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Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking

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Yang, Yifang and Breadmore, MC and Thormann, W (2005) Analysis of the disaccharides derived from hyaluronic acid and chondroitin sulfate by capillary electrophoresis with sample stacking. Journal of Separation Science, 28 (17). pp. 2381-2389. ISSN Print 1615-9306

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Abstract

CE conditions for monitoring the unsaturated disaccharides of hyaluronic acid (di-HA) and chondroitin sulfate (di-CS) using an alkaline tetraborate buffer, electrokinetic sample injection, and UV absorption detection at 232 nm are reported. Separations were performed in an uncoated fused-silica capillary having reversed polarity and reversed electroosmosis generated with the addition of CTAB to the buffer. The influence of various separation parameters, including the concentration of CTAB, buffer pH, concentration of tetraborate, and applied voltage, on the resolution of the two disaccharides was investigated. Baseline separation was obtained with 25 mM tetraborate at pH 10.0 and having 0.05 mM CTAB. Chloride and phosphate in the sample are beneficial for the stacking of the disaccharides, with di-HA forming a much sharper peak than di-CS. Using samples prepared in 25 mM Tris-HCl (pH 7.5) and electrokinetic injection at the cathode at -10 kV for 40 s, linear relationships between the corrected peak area and the concentration of the disaccharides have been found in the ranges of 1.0-400.0 and 0.1-1.0 {micro}g/mL (0.2-1.0 {micro}g/mL for di-CS), with correlation coefficients being >0.9933 in all cases. The RSDs of detection times and corrected peak areas were between 1.13-1.24 and 1.57-2.13%, respectively. Applied to human serum samples that were prepared by ethanol precipitation and depolymerization of the two polysaccharides with chondroitinase ABC reveals comigration of endogenous compounds with di-HA and a sample-dependent detection time. The di-HA content in the serum sample can be estimated via subtraction of the blank peak that is obtained without enzymatic hydrolysis.

Item Type: Article
Keywords: Chondroitin sulfate • Electrokinetic injection • Hyaluronic acid • Serum • Stacking
Journal or Publication Title: Journal of Separation Science
Page Range: pp. 2381-2389
ISSN: Print 1615-9306
Identification Number - DOI: 10.1002/jssc.200500181
Additional Information: Copyright © 2005 John Wiley and Sons
Date Deposited: 05 Jun 2008 12:49
Last Modified: 18 Nov 2014 03:42
URI: http://eprints.utas.edu.au/id/eprint/6636
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