Quantifying Chondroitin Sulfate in Complex Matrices
Explore new scientific developments happening at Eurofins in Des Moines, IA for quantifying chondroitin sulfate in complex matrices like pet food and treats.
Osteoarthritis (OA) is a degenerative disease found in humans and animals. It involves the breakdown of cartilage where those with it describe symptoms of joint pain, bone spurs, and reduced flexibility.(1-3) Owners or individuals might turn to nutraceuticals that may support relief in some with these symptoms. One such nutraceutical is chondroitin sulfate.
Chondroitin sulfate (CS) is an anionic, glycosaminoglycan macromolecule found in cartilage and the extracellular matrix of a wide variety of animals. CS has been used in conjunction with glucosamine to potentially help with these symptoms in both humans and animals. (4)
There are several methods for analyzing CS used today. The first is a cetylpyridinium chloride (CPC) titration that determines the amount of CS present based on the turbidity of the solution. The method is a straightforward, cost effective, and quick analysis for pure CS and raw materials. However, it is easily prone to adulteration by other similar molecules such as glucuronic acid or carrageenan. The second method is the official AOAC 2015.11 method using LC-UV disaccharide profiling after digestion with Chondroitinase. The analysis uses UV wavelength ranges from 220-240 nm. This method is a significant improvement from titration as it allows for targeted analysis of CS and the disaccharide profile information for clients based on their source. Adulteration and fraud for this method is less common as the Chondroitinase enzyme allows analysts to specifically digest and analyze during LC separation. The AOAC method has been successful for supplements and pure materials but falls radically short in complicated matrices. Primarily because other analytes in the matrix will absorb the same wavelength of light creating interferences. Without a sufficient extraction or clean-up steps taken, CS disaccharides can get lost in the noise of the method. This has been a significant problem for pet food and treat clients who need to make label claims on their formulations before they can send them to market.
Eurofins Nutrition Analysis Center in Des Moines, IA has recently developed a method for CS using LC-FLR to meet the challenges that complicated matrices, like pet food and treats, bring to CS quantification. The method performs a gentle extraction followed by digestion with Chondroitinase to produce the disaccharides of interest. From there, the disaccharides are derivatized and cleaned using SPE. The isolated, fluorescently tagged disaccharides are analyzed using HILIC column technology compared to the ion-pairing reverse phase columns currently used in the official AOAC method. The combined improvements allow analysts to quantify low levels of chondroitin down to 50 mg/kg for total chondroitin in pet foods, wet or dry, and pet treats.
Figure 4: Chromatograms of chondroitin sulfate during analysis. Top: Standards of chondroitin sulfate. Middle: Pet food sample containing chondroitin. Bottom: Chicken meal containing chondroitin.
The new CS method developed at Eurofins is a novel development that tackles industry needs with newer technology and methodology. It opens the door for newer joint formulations for pet treats that cannot be performed by any other lab currently. These new developments and improvements at Eurofins are the tip of the spear to serve our clients for their quality and label claim needs. The method is approved for pet foods, treats, and meals containing terrestrial sources of chondroitin sulfate, such as chicken, porcine, and bovine.
Reach out today to learn about other joint nutraceuticals, like glucosamine, and how we can meet your testing needs.
Meet the Author
Dr. Luis A. Camacho III
Dr. Luis A. Camacho III is a method development scientist within the Vitamins and Special Analysis business unit of Eurofins Nutrition Analysis Center. Luis is a leading method development scientist for chondroitin sulfate analysis and assisted with glucosamine method development. His approach to method development examines what chemical properties can be used for easier and quality analysis during extraction, isolation, and UPLC analysis.
Luis earned his PhD at Iowa State University in physical organic chemistry. He specialized in solid-phase peptide chemistry and used UPLC and HPLC instrumentation to analyze his final peptide products. His work was instrumental for developing thioamide protecting groups and installation of amidine functionalities along the backbone of single peptide chains.
Resources
- National Institute of Arthritis and Musculoskeletal and Skin Disease – National Institute of Health; Health Topics - Osteoarthritis (https://www.niams.nih.gov/health-topics/osteoarthritis)
- Center for Disease Control (https://www.cdc.gov/arthritis/osteoarthritis/)
- National Institute on Aging (NIH) (https://www.nia.nih.gov/health/osteoarthritis/osteoarthritis)
- National Center for Complementary and Integrative Health (NIH) (https://www.nccih.nih.gov/health/glucosamine-and-chondroitin-for-osteoarthritis-what-you-need-to-know)
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