Simultaneous determination of uronates found in polysaccharides from natural products by HPLC with fluorometric detection

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of uronates isolated from polysaccharides found in natural products such as glycosaminoglycans and alginate is described. Preparation of iduronate, guluronate, and mannuronate as analytical standards for high performance liquid chromatography was achieved by depolymerization of dermatan sulfate and alginate in 2.5 mol L−1 trifluoroacetic acid at 100 °C for 6 h. Structures of resulting products (iduronate, guluronate, and mannuronate) were characterized by 600 MHz 1H NMR. Five uronates (glucuronate, iduronate, mannuronate, guluronate, and galacturonate) were separated on a Dionex CarboPac PA1 column using an isocratic elution with 8 mmol L−1 acetate buffer (pH 4.84) and were monitored by fluorescence detection using 1.5% 2-cyanoacetamide as a post-column fluorogenic reagent. As little as 50 pmol of each uronate could be detected with excitation at 331 nm and emission at 383 nm.

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► We purified commercially unavailable uronates.
► We identified each uronate by proton NMR.
► We established a new HPLC method for determination of uronates from natural products.