Several years ago, we developed an anion-exchange HPLC with pulsed amperometric detection (PAD) method to quantify polyribosylribitol phosphate (PRP) content in vaccines containing Hib conjugates (Vaccine 12:700-706,1994). This HPLC method has been adapted and used by two vaccine manufacturers in quality control of their licensed Hib conjugate vaccines. O-acetyl and phosphate are non-sugar components in many bacterial polysaccharide (PS) and PS-conjugate vaccines. O-acetyl group is known to be important epitope in certain PSs such as pneumococcal 9V PS and Salmonella typhi Vi PS. Currently, we are exploring an HPLC method to quantify the O-acetyl content of bacterial PSs. The PSs were first treated with mild alkali to hydrolyze O-acetyl group. The acetate released in the hydrolysates was then subjected to HPLC analysis and was monitored with a conductivity detector. We have quantified the O-acetyl content of meningococcal A, C, Y, and W-135 PSs, pneumococcal 9V and 18C PSs, and Vi PS. The results obtained by HPLC method were comparable to those by the colorimetric Hestrin assay.
