Method Development and Validation for Determination of Total and Free Polysaccharide Content in Heamophilus influenzae Type B Vaccine by HPAEC-PAD
Method Development and Validation for Determination of Polysaccharide Content in Heamophilus influenzae Type B Vaccine by HPAEC-PAD
Keywords:
The Haemophilus influenzae type b vaccine, Polysaccharide content, HPAEC-PAD, Method validationAbstract
The Haemophilus influenzae type b (Hib) vaccine is used to prevent meningitis and pneumonia in young children. It is produced from the polysaccharide component of Haemophilus influenzae type b. In the quality control of the Hib vaccine, determining the total and free polysaccharide contents is a crucial parameter, and the World Health Organization (WHO) recommends using the HPAEC-PAD method for analysis due to its accuracy and precision, which is well-suited for detecting polysaccharides. This study, the method involved using an anion exchange column with a column temperature of 30°C, a mobile phase of 580 mM NaOH, a flow rate of 0.4 mL/min, and an injection volume of 50 μL. Detection was performed using a PAD detector. The results showed that the standard curve was linear in the concentration range from 0.15 to 10.50 μg/mL, with a correlation coefficient of 0.9999. The precision on the same day showed the relative standard deviations for total and free polysaccharide contents of 3.38% and 1.61%, respectively, while intermediate precision showed the relative standard deviations of 4.47% and 3.22%, respectively. The method was accurate, with the recovery of standard substances added to the DTP-HB-Hib combined vaccine ranging between 96.74% and 103.37%. The method demonstrated specificity and capability of analyzing polysaccharide content in the vaccine from 0.15 μg/mL. Additionally, column temperature and acid hydrolysis duration were found to affect the analysis. In conclusion, this method can be used as an analytical method in the laboratory of the Institute of Biological Products, Department of Medical Sciences, Thailand for the Hib vaccine quality control purposes.
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