Analysis of Chondroitin/Dermatan Sulphate Disaccharides Using High-Performance Liquid Chromatography
Ivan Mikšík,
Šárka Kubinová,
Marine Morvan,
Karel Výborný,
Ameneh Tatar,
Vladimír Král,
Kamil Záruba,
David Sýkora
Affiliations
Ivan Mikšík
Institute of Physiology of the Czech Academy of Sciences, Vídenska 1083, 142 20 Prague 4, Czech Republic
Šárka Kubinová
Institute of Experimental medicine of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic
Marine Morvan
Institute of Physiology of the Czech Academy of Sciences, Vídenska 1083, 142 20 Prague 4, Czech Republic
Karel Výborný
Institute of Experimental medicine of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic
Ameneh Tatar
Department of Analytical Chemistry, Faculty of Chemical Engineering, University of Chemistry and Technology, Technicka 5, 166 28 Prague 6, Czech Republic
Vladimír Král
Department of Analytical Chemistry, Faculty of Chemical Engineering, University of Chemistry and Technology, Technicka 5, 166 28 Prague 6, Czech Republic
Kamil Záruba
Department of Analytical Chemistry, Faculty of Chemical Engineering, University of Chemistry and Technology, Technicka 5, 166 28 Prague 6, Czech Republic
David Sýkora
Department of Analytical Chemistry, Faculty of Chemical Engineering, University of Chemistry and Technology, Technicka 5, 166 28 Prague 6, Czech Republic
Chondroitin sulphates belong to a group of naturally occurring glycosaminoglycans and play a role in many physiological processes including ageing and the effects of various diseases. Research into chondroitin sulphates has found that the most important analytes are 4- and 6-sulphated disaccharides. We developed an HPLC method for the separation and quantification of underivatized chondroitin/dermatan sulphates—unsaturated disaccharides (4- and 6-sulphated disaccharides). This method is based on the separation of disaccharides by amido as well as amino columns under acidic conditions. These columns enabled the successful separation of 4- and 6-sulphated disaccharides using 50 (amido column) and 25 mmol/L (amino column) phosphate buffer, pH 4.25 (detection at 230 nm), at retention times of less than 10 min. The limit of quantification was 0.5 μg/mL. The applicability of this method was demonstrated through analysis of unsaturated disaccharides produced from the enzymatic digestion of chondroitin/dermatan sulphates of the solubilized extracellular matrix produced from porcine urinary bladder and human umbilical cord.
