Quantitative analysis of neutral and acidic sugars in whole bacterial cell hydrolysates using high-performance...

A procedure for analysis of a mixture of neutral and acidic sugars in bacterial whole cell hydrolysates using high-performance anion-exchange liquid chromatography-electrospray ionization tandem mass spectrometry (HPAEC-ESI-MS-MS) is described. Certain bacteria (including bacilli), grown under phosphate-limited conditions, switch from producing a teichoic acid (containing ribitol) to a teichuronic acid (characterized by glucuronic acid content). Bacterial cells were hydrolyzed with sulfuric acid to release sugar monomers. The solution was neutralized by extraction with an organic base. Hydrophobic and cationic contaminants (including amino acids) were removed using C18 and SCX columns, respectively. HPAEC is well established as a high-resolution chromatographic technique, in conjunction with a pulsed amperometric detector. Alternatively, for more selective detection, sugars (as M-H- ions) were monitored using ESI-MS. In HPAEC, the mobile phase contains sodium hydroxide and sodium acetate, which are necessary for chromatographic separation of mixtures of neutral and acidic sugars. Elimination of this high ionic content prior to entry into the ESI ion source is vital to avoid compromising sensitivity. This was accomplished using an on-line suppressor and decreasing post-column flow-rates from 1 ml to 50 microliters/min. In the selected ion monitoring mode, background (from the complex sample matrix as well as the mobile phase) was eliminated, simplifying chromatograms. Sugar identification was achieved by MS-MS using collision-induced dissociation.