Single Lab Validation for Chondroitin Sulfate Completed

A single laboratory validation (SLV) of a method to quantify chondroitin sulfate (CS) in raw materials and dietary supplements has just been completed by David Ji at Analytical Laboratories in Anaheim. Chondroitin sulfate (CS) is a negatively charged polymeric glycosaminoglycan (GAG) consisting of alternating uronic acid and N-acetylhexosamine residues connected by β1-3 hexuronidic and β1-4-N-acetylhexosaminidic bonds, and is a major component of connective tissue. Dietary supplements containing CS are widely available and are often used to maintain joint health, and there is some clinical evidence that CS might help treat symptoms of osteoarthritis.

Quantitative analysis of chondroitin sulfate in CS raw materials and dietary supplement raw materials has been extremely challenging owing to the wide molecular weight variation of CS polymers, its poor UV absorbance, and strongly ionic nature. Other related GAGs may be present as impurities or adulterants in CS materials, and thus any analytical methodology designed to quantify CS must be selective for CS in the presence of these other GAGs.
Carbazole reaction, cetyl pyridinium chloride (CPC) titration, and size exclusion chromatography have been used to characterize CS, however these methods can not distinguish between CS and related GAGs, and are subject to interferences in dietary supplement finished products. CPC titration, in particular, has become a popular method for determining chondroitin sulfate purity, however this method not only can not distinguish between CS and other GAGs, but will give positive results for any large moleculer anion, such as carrageenan, proteins, and surfactants, thus making economic adulteration possible.

In the method developed by Ji, the chondroitin sulfate is first extracted in water, then selectively hydrolyzed by chondroitinase ACII enzyme to form unsaturated disaccharides; the resulting disaccharides are then quantified by ion-pairing HPLC with UV detection and summed to yield the amount of chondroitin sulfate in the material. A total of 6 disaccharides are quantified, each differing in either their degree of sulfonation and/or position of sulfonation. The single laboratory validation work determined the repeatability, accuracy, selectivity, LOD/LOQ, ruggedness and linearity of the method, with very good results. In addition to being able to quantify chondroitin sulfate in raw materials and dietary supplements, the profile of the resulting disaccharides may be used to help identify the source of the chondroitin sulfate, which typically comes from bovine trachea, porcine cartilage, or shark cartilage.

Development and validation of the method was funded by NIH-ODS. The SLV manuscript will be submitted to the Journal of AOAC International for publication.

David Ji and Dr. Mark Roman from Tampa Bay Analytical Research will then co-direct the AOAC collaborative study involving twelve international laboratories on this method. If results from the collaborative study are acceptable, the method will be recommended for adoption as an AOAC official method. It is anticipated that the collaborative study will be completed by Spring of 2007.

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