Chondroitin Sulfate Testing (AOAC)

Chondroitin sulfate is a major component of connective tissue and is widely used in dietary supplements to promote healthy joints and flexibility. Owing to the complex nature of chondroitin sulfate, many different analytical methods have been developed and used for characterization and determination of purity and label claim verification in finished supplement forms such as cetyl pyridinium chloride (CPC), reverse phase HPLC without hydrolysis, size exclusion HPLC, and carbazole reaction. However, these methods suffer from lack of specificity and selectivity, often resulting in positive interference from impurities and adulterants.

Eurofins’ Success

Eurofins Petaluma has performed an in-house validation of the methodology published in the Journal of AOAC for determination of chondroitin sulfate content in raw materials and dietary supplements by HPLC with UV detection after enzymatic hydrolysis (Enzymatic HPLC). Eurofins has applied this method for determination of chondroitin sulfate in raw materials, human supplements in tablet/capsule/drink forms, and animal supplements in tablet/powder/biscuits/chewables/drink forms with high degree of accuracy and precision.

Method Summary

Chondroitin sulfate in raw materials and dietary supplements is hydrolyzed with chondroitinase AC enzyme after aqueous extraction. This treatment selectively hydrolyses chondroitin sulfate A and C to its unsaturated disaccharide units. The resulting disaccharide units are then separated and quantitated using gradient elution ion-pairing HPLC with UV detection at 240nm. The specific unsaturated disaccharides that respond to the enzyme are found only in chondroitin sulfate A and C. The presence of the disaccharides is confirmation for identity. Disaccharides are summed to determine the level of chondroitin sulfate. Due to it high selectivity, Enzymatic HPLC assay is not affected by possible contaminants and adulterants such as carragennan, dextran sulfate, other related GAGs and proteins, thereby resulting in accurate quantitation of chondroitin sulfate content in the test materials.

Due to it high selectivity, Enzymatic HPLC assay is not affected by possible contaminants and adulterants such as carragennan, dextran sulfate, other related GAGs and proteins, thereby resulting in accurate quantitation of chondroitin sulfate content in the test materials.

Why Enzymatic HPLC?

Enzymatic HPLC method is highly selective, rugged, accuarate, repeatable and precise for quantitation and identification of chondroitin sulfate in raw material and dietary supplement matrices.

1 "Determination of Chondroitin Sulfate Content in Raw Materials and Dietary Supplements by High Performance Liquid Chromatography with Ultraviolet Detection after Enzymatic Hydrolysis", Ji et tal., Journal of AOAC International, Vol. 90, No. 3, 2007, 657-669.

Chondroitin Sulfate Testing (USP)

Chondroitin Sulfate belongs to a group of complex polysaccharides known as glycosaminoglycans (GAGs) or mucopolysaccharides. GAGs are present in the tissue of vertebrate animals (e.g., bovine trachea) and have mainly a linear structure with a repeating unit containing D-glucuronic acid and N-acetylgalactosamine. The N-acetylgalactosamine is either sulfated in the C-4 position (Chondroitin Sulfate A) or the C-6 position (Chondroitin Sulfate C). Chondroitin Sulfate, in conjunction with glucosamine and MSM, is used as a dietary supplement to slow the progression of osteoarthritis.

Due to the complexity of Chondroitin Sulfate, many different analytical techniques are used for its characterization and determination of purity. Eurofins Scientific offers the following tests for Chondroitin Sulfate to clients requiring characterization of their material according to the USP monograph:

The content of chondroitin sulfate (total glycosaminoglycans) are determined using a Brinkmann / Metrohm titrator and probe colorimeter. This instrumentation has been set up to do the CPC (cetylpyridinium chloride) turbidimetric titration. In this procedure the anionic Chondroitin Sulfate forms an insoluble complex with the cationic CPC. After the end point, where the Chondroitin Sulfate is completely complexed, the addition of more CPC causes flocculation. Typically a 5g sample is sufficient for analysis using this method. In addition, the titration can be used to analyze simple finished products, for example chondroitin sulfate / glucosamine tablets. Since we started using the titration, over 10 years ago, the results for a raw material control sample have shown a precision of 1.5% (RSD).

The CPC titration effectively compliments other Chondroitin Sulfate purity determinations offered by Eurofins Scientific. Specifically, these are AOAC enzymatic HPLC and carbazole reaction. These techniques can be used to confirm the results from the CPC titration or help resolve differences when comparing data from other methods. For a complex natural material like Chondroitin Sulfate, the highest quality results are obtained when data from different methods / techniques agree.

Chondroitin Testing Package

Chondroitin sulfate belongs to a group of complex polysaccharides known as glycosaminoglycans (GAGs) or mucopolysaccharides. GAGs are present in the tissue of vertebrate animals (e.g., bovine trachea) and have mainly a linear structure with a repeating unit containing D-glucuronic acid and N-acetylgalactosamine. The N-acetylgalactosamine is either sulfated in the C-4 position (chondroitin sulfate A) or the C-6 position (chondroitin sulfate C). Chondroitin sulfate, in conjunction with glucosamine and MSM, is used as a dietary supplement to slow the progression of osteoarthritis.

Due to the complexity of chondroitin sulfate, many different analytical techniques are used for its characterization and determination of purity.

The CPC titration cannot differentiate between chondroitin sulfate and other GAGs or large molecular anions, such as carrageenan, proteins, and surfactants. Using CPC alone does not ensure the quality of your product. A combined testing package of CPC and enzymatic HPLC will assure identity and confirm the purity of materials. Enzymatic HPLC is highly specific and not subject to positive interferences. Raw material analyses are reported on the dry weight basis (following moisture determination) in accordance with the USP monograph.

To determine purity and identification of chondrotin sulfate Eurofins is offering the following packaged tests:

By offering batched analyses Eurofins can offer competitive pricing while ensuring purity and identity of chondroitin raw materials. Methods can be applied to most dietary supplement matrices.

 

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