Validation of a Rapid GC-MS Procedure for Quantitative Distinction between 3-O-Methyl- and 4-O-Methyl-Hexoses and Its Application to a Complex Carbohydrate Sample

Methylation of one hydroxyl group of monosaccharides occurs in some bacteria, fungi, worms, molluscs, and also in plants. Although knowledge on the exact functions of this process is missing, methylation is an option to modulate glycan structures thereby leading to new biological activities. In plants, methylated monosaccharides are often present in minor amounts and, therefore, overseen in analytical investigations. A special difficulty is the distinction between 3-O-methyl- and 4-O-methyl-hexoses, due to similar fragmentation patterns of methylated alditol acetates in gas-chromatography with mass spectrometric detection and, in the case of galactose, identical retention times due to symmetry. We, therefore, developed and validated an easy method for the quantitative distinction between 3-O-methyl- and 4-O-methyl-hexoses and showed its functionality by quantification of 3-O-methyl galactose in a high molecular weight polysaccharide mixture from the charophyte Spirogyra. A systematic search for methylated monosaccharides in different plant lineages may offer new insights in plant cell wall evolution.