Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD.
A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.
Kim
John S
JS
Wyeth Vaccine, Research & Development, 4300 Oak Park, Sanford, NC 27330, USA. kimj9@wyeth.com
Laskowich
Erin R
ER
Arumugham
Rasappa G
RG
Kaiser
Raymond E
RE
MacMichael
Gregory J
GJ
D002240:Carbohydrate Sequence; D002623:Chemistry Techniques, Analytical; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000592:standards; D000432:Methanol; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009005:Monosaccharides / Q000032:analysis* / Q000592:standards; D022242:Pneumococcal Vaccines / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000276:immunology; D012015:Reference Standards; D012703:Serotyping; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000145:classification / Q000276:immunology; D018074:Vaccines, Conjugate / Q000737:chemistry
D009005:Monosaccharides; D022242:Pneumococcal Vaccines; D011135:Polysaccharides, Bacterial; D018074:Vaccines, Conjugate; D000432:Methanol