Moving beyond reference standards

Along the path to substantiating claims for a product and achieving conformance with GMPs, savvy manufacturers turn to two additional tools: botanical and analytical reference materials. Jim Lassiter explains.

A reference standard is a highly purified compound that is well characterized.?

This quote comes from a document published by the US Food and Drug Administration titled: ?Reviewer Guidance, Validation of Chromatographic Methods.? The date of the document is as important as the title and reference—it was issued shortly after the passage of DSHEA in October of 1994; however, this same definition remains in existence today in other guidance documents and discussions.

The definition is an important beginning because it reflects not only the regulatory perspective but also the understanding of the scientific community at large. Knowing what a ?reference standard? is—and what it is not—is the basis for this discussion.

Staking your claim
Looking back, we find that along with DSHEA 10 years ago, the dietary supplements industry in the US received a host of opportunities. One of the most significant is the opportunity to produce and market beneficial products and make appropriate claims for them. The simple requirement is that you must have substantiation for these claims. The determination of what precisely constitutes ?substantiation? resides somewhere in the regulatory future.

Rest assured, however, that any resulting definition of ?substantiation? will include the obvious—that the claim can apply to your product only if your product is the one that demonstrated the claimed effect. This means that you have work to do if you wish to make any meaningful claims.

The first step in any demonstration or differentiation of the benefits of your product is the characterisation of it. When the FDA published the Proposed Rule on Dietary Supplement GMPs, the agency mandated the testing of products using ?scientifically valid methods.? This brings us all back to the definition of what a reference standard is.

Reference standards are the most commonly used material in establishing scientific validity. They are useful in evaluating process controls for establishing the validity of your test methods and for testing specific chemicals. The problem starts when your product is a botanical or botanical extract, and not a single chemical entity. If it is a complex matrix consisting of hundreds of phytochemicals, the levels of which vary from harvest to harvest, how do you test and characterise it?

Testing and characterising
One logical response the industry employs in order to characterise a botanical is to use ?standardised? extracts. These extracts borrow a bit from the reference standards model in that they typically focus on one of the phytochemicals present and dismiss the rest. While it is true that you can use reference standards to develop test methods to quantify a single chemical marker in a botanical material, this is probably not enough.

Without debating the wisdom of using these standardised extracts, the point is that the effort to create them focuses on testing only one thing present in a complex extract. However, your product is not one chemical, so testing it therefore poses a challenge.

Next is the matter of ensuring that the method for testing your product is ?scientifically valid.? When looking at your method, you must remember that the material is a highly complex composition. Using a reference standard here provides validity but the real issue is that the matrix in which that single chemical exists (ie, the single chemical that is by definition the reference standard) is complex and can pose some interesting challenges in creating this scientifically valid method. The mandate for using scientifically valid test methods will not go away when the final rule on GMPs passes, probably later this year.

Additional tools
A real solution to the problem is a multilayered approach. Along with using a reference standard, you should use BRMs and ARMs. Never heard of them? Well, the concept is relatively new.

BRMs are Botanical Reference Materials and ARMs are Analytical Reference Materials. These are the additional tools necessary to achieve conformance with GMPs while allowing you to better characterise, understand and differentiate your product. BRMs and ARMs work in concert with, but not in place of, reference standards. In other words, they are not interchangeable.

This multilayered approach results not only in a scientifically valid method for testing your product but you also gain legitimate characterisation of your product. A last benefit is that when you use BRMs and ARMs, you maximise the opportunity to identify any adulteration of the product.

Recall that the FDA repeatedly cited the digitalis-contaminated plantain material in its discussion of why GMPs are necessary. Being able to show that your methods are valid goes a long way toward finding those adulterants. Therefore, a reference standard is likely not the complete answer to your situation.

The dietary supplements industry needs all three tools in order to conform to the upcoming regulations. To see how it all works, let?s look at the definition of reference material:
?Material or substance one or more of whose property values are sufficiently homogeneous and well established to be used for the calibration of an apparatus, assessment of a measurement method or for assigning values to materials.? (From the National Institute of Standards and Technology)

While the wording may appear awkward, the key phrase is ?one or more of whose property values are sufficiently homogeneous and well established.? In other words, reference materials are well characterised. To see how the definitions apply to BRMs, ARMs and reference standards, we begin at the least-specific end of the spectrum—BRMs—and proceed through ARMs to reference standards.

Botanical reference materials
BRMs most effectively work on botanical materials that have not gone through an extraction process. Using BRMs provides you a piece of assurance that the plant or raw material you purchased matches what you wanted. As a first step you would use a BRM to compare your material either macro- or microscopically. BRMs can exist as voucher specimens of specific botanicals and therefore are useful as comparatives to the plant you received. The characterisation here is in the known source of the voucher specimen and known genus, species and site where the plant was grown.

A well-documented BRM will allow microscopic evaluation. In addition, because it is frequently not possible even microscopically to discern whether the fine brown powder you purchased is from a specific plant, you can still use the BRM to assess your material via thin layer chromatography (TLC). How is this possible? BRMs meet the definition of reference materials in that their properties are ?homogeneous and well established.?

The BRM comes with the information you need regarding its characterisation. This information should include the assurance that the material is a specific genus and species of the botanical, the macro- and microscopic evaluation, and a TLC presentation of the BRM (the fingerprint) for this specific plant.

Remember, though, that the BRM is not an end in itself. Its characterisation cannot take into account all the variables of growing conditions from which the plant emerged, nor does it need to. However, it is homogeneous and more than one of its properties is well established.

Is this evaluative tool quantitative? Not really. You may see intensity differences in TLC bands when comparing your material to the BRM, but this is not necessarily an indication of superiority or a problem. You may also see different bands in the TLC comparison of your material to the BRM. However, the fact remains that multiple properties of the BRM are well established. Thus, the BRM is indicative of what a specific genus and species of plant should look like. BRMs become a vitally important first step in assessing your material.

Analytical reference materials
ARMs are useful on two fronts. First, they help you determine the scientific validity of the method you choose to employ in testing the product you purchased. While you may apply ARMs to whole-plant materials, they are better suited to assess the extracts. Second, using ARMs assists you in characterising your products. Just like BRMs, ARMs come with full characterisation of their well-established property values. You also use ARMs to check your analytical methods, thus assuring the long-term integrity of quality assurance programmes.

Looking at what the National Institute of Standards and Technology identifies as their standard reference materials, we find that they have ?carefully assigned values for chemical composition and physical properties.? ARMs fit neatly into this definition. Using ARMs gives you both a method assessment capability and a product comparison. ARMs come with defined levels of the chemicals present in a matrix. Thus you could discover that the material you received contains twice the amount of valerenic acid (for example) compared to the ARM you used. While this does not provide a judgement as to your material?s efficacy, it is an indicator of what you have on a quantitative basis.

If you have nonextracted material, you can use the ARM to determine whether your test method is suitable for this type of material. You can also see what is present in your material at relative levels. Comparing the various peaks from the ARM to the peaks from your purchased product affords you better understanding of what you have, and how good your analytical methods are. Even better than that, you also use ARMs to determine the consistency of the supply you receive because ARMs tell you whether your method remains valid and that the results you get in testing your product are equally valid.

Reference standards
Last comes the reference standard. This highly purified single compound is used to quantify a specific phytochemical present in either the plant or the extract you purchased. Reference standards are the tools necessary to ensure that your method works for the single chemical.

Reference standards are the tools that simultaneously mean the most and the least. Specific focus on a single chemical entity selected from the dozens normally present in a plant often flies in the face of the reason botanical supplements exist. Moreover, the fact that a reference standard is a single chemical entity means you do not have in-depth information about the effects of the botanical matrix on your method.

Still, the use of reference standards is a vital component to accurately identifying the levels of the single chemical and whether the method you use to make this determination is, in fact, appropriate.

International ramifications
These tools know no borders. As you think about the use and application of BRMs and ARMs, you should think to the future not only in the US but internationally. The concept of using these new reference materials in addition to the appropriate application of reference standards is gaining momentum. Consider that Australia is reviewing its assessment criteria for botanical supplements. There is a strong likelihood that the methods used in assessment by the Australian Therapeutic Goods Administration will mandate method assessment to ensure accurate characterisation of product. This means you will need to use BRMs and ARMs.

Canada is developing regulations and preparing to implement monographs for the class of goods known there as Natural Health Products. Using BRMs and ARMs allows you to demonstrate that your product conforms to the upcoming Canadian monographs. The use of these important tools is likely to become an important factor to international regulatory compliance—let alone what you will need in the US. As to the future, the methods you use to test your product will come under increasing scrutiny. Likewise, adequate characterisation of your product or the material you wish to incorporate in your product will come more into the regulatory spotlight. In the end, using BRMs and ARMs is more than desireable; it will become mandatory as the regulatory noose tightens around the dietary supplements industry.

Jim Lassiter has more than 25 years of experience in regulatory affairs and is currently director of consulting and regulatory services at ChromaDex, a global provider of high-purity reference standards and regulatory consulting services. Respond: [email protected] Correspondences will be forwarded to the author.

Creating a marketable difference

If you apply botanical and analytical reference materials and reference standards, you have the opportunity to better promote your product and gain the market edge you seek. You only need to be willing to put in the necessary effort to verify the efficaciousness of your product. As a case study, let us assume you know of a botanical that you believe can address stress-related sleep disorders. You obtain some of the botanical material of interest and first test it using a BRM to determine that what you received is what you want.

Next, whether you extract it yourself or have the extraction performed on your behalf, you use the ARM to see if your method for testing the extract works. Now you can validly determine the relative levels of various chemical components in the extract. Thus, you use the BRM and ARM as tools to attain regulatory compliance against the ?scientifically valid? requirement while verifying what you have in the extract is what you want.

If you really are with it, you use these data to insist on a relatively narrow range of variation among the phytochemical ratios present in your extract. This provides assurance that your product is sufficiently well characterised and consistent enough to go forward with substantiation.

As you perform evaluative studies to generate your substantiation, reference standards come more into play. These allow you to develop and apply methods that focus on the quantity of specific chemicals present in the product. This knowledge may lead you to understand if any one or more of these are responsible for the benefits derived.

In the end, you are able to demonstrate the scientific validity of your method and have knowledge that your well-characterised product works. This characterisation is what sets your product apart and is the one demonstrated to work. You also have a potential opportunity for focus on a single chemical in the product for future endeavours. All of this knowledge initiates with the use of BRMs and ARMs.


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