Quantification of 2-keto-3-deoxyoctonate in (lipo)polysaccharides by methanolytic release, trifluoroacetylation and capillary gas chromatography.
Several conditions of acidic anhydrous methanolysis were examined to optimize the release and minimize the degradation of unphosphorylated 2-keto-3-deoxy-D-manno-octonic acid (KDO) from bacterial lipopolysaccharides and polysaccharides. The reaction was monitored by capillary gas chromatography after derivatization by trifluoroacetic anhydride. The best results were obtained by use of 2 M hydrochloric acid at 60 degrees C for 2 h. Under these conditions a single KDO component appeared, and KDO was quantitatively released from all model compounds except when glycosidically linked to hexosamines. For quantitative cleavage of this linkage a reaction time of 6 h was required at 60 degrees C, giving rise to 5-10% of secondary KDO products. The KDO detection limit was about 250 pmol (50 ng) and the molar response was the same as for glucose. The KDO derivative gave a mass spectrometric fragmentation pattern consistent with a pyranosidic methylketoside methyl ester structure. Differentiation of KDO linkage types could be obtained by determination of the rates of KDO release by mild methanolysis.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002849:Chromatography, Gas; D004355:Drug Stability; D008401:Gas Chromatography-Mass Spectrometry; D008070:Lipopolysaccharides / Q000032:analysis*; D000432:Methanol; D013400:Sugar Acids / Q000032:analysis*; D014269:Trifluoroacetic Acid
D008070:Lipopolysaccharides; D013400:Sugar Acids; C002532:2-keto-3-deoxyoctonate; D014269:Trifluoroacetic Acid; D000432:Methanol