June 10, 2007
The recent ConsumerLab.com report that 7 out of 11 chondroitin products failed to meet label claims did not come as a surprise to analytical experts who have been lamenting a common practice in the supplement industry - the use of inappropriate methods or methods that are not fit for their intended purpose. With so much media focus on poor quality, unsafe and non-effective products, this should serve as a wake-up call to our industry. Simply put, the use of non-specific methods of analysis and reliance on supplier Certificates of Analyses (C of A’s) without supporting data are not sufficient standards of evidence for identity and purity when evaluating raw materials.
It has been known for several years that using the cetyl pyridinium chloride (CPC) titration method alone is not adequate for identification or quantification of chondroitin materials. Many people have been under the false impression that by using the CPC method they are being compliant with the USP monograph for chondroitin sulfate (CS). This is not the case. Unless all the tests mandated in the monograph are being performed together, you cannot claim USP compliance. The analytical methods within the USP monographs are not “stand alone” methods. The monographs are a compilation of several different testing methodologies, each with a specific purpose, that together serve to set and test product specifications for identity, content and purity.
As people began to realize the CPC method alone was not adequate for testing finished products. they shifted to size exclusion chromatography (SEC) methods. Some laboratories claim that these methods are more specific, however SEC is based only on the size of the molecule of interest, so any molecules that fall within the same molecular weight range as CS will give a response similar to that given by chondroitin itself. Again, this is a non-specific method. The use of dimethyleneblue (DMB) as a derivatization reagent in combination with SEC can improve specificity by narrowing results to only those polyanionic polymers that fall within the correct molecular weight range. Unfortunately, this is not much help since the most common adulterants of CS are carrageenan and dermatan sulfate, both of which are polyanionic polymers that have similar molecular weights.
Thus, when used alone, this method is still not adequate for positively determining the identity, content or purity of CS raw materials or finished products. In fact, a review panel of industry and government experts convened in November 2003 by AOAC International reported that the CPC and SEC methods demonstrated “poor specificity, an inability to separate CS from CS-related impurities such as dermatin or heparain, and was subject to significant interference by other water-soluble polymers.”
The confusion does not end there; as methods became suspect, the industry continued to look for other analytical options, but unfortunately these “new” methods also lacked specificity for CS.
Capillary electrophoresis (CE) separates on charge-to-size ratio, with many related GAGs possessing approximately the same charge-to-size ratio, so this method does not afford much more selectivity than other methods. An isocratic reverse phase HPLC method run at a low wavelength has also been employed. The rationale for this method still remains a mystery, as the target analytes elute in or near the void volume – which fundamental Analytical Chemistry 101 teaches us is not good if you are actually trying to measure the analyte with any degree of accuracy or certainty.
The bottom line is that of all these methods lack specificity or are simply not suitable for determination of CS. They are not appropriate to establish identity nor should they be used to quantify CS content in raw material and finished products.
Despite the fact that almost a decade ago, enzymatic digestion followed by HPLC-UV analysis was identified as a specific, stand alone method, it has never been widely accepted by industry. Perhaps the accurate but lower yielding results were simply not palatable for industry. When coupled with the added cost and time required to run an enzymatic digestion, it seems industry has opted to continue to employ unsuitable methods that tend to produce meaningless, but high, numbers.
Some companies have even adopted the argument, (and myth) that, “enzymatic hydrolysis might not be accurate if the molecular weight is not taken into consideration and thus, some molecular weight formulations of chondroitin are not suitable for this analysis.” This is simply untrue. The ability of the enzyme to hydrolyze chondroitin is based on a total weight to weight ratio and is not dependent on polymer size. It simply does not matter whether your material is one giant polymer or composed entirely of small oligomers, this method will work.
Why are we so confident? How do we know the method works?
In 2003, an AOAC expert review panel for chondroitin sulfate met to evaluate the existing methods being used in the industry for CS. By consensus it was determined that the enzymatic digestion HPLC-UV Method was the only method that was suitable to, “accurately quantify chondroitin sulfate A and chondroitin sulfate C in dietary supplements and in-process materials, as a neat product and in the presence of potential interferences including heparin, heparin sulfate, keratan sulfate, dermatan sulfate (i.e., chondroitin sulfate B), hyaluronic acid, carrageenin, glucosamine, MSM, and minerals such as calcium and manganese”.
In the enzymatic digestion HPLC-UV method, the chondroitin sulfate is first selectively hydrolyzed by chondroitinase AC enzyme, which is specific for chondroitin sulfate. With the exception of hyaluronic acid, chondriotinase AC only reacts with CS and will not react with carrageenan or other GAGs.
The digestion products are unsaturated non-sulfated and sulfated disaccharides, which can be separated and quantified by reverse phase HPLC using an ion-pairing agent in the mobile phase. The resulting disaccharides are unsaturated so selective UV detection at 240 nm is possible. This method has been shown to be accurate and precise for the quantitation of CS in both raw materials and dietary supplement finished products. Through conducting a single laboratory validation (SLV, see AOAC Guidelines) of the method, it was established that the assay is not affected by the presence of many other ingredients or adulterants that may be present. This is the only method that has been proven as fit for the intended purpose of establishing the identity and quantity of CS in these materials.
So why did 7 of 10 CS Products fail to meet Label Claim?
Perhaps manufacturers had ill-fated confidence in C of A’s generated by using the wrong method. Chondroitin is not the exception, it is the norm. This situation exemplifies what can happen when a test is used outside of its scope and applicability. The most widespread version of this practice is the extraction of an analytical test method from a Monograph, such as the USP.
Should the industry wait for another watchdog group to expose bad science (misbranded products) in the industry or have we finally had enough?
Shall we continue to be reactive and watch the negative press erode our profits as uncertain consumers turn away from supplement use or is it time to become proactive and invest in the science needed to ensure longevity?
It is time for the Dietary Supplement Industry to implement a Workplan for Quality. To do this in a cost effective, efficient and informed manner we need to build stronger bridges between the raw ingredient suppliers, manufacturers, Contract Analytical Labs (CALs) and scientists.
To this end, a new group, the Analytical Research Collective (ARC) was established by scientists with expertise in dietary supplement quality assessment for the purpose of supporting Industry in the development and implementation of a quality workplan. Fundamentally, ARC believes that a team approach is needed to solve quality problems in a cost effective and efficient, yet scientifically rigorous manner. Nobody vested in the Dietary Supplement Industry can afford the continued bad press resulting from inferior products.
This issue obviously harms everyone’s bottom line. If consumers lose confidence in our industry and stop buying, there will not be a need for any of us anymore. We won’t need raw material suppliers, CMO’s, testing labs, certification bodies, monograph authorities or distributors. However, by working together and addressing the issues head-on, we can solve many of these problems. They are not all easy and not all fast. No one company can do it alone. Many hands make light work and by sharing the costs of research and development, the industry can thrive as a whole, on an even playing field.
Let’s face it though; many products have a rapid life cycle in our industry. St. John’s Wort does not have the same market share it had in the late 90’s. Products come and go. In these cases, it may not be reasonable to take a method through a full collaborative study to OMA status. A USP monograph might not make sense for a product that has an uncertain future. However, a peer-validated method by independent scientists would be an excellent solution. In some cases, only a Single Laboratory Validation (SLV) is needed. However, in all cases, sound science, method development, validation and fitness for purpose must be addressed.
It does not matter if you are developing a method for a proprietary formulation or for an industry mainstay. The basic ground rules are the same. ARC understands the issues facing our industry and we care. That is why we came together to offer our expertise to the industry as a whole. We are here to help the grower, distributor, CMO, independent testing labs, monograph authorities and certification bodies and, yes, even the FDA. It is only by working as a team that we can all thrive in the future.
We are ready to do our part, in fact, we’ve been doing it for years, are you?
For more information on Chondroitin test methods and ARC, please contact Jana Hildreth at 310-920-4517.
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