QUANTITATIVE-ANALYSIS OF NEUTRAL AND ACIDIC SUGARS IN WHOLE BACTERIAL-CELL HYDROLYSATES USING HIGH-PERFORMANCE ANION-EXCHANGE LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION TANDEM MASS-SPECTROMETRY

A procedure for analysis of a mixture of neutral and acidic sugars in bacterial whole cell hydrolysates using high-performance anion-exchang e liquid chromatography-electrospray ionization tandem mass spectromet ry (HPAEC-ESI-MS-MS) is described. Certain bacteria (including bacilli ), grown under phosphate-limited conditions, switch from producing a t eichoic acid (containing ribitol) to a teichuronic acid (characterized by glucuronic acid content). Bacterial cells were hydrolyzed with sul furic 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 C-18 and SCX columns, resp ectively. HPAEC is well established as a high-resolution chromatograph ic technique, in conjunction with a pulsed amperometric detector. Alte rnatively, for more selective detection, sugars (as M-H- ions) were mo nitored using ESI-MS. In HPAEC, the mobile phase contains sodium hydro xide and sodium acetate, which are necessary for chromatographic separ ation of mixtures of neutral and acidic sugars. Elimination of this hi gh ionic content prior to entry into the ESI ion source is vital to av oid compromising sensitivity. This was accomplished using an on-line s uppressor and decreasing post-column flow-rates from 1 mi to 50 mu l/m in. 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 colli sion-induced dissociation. (C) 1997 Elsevier Science B.V.