Microscale analysis of glycosphingolipids by methanolysis, peracetylation, and gas chromatography

A method for the analysis of pure samples of individual glycosphingolipids by microscale methanolysis, peracetylation, and gas chromatography is described. Solvolysis of glycosphingolipids in dry methanolic HCl and peracetylation were conducted in a single 4.5-cm sealed capillary tube (2 mm i.d.), after which the products were directly injected into a gas chromatograph. Total-component analysis (i.e., analysis of the sugar, fatty acid, and sphingosine moieties) was possible after a 45-min chromatographic run. Time-course studies of the acid-catalyzed methanolysis of Gal beta 1-4GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer ganglioside at 80, 110, and 150 degrees C showed that methanolysis was complete after 2 h at 110 degrees C. Rates of methanolysis of individual components were compared and the release of the fatty acid moiety from the long-chain base was shown to be the slowest reaction. The methanolysis of all glycosidic bonds were complete in 0.5 h. Peracetylated methanolysis products were very stable over time and provided for good gas chromatographic detection of subnanomolar amounts of hexose, hexosamine, fatty acid, sialic acid, and long-chain sphingoid base components. Recoveries of fucose and N-acetylglucosamine were determined with reference samples of Fuc alpha 1-2Lac and lacto-N-fucosylpentaose II. Applications of the method are presented for the component analysis of a gift mixture of NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-1Cer ganglioside and NeuAc alpha 2-6Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer ganglioside and analysis of NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer isolated from human plasma.