Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography

The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.