3%~6.5%之间,不同浓度样品回收率在93.5%~105.3%之间;两种方法检测的3批Hib荚膜多糖中核糖含量无明显差别,均在《中国药典》三部(2010版)规定的320~410 mg/g之间。结论 1H-NMR定量方法取样少,可直接定量,该方法精密度高、准确度高、重现性好,且不破坏多糖结构,可应用于细菌荚膜多糖质量控制的核糖定量。
Objective To establish a method for the determination of ribose content in capsular polysaccharide of Haemophilus influenzae type b (Hib) by nuclear magnetic resonance (NMR). Methods The quantification of hydrogen nuclear magnetic resonance spectrum (1H-NMR) in NMR was used to quantitatively analyze the D-ribose at different concentrations. The precision and accuracy of the method were verified and compared with the traditional chemical methods. Results The relative standard deviations (RSDs) were all less than 1.0%. The relative errors were all within 1% ~ 3% with the lowest concentration of 20 mmol / L being 7.4%. The recoveries of samples with different concentrations were 97.2% % To 107%. The RSD of chemical quantitative results was between 0.6% and 7.9%, the relative error was between 3% and 6.5%, and the recoveries of different concentrations were between 93.5% and 105.3% The three batches of Hib capsular polysaccharides in the ribose content was no significant difference, are in the “Chinese Pharmacopoeia” three (2010 edition) of 320 ~ 410 mg / g. Conclusion The method of 1H-NMR quantification is simple and can be directly quantified. The proposed method has high precision, high accuracy and good reproducibility, and does not destroy the structure of polysaccharides. It can be used for quantitative determination of ribose in quality control of bacterial capsular polysaccharides.