肺炎球菌荚膜多糖中C多糖的定量1H NMR和31P NMR法测定

Determination of Pneumococcal C-polysaccharide in Capsular Polysaccharides from Streptococcus pneumoniae by the Quantitative 1H and 31P NMR Method

关键词:  肺炎链球菌  C多糖  定量1H NMR  定量31P NMR  六甲基磷酰胺

Key words:  Streptococcus pneumoniae  C-polysaccharide  quantitative 1H NMR  quantitative 31P NMR  HMPA

摘要:

目的 利用定量1H NMR和31P NMR核磁定量法测定肺炎球菌荚膜多糖中C多糖残留量。方法 采用Bruker AVANCE NEO 600 MHz核磁共振波谱仪，以六甲基磷酰胺（HMPA）作为内标物，对不同血清型及不同浓度荚膜多糖溶液中C多糖进行定量1H NMR和31P NMR分析。结果 在定量1H NMR中二甲基亚砜（DMSO）的回收率为96.41%~104.16%，相对标准偏差（RSD）值均小于2.00%，表明定量1H NMR具有较好的准确度和精密度。在定量1H NMR中，当荚膜多糖样品浓度大于9 mg·mL-1时满足信噪比要求，而定量31P NMR中测定浓度需大于15 mg·mL-1。对于7F型荚膜多糖在浓度15~25 mg·mL-1时两种方法测定结果无显著性差别。而对于4型荚膜多糖在浓度为3~15 mg·mL-1时定量1H NMR测定结果无显著性差异。通过定量1H NMR和31P NMR法测定多种不同血清型荚膜多糖中C多糖含量，结果显示，19种血清型荚膜多糖中C多糖含量为0.57%~5.07%。结论 以HMPA为内标建立的C多糖定量1H NMR和31P NMR测定法，其操作简单，准确性高，重复性好，可作为肺炎球菌C多糖残留量检测的一种有效手段。

Abstract:

To establish a method for detection of the content of residual C-polysaccharide (C-Ps) in the capsular polysaccharide by the quantitative 1H NMR and 31P NMR method. METHODS All NMR experiments were carried out on a Bruker AVANCE NEO 600 MHz spectrometer using HMPA as internal standard. Quantitative 1H NMR and 31P NMR analysis of C-Ps in different concentrations solution of multiple serotypes capsular polysaccharide were performed. RESULTS The recovery of DMSO in quantitative 1H NMR were 96.41%~104.16%, and the relative standard deviation (RSD) values were all less than 2.00%, indicating that the quantitative 1H NMR possessed good accuracy and precision. The signal-to-noise ratio (SNR) met requirement when the concentration was greater than 9 mg·mL-1 in 1H NMR, while 15 mg·mL-1. for 31P NMR. For serotype 7F capsular polysaccharides, there was no significant difference between the two methods under concentrations of 15~25 mg·mL-1. And for serotype 4 capsular polysaccharides, there was no significant difference under concentrations of 3~15 mg·mL-1 in 1H NMR. The content of C-Ps in multiple serotypes were determined by quantitative 1H NMR and 31P NMR, and the results showed that the content of C-Ps in 19 serotypes capsular polysaccharides was 0.57%~5.07%. CONCLUSION The quantitative 1H NMR and 31P NMR methods established with HMPA as the internal standard for the determination of pneumococcal C-Ps were simple, accurate and reproducible, and could be used as an efficient method for the detection of pneumococcal C-Ps.