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Reagents for Chondroitin Sulfate Analysis

Analysis for the presence of chondroitin sulfates (CS) in raw materials and commercial products is important in areas such as pharmaceuticals, nutraceuticals,1 and dietary supplements. Accurate determination of CS amounts is difficult because of several intrinsic properties of chondroitin sulfates, such as wide variation in molecular weights of CS polymers, their strongly anionic nature, and their very low UV absorbance. Various historical analytical methods for the determination of CS content include cetylpyridinium chloride titration, the Alcian Blue method,2 and the carbazole method, as well as techniques that do not chemically modify CS such as size exclusion chromatography. However, these methods do not readily distinguish CS from other, similar glycosaminoglycans such as heparan sulfate and keratin sulfate.3

More specific analytical methods for CS determination have made use of chondroitinases, enzymes specific for digestion of chondroitin sulfates, to give smaller oligosaccharides that can be analyzed by subsequent methods. Various procedures have been developed that couple chondroitinase digestion of CS to downstream methods such as ion-pairing liquid chromatography,3 a colorimetric thiophene microplate assay,4 and strong anion-exchange HPLC.5

1. Volpi, N., Quality of different chondroitin sulfate preparations in relation to their therapeutic activity. J. Pharm. Pharmacol., 61, 1271-1280 (2009).
2. Frazier, S.B., et al., The Quantification of Glycosaminoglycans: A Comparison of HPLC, Carbazole, and Alcian Blue Methods. Open Glycoscience, 1, 31-39 (2008).
3. Ji, D., et al., Determination of Chondroitin Sulfate Content in Raw Materials and Dietary Supplements by High-Performance Liquid Chromatography with Ultraviolet Detection After Enzymatic Hydrolysis: Single-Laboratory Validation. J. AOAC Int., 90(3), 659-669 (2007).
4. Toby, T.K., et al., Detection of native chondroitin sulfate impurities in heparin sodium with a colorimetric micro-plate based assay. Anal. Methods, 4, 1488-1491 (2008).
5. Sim, J.-S., et al., Quantitative analysis of chondroitin sulfate in raw materials, ophthalmic solutions, soft capsules and liquid preparations. J. Chromatogr. B, 818, 133-139 (2005).