Liquid chromatography "on-flow" H-1 nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs

Glycosphingolipids were prepared from pig lung and pooled into two fraction s with (i) less than or equal to 3 sugar residues, and ( ii) greater than o r equal to 3 sugar residues. Oligosaccharides were prepared and used for ga s chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i a nd ii were further characterised and purified using a novel method based on high performance liquid chromatography "on-flow" proton nuclear magnetic r esonance. The LC "on-flow" NMR technique showed good chromatographic separa tion and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional H-1 NMR, thin la yer immunostaining, gas chromatography, gas chromatography/mass spectrometr y and matrix-assisted laser desorption/ionization mass spectrometry were us ed to characterise the glycolipids and to validate LC-NMR spectral data.