The Laboratory Notebook

Answering important questions about quality.

By Robert Green

Over the last few months the most frequently raised topic has been the analysis of chondroitin sulfate. With several analytical methods currently in use, confusion reigns supreme. In an attempt to sort matters out, we devote this column to a discussion of the product and its analysis, including a review of the analytical methods now in use.

Chondroitin sulfate is a major constituent of cartilage. It provides structure, holds water and nutrients, and allows other molecules to move through cartilage. The letter property is of particular importance since there is no blood supply to cartilage. In degenerative joint disease, such as osteo­­arthritis, there is a loss of chondroitin sulfate as the cartilage erodes. With an estimated 21 million adult suffers in the U.S., the interest in chondroitin sulfate is no surprise.

Chondroitin sulfate belongs to a family of heteropolysaccharides called glycosaminoglycans or GAGs. Glycosaminoglycans are also known as mucopolysaccharides. This macromolecule naturally co-exists with several other known GAGs, including keratan sulfate, heparin, heparan sulfate and dermatan sulfate.

Chondroitin sulfate is not one molecule, but rather, a polymer made up of linear repeating units containing D-galactosamine and D-glucuronic acid. This factor is critical in the analysis of chondroitin, which is de­scribed later. As a polymer, chondroitin sulfate does not have a single molecular weight, but rather, a distribution of molecular weights. Therefore, a proper reporting of the molecular weight of a sample is at or above a specified dalton amount (e.g., > 5000 daltons) and not one number. The molecular weight of chondroitin sulfate in the human body is > 50,000 daltons, although for supplements, material above 5000 daltons is considered acceptable. Chondroitin contains about 15 to 150 basic units of D-galactosamine and D-glucuronic acid.

Chondroitin sulfate is found in humans in the cartilage, bone, cornea, skin and the arterial wall. This type of chondroitin sulfate is sometimes referred to as chondroitin sulfate A or poly-galactosaminoglucuronoglycan sulfate. Chondroitin sulfate C, primarily found in fish and shark cartilage, but also in humans, is also made up of linear repeating units of D-galactosamine and D-glucuronic acid. Chondroitin sulfate C is sometimes called chondroitin 6-sulfate. Chondroitin sulfate B is found in skin, heart valves, tendons and arterial walls. It is also referred to as dermatan sulfate, and is made up of linear repeating units containing D-galactosamine and either L-iduronic acid or D-glucuronic acid.

The major source of chondroitin sulfate used in nutritional supplements consists of bovine trachea and pork by-products. Shark cartilage is also used. Supplements are usually isomeric mixtures of chondroitin sulfate A and chondroitin sulfate C.

Various methods exist for the extraction and purification of chondroitin sulfate, all designed to selectively concentrate it to the desired purity level. This is an exacting pro­cess, for over-processing breaks down the chondroitin below 5000 daltons, to a point where it should no longer be considered chondroitin. We see this frequently, and this is what usually constitutes the “cheap stuff.”

The process usually starts with hammer milling, followed by acid-pepsin digestion to remove the animal protein, and acetone extraction to eliminate the fat. The material is then pulverized by ball milling into fine powder, which is then dried on large racks.

There are many analytical methods used to analyze chondroitin sulfate, and unfortunately several are in wide use today. Some are simply not appropriate in today’s high-tech analytical world. Before reviewing these methods, let’s keep two principles in mind. First, chondroitin is not a single entity, but rather, a polymer, so techniques designed for single component products are not appropriate. Second, the analytical goal is to use a method that is selective for the item of interest. That is, the method reports the de­sired analyte and not anything else that may be present, either naturally or added to “fool the tester.” Here are the methods most in use today with our editorial comments:

Carbozole. This method uses strong acid to break the components apart to separate glucuronic acid. It is then measured by a simple color reaction. This method is not specific. Other GAGs containing glucuronic acid, such as heparin, or even free glucuronic acid on its own, will have the same response as chondroitin. So you cannot be sure that the reported results are reflective of the true chondroitin sulfate content. This method leaves the door wide open for cheaters who might add glucuronic acid from non-chondroitin sulfate sources in an attempt to augment the true quality of their product. Thus, it fails our specificity requirement.

Reversed Phase HPLC. Here the most commonly used analytical technique for nutritional supplements is ap­­­­­plied. The problem is chondroitin sulfate is not a common supplement. As described above, it is not a single entity with one molecular weight, but rather, a polymer composed of a mass of repeating units with many molecular weights. The chromatogram generated from this method shows a single peak (indicating a single molecular weight) but we know this is not reflective of the material. In addition, chondroitin lacks chromophores, which are light absorbing groups required for HPLC analysis. Octane-sulfonic acid is frequently added to improve the chromophore, but this adversely affects the sample. This method fails the polymer principal.

CPC Titration. This is now referred to as the “proposed USP” method. It is based on the formation of turbidity when a reagent called CPC (cetyl pyridinium chloride) reacts with organic anions (negatively charges ions). The cloudier the sample, the higher the score, which is interpreted as a higher chondroitin purity. The method is not specific for chondroitin sulfate, since other GAGs have the same response as chondroitin. In addition, molecules of heavier molecular weight (such as chondroitin sulfate at 50,000 daltons) fall out of solution sooner than lighter materials, so the heavier weight chondroitin (which more closely resembles chondroitin found in the body) tests inferior to lighter weight chondroitin. This method fails the specificity principal. It also can only be used for independent raw materials and not finished products.

Size Exclusion HPLC. Here the material is sent through a column filled with beads having pores of many sizes. Polymer molecules of different sizes take different amounts of time to get through the column, thereby separating them by size. The range of mo­lecular weights is determined by the elution time. Using a suitable derivatizing reagent, chondroitin sulfate will form a chromophore enabling the various sized polymers to be identified using a UV detector. This method passes both the polymer and specificity test, and is our method of choice. It works for both raw materials and finished products.

Because chondroitin sulfate is a polymer and not a single entity, its characterization is very difficult, and no one method can yield the definitive answer. Absolute certainty can only be had by running several analytical tests. If, on account of practical expedience, you are limited to one test, we think size exclusion HPLC is the way to go.NW