Analysis of Monosaccharides and Uronic Acids in Polysaccharides by Pre-column Derivatization with p-Aminobenzoic Acid and High Performance Liquid Chromatography
HAO Guitang1, CHEN Shangwei2, ZHU Song2, YIN Hongping3, DAI Jun2, CAO Yuhua1
1.School of Chemistry and Material Engineering of Southern Yangtze University, Wuxi 214036, China; 2.Testing & Analysis Center of Southern Yangtze University, Wuxi 214036, China; 3.School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
An ion-pair reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of carbohydrate and uronic acids was developed. p-Aminobenzoic acid (p-AMBA) was used for pre-column derivatization of the analytes, enabling fluorescence (λex=313 nm, λem=358 nm) or ultraviolet (UV at 303 nm) detection. Reaction conditions such as reaction temperature and reaction time were optimized. Atlantis dC18 column with hydrophilic end capping was selected for the separation of derivatives. Effects of mobile phase compositions such as ion pairs and their concentrations and pH on the retention behaviors and separation results of 9 monosaccharides and 2 uronic acids were investigated. Derivatives of fructose, galactose, glucose, mannose, xylose, arabinose, ribose, galacturonic acid, fucose, glucuronic acid and rhamnose were separated within 42 min, applying tetrabutyl ammonium hydrogen bisulfate (TBAHSO4) as the ion pair reagent. The detection limits were between 3.38×108 mol/L and 176×108 mol/L for fluorescence detection and between 2.55×107 mol/L and 13.4×107mol/L for UV detection. Good linearities were obtained with correlation coefficients (r2) above 0.99. The relative standard deviations (RSDs) of the peak area of the derivatives in 1251 h after derivatization were from 2.5% to 3.9%. This method has been applied for the determination of mono-/disaccharides and uronic acids in spirulina polysaccharide after dissolved in trifluoroacetic acid solution (2 mol/L). The results showed this method is suitable for the analysis of monosaccharide compositions in polysaccharides.