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Analysis of dietary supplements: turbidimetric titration of chondroitin sulfate
Chondroitin sulfate is a dietary supplement which is available as the sodium salt in the form of capsules or tablets. In the field of alternative medicine, the intake of chondroitin sulfate is recommended as treatment for degenerative joint diseases such as osteoarthritis. According to the United States Pharmacopeia, the determination of sodium chondroitin sulfate in dietary supplements is performed by turbidimetric titration using the DP5 Phototrode.
What are dietary supplements?
Dietary supplements are commonly used to complement the daily diet by increasing the intake of ingredients such as vitamins, minerals, amino acids, and metabolites. Generally, they are ingested in the form of capsules, effervescent tablets or syrups. Well known examples of dietary supplement include multivitamins and minerals.
Today, a wide range of products are available whose composition is not only limited to a single active ingredient but includes vitamins, minerals, other nutrients, botanical supplements, as well as ingredients and extracts of animal and plant origin [1]. People use nutritional supplements with the aim of improving general health and also in the hope of treating a specific disease, since it is also believed that their use results, in some cases, in a cure. Recently, an increasing demand by consumers as well as evidence that some of these supplements actually help patients, has increased the production of dietary supplements. In parallel, the need for accurate quality control in the production plant has also increased: rapid, robust and accurate analytical techniques are needed to achieve efficient control in the production line.
Chondroitin sulfate, CS, is a dietary supplement which is available as the sodium salt from drugstores, groceries and pharmacies in the form of capsules or tablets. Although the medical community is not yet convinced of its effectiveness, it is currently used by consumers to treat the pain resulting from osteoarthritis. In fact, it has been shown in medical studies that chondroitin sulfate is helpful in treating this disease, and it was also reported that patients with back and neck pain have also considered the use of this nutritional supplement as an alternative treatment to traditional medicine [1].
Why chondroitin sulfate as a dietary supplement?
Chondroitin sulfate is a constituent of cartilage, i.e. of the structure covering the ends of the bones, which protects them from mechanical friction and absorbs shocks. In particular, cartilaginous proteins lubricate the joints to prevent damage during movement. To improve their shock absorption and lubricating characteristics, polysaccharides are bound to cartilaginous proteins, with each polysaccharide consisting of hundreds of sugar molecules. The function of chondroitin sulfate, a complex polysaccharide, is to provide structure, hold water and nutrients, and to allow other molecules to move through the cartilage. This is an essential property, since no blood is present in the cartilage to allow for exchange of metabolites.
Cartilage proteins are continuously produced by the cells in the joints to replace deteriorated cartilage. As the cartilage degenerates, so the lubricating function is decreased.
As a consequence, the bones are no longer protected by the cartilage, are exposed and can rub together. This may lead to acute pain, a symptom of osteoarthritis, the gradual degeneration of cartilage. Until now, the exact cause of this degenerative disease is unknown and traditional medicine does not yet have an effective treatment to completely stop the cartilage degradation process. Usually, medical treatment simply involves pain management, but sometimes the therapy may not be completely successful and undesired side effects may occur.
In degenerative joint diseases, such as osteoarthritis, there is a loss of chondroitin sulfate as the cartilage erodes. Studies have indicated that chondroitin sulfate may promote healing of bone. In particular, trials have shown that chondroitin sulfate alleviates joint pain and possibly slows the progression of osteoarthritis. The intake of chondroitin sulfate as dietary supplement may be an alternative treatment for patients suffering from this disease. In fact, chondroitin sulfate is not found in the diet in appreciable amounts the primary source being animal cartilage (such as the trachea of cows). The treatment is based on two theories: chondroitin sulfate may provide the raw material to nourish joint cartilage and chondroitin sulfate may block the activity of enzymes that break down cartilage.
Analysis of chondroitinsulfate
Chondroitin sulfate falls into a category of chemical compounds known as glycosaminoglycans, long chains of specialized polysaccharides (or sugars). In particular, chondroitin sulfate is made of repeated units consisting of two compounds, glucuronic acid and N-acetylgalactosamine. In dietary supplements, chondroitin sulfate is present as the sodium salt. The raw material for the pro¬duction of sodium chondroitin sulfate is obtained from the cartilage of healthy, domestic animals used for food by humans, such as cows, pigs and chickens. Sodium chondroitin sulfate contains from 90.0 up to 105.0 culated on the dried basis [2].
According to the United States Pharmacopea, the chondroitin sulfate content is determined by turbidimetric titration at 420, 550 or 660 nm using cetylpyridinium chloride (hexadecyl-pyridinium chloride, CPC, C21H38ClN) as titrant [2]. The addition of CPC to the sample solution leads to the formation of an ion-pair complex with the chondrotin sulfate. The formed product precipitates in the solution leading to an increase in turbidity which can be monitored using a METTLER TOLEDO DP5, DP550 or DP660 Pho-totrode™ immersed in the sample solution.
The Phototrode is a photometric sensor, allowing the monitoring of turbidity and color changes in the sample solution by measuring the attenuation of the light beam passing through the sample due to absorption and light scattering during titration. The attenuated light beam is reflected back to a detector by a concave mirror located at the bottom of the measuring cell.
The exact stoichiometry, i.e. the ratio between the products and the educts, of the precipitation reaction between chondroitin sulfate and the titrant CPC is unknown. A calibration titration is therefore first performed with a standard solution of known concentration (mg/mL) of pure chondroitin sulfate before sample determination. From the titrant con¬sumption to the equivalence point, a factor F in mg/mL can be calculated indicating the amount of chondrotin sulfate in mg titrated with 1 mL of CPC solution. Subsequently, this factor is used to determine the amount of chondroitin sulfate in the samples.
As an example, the content in 400 mg chondroitin sulfate capsules was determined by CPC titration based on the procedure described. To account for inhomogeneity in each capsule, the contents of five of them were mixed together. After accurately mixing the powder, enough samples were taken to obtain approximately 100 mg chondroitin sulfate in a 100 mL volumetric flask. Subsequently, a pH 7.2 buffer and water was added, and the sample was sonicated since the powder is not completely soluble due to the presence of filler, e.g. calcium carbonate, and other additives. The flask was filled up to the mark with water, mixed and finally centrifuged. Aliquots of 5 mL were carefully taken after decantation and they were then titrated with CPC.
The results, given as recovery rate in percentages, were obtained by measuring at different wavelengths, i.e. at 555 and 660 nm using the METTLER TOLEDO Phototrode, and monitoring the turbidity at 420 nm. In addition, the system was tested by titrating a standard solution of pure sodium chondroitin sulfate (Bioiberica, certified value: 101%). The recovery is essentially the same at all three different wavelengths since the deviation lies within the measurement uncertainties (accuracy). Also the repeatability (precision), given as relative standard deviation in %, shows reasonable values, varying from approximately 1 down to 0.4%.
Summary
The determination of sodium chondroitin sulfate in dietary supplements can be performed by titration with CPC, leading to precipitation of CS in the sample solution and by monitoring the turbidity increase during the titration. The analysis can be automated using the DP5 Phototrode. If a large number of samples need to be determined then fully automatic analysis can be achieved using the Rondo 60 sample changer in combination with the titrator.
About the Author
METTLER TOLEDO specializes in the area of precision instruments for professional use. METTLER TOLEDO laboratory instruments are used in research, scientific, drug discovery, and quality control labs, amongst many others in the pharmaceutical, chemical, food and cosmetics industries.
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