Glucosamine Testing in a Forest of Complexity
Glucosamine (GlcN) is a common amino sugar found in a variety of glycosaminoglycan and glycoprotein macromolecules. It serves as a building block for many of these important macromolecules found in the extracellular matrix necessary for the lubrication of joints. More specifically, GlcN is a key component to the biosynthesis of hyaluronic acid and aggrecan which attract water due to their high anionic character.
In pets, as in humans, GlcN has been used with chondroitin sulfate in nutraceuticals to support joint health and mobility. GlcN supplementation supports joint health by encouraging hyaluronic acid production for joint lubrication and acting as an anti-inflammatory (4). These promising results give both humans and animal owners possible options for supplemental joint health support.
Despite promising results, historically GlcN testing has been limited to pure materials and supplements. AOAC 2005.01 and USP-NF monographs are the current methods used for analyzing GlcN in pure materials and supplements. Both methods use HPLC-UV to quantify the amount of GlcN in matrices. While the methods are very useful, they do have some limitations when applied to more complex matrices. Complicated matrices or matrices that are highly processed with long ingredient lists, like pet food and pet health supplements, will produce many interferences that increase noise and reduce sensitivity. This is normally not an issue with the original AOAC or USP methods as they were designed for cleaner matrices that do not require complicated clean-up procedures. GlcN can remain bound in matrices and other macromolecules. Distinguishing bound and free GlcN is important for pet food producers to know what is naturally available in meals and the amount they add to their formulations.
Third, if an extraction is performed and derivatization is required to distinguish GlcN from the matrix as in AOAC 2005.01, then other nucleophilic molecules (galactosamine or amino acids) can also attach to the more commonly used succinimide reagents. All these problems put together make analyzing GlcN a challenge for pet food and pet health supplement clients to meet label claims on their formulations.
Eurofins Nutrition Analysis Center in Des Moines, IA, has recently developed a method for testing GlcN using LC-MS. The method allows for selective mass detection and resolution of GlcN in a wide variety of matrices. GlcN is extracted by acid hydrolysis and derivatized for retention on reverse-phase column technology. Pet foods and pet health supplements containing GlcN and chondroitin are capable of being separated from one another during analysis. Even mannosamine, another amino sugar used in glycoproteins, is resolved to prevent interferences and give accurate results down to 50 mg/kg in a wide variety of matrices. Matrices validated for this method include dry and wet pet foods, soft treats and chews, meals, supplements, and pure materials.
Reach out today to learn about joint formulation testing, including chondroitin sulfate, and how we can meet your testing and labeling needs.
Meet the Authors
Ben Pointer is a Method Development Scientist at Eurofins Nutrition Analysis Center (ENAC) in Des Moines, IA. Ben specializes in liquid chromatographic methods, with a focus on amino acid, sugars, and other food additives in human and pet food. Ben has developed several methods at ENAC, including the glucosamine by LC-MS method presented here.
Dr. Luis A. Camacho III is a Method Development Scientist within the Vitamins and Special Analysis business unit of ENAC. Luis is a leading method development scientist for chondroitin sulfate analysis and assisted with glucosamine method development.
References
- 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)
- Jerosch, Jörg, Effects of Glucosamine and Chondroitin Sulfate on Cartilage Metabolism in OA: Outlook on Other Nutrient Partners Especially Omega-3 Fatty Acids, International Journal of Rheumatology, 2011, 969012, 17 pages, 2011 (https://doi.org/10.1155/2011/969012)
