Determination of saccharides labelled with a fluorescent reagent, DBD-ProCZ, by liquid chromatography

Saccharides were determined by high-performance liquid chromatography (HPLC) after precolumn tagging with 4-(2-carbazoylpyrrolidin-1-yl)-7-(N,N-dimethylaminosulphonyl )-2,1, 3-benzoxadiazole (DBD-ProCZ). The tagging conditions were optimized with D-glucose. Monosaccharides and oligosaccharides were efficiently labelled under conditions of 65 degrees C for 180 min in water: acetonitrile mixture containing 0.5% trichloroacetic acid (TCA). The resulting derivatives were detected at 540 nm (excitation at 450 nm) after separation by HPLC. The separation of individual saccharides was possible using water: acetonitrile as the mobile phase using a reversed-phase column (Inertsil ODS-80A) and a normal-phase column (TSKgel Amide-80). N-Acetyl-D-glucosamine (GluNAC), N-acetyllactosamine (LacNAC) and tri-N-acetyl-chitotriose (tri-N-AceChito), which are components of glycoconjugates, were completely separated with an isocratic elution using as Inertsil ODS column. The on-column detection limit with the proposed HPLC separation and fluorescence detection at the low pmol level.