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

OBJECTIVE  To establish a method for detection of the content of residual C-polysaccharide(C-Ps) in the capsular polysaccharide from Streptococcus pneumoniae by the quantitative ¹H-NMR and ³¹P-NMR method.

METHODS  All NMR experiments were carried out on Bruker AVANCE NEO 600 MHz spectrometer using hexamethylphosphoramide(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 96.52%−103.31%, and the RSD values were all <2.00%, indicating that the quantitative ¹H-NMR possessed good accuracy and precision. The signal-to-noise ratio met requirement when the concentration was >9 mg·mL⁻¹ in quantitative ¹H-NMR, while 15 mg·mL⁻¹ for quantitative 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 quantitative 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 quantitative¹H-NMR and ³¹P-NMR methods established with HMPA as the internal standard for the determination of pneumococcal C-Ps are simple, accurate and reproducible, and can be used as an efficient method for the detection of pneumococcal C-Ps.