A new method for the analysis of amide-linked hydroxy fatty acids in lipid-As from gram-negative bacteria.

Lipid-A represents the ubiquitous, covalently bound hydrophobic component of bacterial lipopolysaccharides (endotoxins). Lipid-As isolated and characterized from rhizobial species have large variations in their backbone sugars, as well as in their hydroxy fatty acid substituents. The sugar backbones consist of either glucosamine and galacturonic acid or glucosamine and 2,3-diaminoglucose. The published procedures for characterizing amide-linked fatty acids do not release all these fatty acids, hence a new method was developed to characterize the amide-linked hydroxy fatty acids. This method involves a mild methanolysis procedure to release glucosamine methyl glycosides which still contain the amide-bound hydroxy fatty acids. The products were analysed by fast atom bombardment mass spectrometry (FAB-MS) and, after trimethylsilylation, by electron impact (E.I.) and chemical ionization (C.I.) gas chromatography-mass spectrometry (GC-MS). The procedure was applied to lipid-A preparations from several gram-negative bacteria. This method allows the unequivocal identification of amide-linked hydroxy fatty acids and also allows determination of the microheterogeneity of the N-acyl substituents in lipid-As from gram-negative bacteria.