Consecutive Analysis of Sphingoglycolipids on the Basis of Sugar and Ceramide Moieties by High Performance Liquid Cromatography

Abstract

A quantitative consecutive method was developed for analysis of sphingoglycolipids in bio-logical materials by high performance liquid chromatography (HPLC). Crude lipid extracts were separated into neutral and acidic fractions on a DEAE-Sephadex column. Glycolipid fractions were obtained by acetylation and Florisil column chromatography, and the acetylated glycolipids were N-p-nitrobenzoylated by treatment with p-nitrobenzoyl chloride in pyridine at 60°C for 6 h. Excess reagent and by-products were removed by solvent partition and gel filtration. The glycolipid derivatives were analyzed by their absorption at 254 nm on Zorbax SIL, a silica gel column, with a gradient of 0.5-7 % isopropanol in hexane-chloroform (2 : 1, v/v) at a flow rate of 0.5 ml/min. The detector response was linear with up to 60 nmol of injected glycolipids. The practical lower limit of detection was about 50 pmol. The deriva-tives were separated on the basis of their sugar chains. Effluents corresponding to each peak were collected and analyzed further on the basis of their lipid portion on μ-Bondapak C18, a reversed phase column. This combined procedure was applied to the analysis of erythrocyte glycolipids. Samples containing as little as 20μg of glycolipids could be analyzed by this method.