Monitoring activation sites on polysaccharides by GC–MS

A method has been developed to determine the location and order of activation for potential saccharide antigens used in conjugate vaccine development. Saccharides were monitored for activation by sodium periodate oxidation and subsequent analysis by gas chromatography–mass spectrometry (GC–MS). Pneumococcal serotype polysaccharides 7F and 18C were evaluated as polysaccharides containing multiple potential sites for activation. Sialyllactose was used as a model oligosaccharide compound to evaluate oxidation of terminally linked sialic acids and reducing sugar residues. Oxidized saccharides were analyzed by monosaccharide composition and/or linkage analysis to elucidate specific activation of cis versus trans diols, as well as diols containing primary versus secondary alcohols, at specified levels of periodate. Samples (100–500 μg) were sequentially oxidized, reduced, methanolyzed, and derivatized in a single reaction vial for routine analysis by GC–MS.