A sensitive HPLC method has been developed for the quantitation of polysaccharide (PS) in Haemophilus influenzae type b (Hib) conjugate vaccines. Hib PS is readily depolymerized in 0.1 N sodium hydroxide into its subunit. The major depolymerized product (82%) was isolated and characterized by composition analysis and 31P NMR to be a single repeating unit of the PS, ribitol-ribose-phosphate. The single repeating unit in NaOH-treated vaccines was analyzed by high-performance anion-exchange chromatography using CarboPak PA-1 column and quantitated by pulsed amperometric detection. The PS contents of three conjugate vaccines and three PS vaccines from different manufacturers were determined. Their contents were in the ranges of expected values. This method is particularly useful for a vaccine with a sugar stabilizer such as lactose which would interfere with the colorimetric orcinol assay currently used for the determination of the PS. The HPLC method can quantitate 0.1 microg of the PS and its sensitivity is at least 30-fold higher than that of the orcinol assay. It may be used for stability studies of the conjugate vaccines since as little as 5% breakdown of PS from the PS-protein conjugates would be detected.

National Institute of Health (NIH)
Food and Drug Administration (FDA)
Intramural Research (Z01)
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