AN INNOVATIVE DERIVATIZATION METHOD FOR SIMULTANEOUS DETERMINATION OF URONIC ACIDS AND NEUTRAL AND AMINO SUGARS IN COEXISTING SAMPLES BY HPLC-ESI-MS/MS²

Tributylamine Facilitated Separations of Fucosylated Chondroitin Sulfate (Fucs) by High Performance Liquid Chromatography (HPLC) into its Component Using 1-Phenyl-3-Methyl- 5-Pyrazolone (PMP) Derivatization

“…The column used is not specific to carbohydrate analysis and the separation is solely based on the oven temperature, where the volatility of the compounds is reflected in the retention time. Failing the distinct separation by using non-specific column is due to the compounds nature, as the interested monosaccharides using triethylamine as a catalyst during PMP derivatization [9]. The basis of choosing tributylamine in this experiment is that longer alkyl chain length showed better compatibility with on-column retention of saccharides for a typical C18 type stationary phase [5].…”

“…Derivatization procedure was adapted from Zhang et al [9] and replacement used NaOH with 50 µl tributylamine as the catalyst. Without NaOH, the neutralization step with HCl was omitted.…”

“…Zhang et al [9] demonstrated the addition of triethylamine can facilitate reaction to completeness in a mixture of sugar standards. In our conventional method, GalNAc-PMP derivative had lower peak intensity than GlcUA-PMP.…”

Development and Validation of HPLC-DAD Method with Pre-Column PMP Derivatization for Monomeric Profile Analysis of Polysaccharides from Agro-Industrial Wastes

“…The reaction is proceeded on the sugar-reducing end, where in a stepwise mechanism two PMP molecules react with one aldehyde group to form a bis-PMP–sugar derivative. The reaction requires an alkaline medium to form reactive PMP enolate ions [ 5 , 12 ] and a high excess of PMP reagent [ 14 ]. As the aldehyde group is required for reaction, alcohol sugars cannot be analyzed as PMP derivatives [ 12 , 15 ].…”

“…In the original procedure, the optimized conditions comprised sodium hydroxide (NaOH) at 0.3 M concentration, PMP methanolic solution (0.5 M), a reaction temperature of 70 °C and a reaction time of 30 min [ 13 ]. In more recent attempts to optimize the reaction, the reaction time and temperature were reconsidered [ 14 , 15 , 16 ], along with the base catalyst concentration and amount of PMP [ 14 ]. It was found that the presence of different functional groups (e.g., carbonyl-, amino-) and their positions in the sugar molecules result in differences in the reaction times needed to obtain maximal yields of a respective PMP derivative [ 15 ].…”