Analysis of chondroitin/dermatan sulphates using high-performance liquid chromatography

Ivan Mikšík, Institute of Physiology CAS, Prague 4, Czech Republic (Ivan.Miksik@fgu.cas.cz)
Šárka Kubinová, Institute Of Experimental Medicine Cas, Prague 4, Czech Republic
Karel Výborný, Institute Of Experimental Medicine Cas, Prague 4, Czech Republic
Kamil Záruba, Department Of Analytical Chemistry, Faculty Of Chemical Engineering, University Of Chemistry And Technology, Prague 6, Czech Republic
David Sýkora, Department Of Analytical Chemistry, Faculty Of Chemical Engineering, University Of Chemistry And Technology, Prague 6, Czech Republic

There was developed an HPLC method for separation and quantification of underivatized chondroitin/dermatan sulphates - unsaturated disaccharides (4- and 6-sulphated disaccharide). This method is based on the separation of disaccharides by amido as well as amino-columns at acidic condition. Amido-column was core-shell particle column with amide polyol with TMS end-capping (2.6 µm particles, 150x2.1 mm) and amino column was 3-aminopropyl silica (5 µm particles, 100x3mm). There was also tested amino column modified by phenylboronate (enables separation of carbohydrates with vicinal-diols at cis-position) but only with slight improvement of separation. C18 reversed phase columns can also separate tested disaccharides but only in presence of ion-pairing agent (tetrabutyl ammonium bromide) and at lower separation efficiency. The amido column as well as the amino column enabled successful separation of 4- and 6-sulphated disaccharides at 50 mmol/L resp. 25 mmol/L phosphate buffer pH 4.25 (detection at 230 nm) at retention time less then 10 minutes. Limit of quantification was 0.5 µg/ml. Applicability of this method was demonstrated by the analysis of unsaturated disaccharides produced after the enzymatic digestion of chondroitin/dermatan sulphates of extracellular matrix gel produced from human umbilical cord.

Figure. Separation of 4-sulphated (4S) and 6-sulphated (6S) disaccharides at amino and amido column; a – standard (20 µg/mL), b - enzymatic digestion of chondroitin/dermatan sulphates of extracellular matrix gel produced from human umbilical cord, c – spiked sample (40 µg/mL + b, 1:1))

Acknowledgements

This work was supported by the Czech Science Foundation (Grant No. 18-02597S), The Czech Academy of Sciences support for long-term conceptual development of research organization RVO: 67985823, which is gratefully acknowledged.

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Abstract Reference & Short Personal Biography of Presenting Author

Prof. Mikšík has a broad spectrum of professional interest: development of new methods for analysis of a wide spectrum of compounds by capillary electrophoresis and chromatography and their applications to the analysis of biological (physiological) functions of the organism. In practice he uses coupling of capillary electrophoresis and chromatography to mass spectrometry and their application to study of proteins, peptides and other physiological important compounds; analysis of steroids and their metabolites in organisms, native and modified proteins; physiology and chemistry of connective tissue and their cross-links; study of nonenzymatic posttranslational modification of proteins during ageing as well as diseases. He also analyses pigments and proteins at eggshell.

He is teaching at University of Pardubice (Czech Republic) - course “Analysis of Biological Materials” at Department of Biological and Biochemical Sciences.

He published 180 papers in the international journals with Impact factor, 9 papers in journals without Impact factor, co-editor of 1 book and 2 Special Volumes of Journal of Chromatography B, 1 Special Issue of American Journal of Analytical Chemistry, 14 chapters in books and 3 CD-ROMs about bibliography of separation techniques, 2 patents. See list at: http://analyt.natureblink.com/publikace_pdf.htm