MICROSCALE ANALYSIS OF GLYCOSPHINGOLIPIDS BY METHANOLYSIS, PERACETYLATION, AND GAS-CHROMATOGRAPHY

A method for the analysis of pure samples of individual glycosphingoli pids by microscale methanolysis, peracetylation, and gas chromatograph y 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 int o 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 met hanolysis of Gal beta 1-4GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4G lc beta 1-1Cer ganglioside at 80, 110, and 150 degrees C showed that m ethanolysis was complete after 2 h at 110 degrees C. Rates of methanol ysis of individual components were compared and the release of the fat ty acid moiety from the long-chain base was shown to be the slowest re action. The methanolysis of all glycosidic bonds was complete in 0.5 h . Peracetylated methanolysis products were very stable over time and p rovided for good gas chromatographic detection of subnanomolar amounts of hexose, hexosamine, fatty acid, sialic acid, and long-chain sphing oid base components. Recoveries of fucose and N-acetylglucosamine were determined with reference samples of Fuc alpha 1-2Lac and lacto-N-fuc osylpentaose II. Applications of the method are presented for the comp onent analysis of a gift mixture of NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-1 Cer gan glioside 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 gangli oside and analysis of NeuAc alpha 2-3GalB1-4GlcB1-1 Cer isolated from human plasma. (C) 1994 Academic Press,Inc.