A Simple Analysis Method for 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid by HPLC-ELSD with Column for Anion Analysis

Abstract

4-Deoxy-l- erythro-5-hexoseulose uronic acid (DEH) is a rare deoxy sugar produced from alginate by the action of an exotype alginate lyase. A simple and rapid method for analyzing DEH using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed in this study. For chromatography, an isocratic elution of ammonium formate buffer including formic acid and a column for anion chromatography were used. In the developed method, DEH was detected at a retention time of 3.038 minutes and limits of detection (signal-noise ratio = 3) and quantification (signal-noise ratio = 10) were 37.5 and 124.9 µg/mL as a sodium DEH, respectively. In addition, separation and detection of alginate unsaturated oligosaccharides were also tested using the method. Within an analysis time of 10 minutes, it was possible to separate and detect unsaturated disaccharide, unsaturated trisaccharide, and unsaturated tetrasaccharide prepared using poly(β-d-mannuronate) lyase and sodium alginate of high mannuronate type. The HPLC-ELSD method established in this study will be applicable for quantitative analysis of DEH and measurement of exotype alginate lyase activity.