Development of an Analysis Method for 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid by LC/ESI/MS with Selected Ion Monitoring

Abstract

This study describes a simple and rapid analytical quantitative method for measuring 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) using liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). For a chromatographic condition, Shodex IC NI-424 column (4.6 mm i.d. x 100 mm, 5 μm) for anion analysis and an isocratic elution of 40 mM ammonium formate buffer including 0.1% formic acid (pH 3.75) at a flow rate of 0.5 mL/min was used. The column temperature was set to 40°C. In the analysis of DEH produced by exo-type alginate lyase (AlyFRB) from Falsirhodobacter sp. alg1, a peak was detected with a retention time of 3.207 min. The prepared calibration curves for DEH analysis using the selected ion monitoring (SIM) mode of a mass spectrometer revealed a good linear relationship (correlation factor: 0.9998) within the test range (0.1–100 μg/mL). The limits of detection (S/N = 3) and quantification (S/N = 10) for DEH in SIM analysis were 0.008 and 0.027 μg/mL, respectively. Using the developed condition of LC/ESI/MS analysis, separation and detection of alginate unsaturated oligosaccharides were also tested. In an analysis time of about 13 min, this method was able to separate and detect an alginate unsaturated disaccharide, a trisaccharide, and a tetrasaccaride produced by poly(β-D-mannuronate) lyase, respectively. The analysis method established in this study will contribute to the quantitative and qualitative analysis of DEH, and the activity measurement of exo-type alginate lyase.