Determination of ribose and phosphorus contents in Haemophilus influenzae type b capsular polysaccharide by a quantitative NMR method using a single internal standard

The ribose and phosphorus contents in Haemophilus influenzae type b (Hib) capsular polysaccharide (CPS) are two important chemical indexes for the development and quality control of Hib conjugate vaccine. A quantitative ¹H- and ³¹P-NMR method using a single internal standard was developed for simultaneous determination of ribose and phosphorus contents in Hib CPS. Hexamethylphosphoramide (HMPA) was successfully utilized as an internal standard in quantitative ¹H-NMR method for ribose content determination. The ribose and phosphorus contents were found to be affected by the concentration of polysaccharide solution. Thus, 15−20 mg·mL⁻¹ was the optimal concentration range of Hib CPS in D₂O solution for determination of ribose and phosphorus contents by this method. The ribose and phosphorus contents obtained by the quantitative NMR were consistent with those obtained by traditional chemical methods. In conclusion, this quantitative ¹H- and ³¹P-NMR method using a single internal standard shows good specificity, accuracy and precision, providing a valuable approach for the quality control of Hib glycoconjugate vaccines.