Determination, by methylation analysis, of the glycosyl-linkage compositions of microgram quantities of complex carbohydrates

Abstract

A methylation-analysis procedure was developed by which the glycosyl-linkage compositions of microgram quantities of complex carbohydrates, including those containing hexosyluronic acid residues, can be determined. The effectiveness of the procedure was demonstrated by correctly determining the glycosyl-linkage compositions of 1 .mu.g of a disaccharide and 5 .mu.g of an acidic polysaccharide whose structures were unknown to the analyst. The development of a new technique, namely, reversed-phase chromatography on Sep-Pak C18 cartridges, to recover and purifiy microgram quantities of per-O-methylated complex carbohydrates from methylation-reaction mixtures, was critical to the success of the microscale procedure. The use of GLC mass spectrometry with multiple, selected-ion monitoring was also essential for identification and semiquantitation of the partially O-methylated alditol acetates derived from 1-5 .mu.g of a complex carbohydrate.