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Meeting 100% Ingredient Testing Requirements for Botanical IdentityMeeting 100% Ingredient Testing Requirements for Botanical Identity

June 4, 2013

11 Min Read
Meeting 100% Ingredient Testing Requirements for Botanical Identity

by James Neal-Kababick and Kathryn Lawrence

Under 21 CFR 111, the federal dietary supplement GMPs (good manufacturing practices), manufacturers must establish an identity specification" and ensure that test and examination method(s) used are fit for purpose". Testing of raw materials to fulfill 100-percent identity, especially with botanical extracts or premixes sourced from around the world, is challenging and, often, controversial since many manufacturers would like to see the accountability lie with suppliers instead of themselves.

True characterization of a material can take a lot of work, and may require multiple techniques, experiments, resources and time. Although there are many scientifically valid techniques, manufacturers must factor in the cost of compliance when choosing a given route for meeting the regulations. Technologies that are often employed in the dietary supplement industry with regard to botanicals include traditional organoleptic analysis (sight, taste, smell, etc.) or microscopy, as well as chemical means such as High Performance Liquid Chromatography (HPLC), High Performance Thin Layer Chromatography (HPTLC), Flash Chromatography, Gas Chromatography (GC), Mass Spectrometry (MS), Infra-red (MIR, NIR, FTIR), UV-VIS and many others. One technique that can be utilized to assist in meeting the requirements of the cGMP and, in particular, the analysis of botanical extracts and complex mixtures, is Advanced Flash Column Chromatography (AFCC). AFCC is a complimentary separation technology that, when coordinated with other technologies such as HPTLC, can provide manufacturers with a significant set of tools for cGMP complianceoften at a more reasonable cost. Use of appropriate identity methods and technologies is critical to ensure dietary ingredients are properly identified and provide a greater chance that adulterated raw materials will be detected before they are incorporated into finished products. A review of some of these methods follows.


Increasingly, companies are seeking higher powered solutions to fully characterize the sample reference set they are going to use for analysis of the routine samples and develop truly validated methods. In particular, implementing advanced next-generation flash chromatography equipped with integrated multi-detection by UV and ELSD to rapidly isolate, purify and characterize new compounds has been key to addressing the majority of identity testing challengesparticularly botanical extracts and pre-mixes. The One Pass Flash" technique, developed by FRL, is used routinely to simplify highly complex premixes and resolve them into distinct sample fractions for immediate qualitative and quantitative evaluations of identity. That equates to higher throughput, less downtime and more reliable data.

Also, samples can be purified to prepare standards for measuring new compounds when no standard is available, the standard is too costly or it is too delayed. A prime example comes from excellent work on kava roots by Paula Brown, et al, who demonstrated the use of AFCC to isolate novel compounds from kava, which resulted in the purification of known cutting edge flavokavains and the discovery that one of the compounds was a novel ingredient not previously reported. Currently, there is no physical or chemical quality specification for commercial manufacture of kava products and it is essential suitable quality specifications are developed. A key objective of the study was to establish purity of the compounds by quantitative nuclear magnetic resonance (q-NMR) for use as a chemical reference material and for further studies. An AFCC method for purifying targeted phytochemicals from P. methysticum leaves, stem peelings, and roots was developed. Fractionation, guided by gas chromatography with mass spectroscopic detection (GC/MS) monitoring of expected masses, was performed.

Kava roots with GCMS

carrageenan and chondroitin

The second image shows the GC/MS spectra for flavokavains A, B, C (compound reassigned by NMR) and pipermethystine.

 Flavokavain A and B, as well as pipermethystine, were successfully isolated and purified using AFCC with a high degree of purity in under an hour. These isolated compounds can be used in future studies and as reference materials for phytochemical comparisons of P. methysticum traditional preparations and commercial products.


Simple screening of incoming premixes by near infra-red (NIR) or mid-infra red (MIR/FTIR) or other fast-scan techniques may not ensure reasonably anticipated contaminants (RACs) are absent in the ingredients, and these techniques often fall short of this requirement due to lack of method validation or inadequate sampling. MIR and NIR can give a picture of the whole sample, but require much up-front work to validate for botanical identity methods. NIR, perhaps the most misunderstood technique in the industry, is problematic for identity testing if not fully validated by a chemometric model. In many instances these techniques are not being properly utilized for botanical identity testing, which can result in failures in identity. The example below demonstrates how easy it is to pass carrageenan in routine NIR analyses as chondroitin.

carageenan and chondroitin

In addition, the GMPs require manufacturers to ensure product specifications (identity, purity, strength, composition, contaminants) are established and, more importantly, met. If a product specification is goldenseal root extract standardized to 5-percent alkaloids, the product must be proven by the manufacturer (not the raw material supplier) that it meets this specification. To accomplish this using MIR, it would not be unusual for a proper MIR method to require a few hundred materials up front to take the place of multiple testing methods such HPLC and HPTLC if one wishes to confirm botanical source identity as well as a quantitative assay and any contaminants (for example, in this case, the RAC synthetic berberine). As a complementary technique and to keep standard sourcing costs low, AFCC can be used to isolate standards for use in validating these spectroscopic methods.


HPTLC is an excellent fundamental means to examine botanical materials & several other dietary ingredients but can be more labor-intensive. HPTLC allows for comparison of a test sample against various reference materials (desired and adulterant) as well as grades of material. AFCC has been effective for quickly identifying markers in standardized extracts, especially when HPTLC profiles are inconclusive compared to monograph methods since they are based on whole botanical extract not standardized extract. Highly concentrated extracts often require removal or reduction of other compound classes that are used in routine HPTLC methods for profiling the species. In order to properly identify these highly processed materials, AFCC is used to isolate and concentrate select markers which allow species assignment. However, an HPTLC analysis of an extract sample of Schizandra berry powder (left lane) and the standardized extract (right lane) shown below demonstrates the challenge to complex mixes.

HPTLC for schizandra

Note the difference in band intensity. In addition, the diagnostic band of interest does not appear in the standardized extract.

This method uses bands in the profiling that are not the same as the extract markers leaving routine identity by HPTLC lacking. However, simplifying the standardized extract by flash chromatographic separation first would essentially concentrate the marker band and allow for identification of new negative markers in other cases.

Below is the result following successful isolation of a pure fraction containing the marker band for Schizandra chinensis from the sample viewed at VU 366nm. In the left lane is starting material while the right lane is the fraction from starting material with concentrated diagnostic compound. This result confirms Schizandra chinensis is present in this complex sample and demonstrates the usefulness of the advance fractionation of the mixture before HPTLC analysis.

Kava root testing

Liquid Chromatography/Mass Spectrometry & Nuclear Magnetic Resonance (LC/MS & NMR)

Liquid chromatography with mass spectroscopy (LC/MS) and NMR techniques employed for identity require significant upfront capital investments, operator know-how, and cost of operation. Implementation challenges for developing truly validated testing protocols could include: botanical identity methods for complex pre-mixed blends, sourcing costly and obscure phytochemical standards, controlling high-level labor hours required for advanced spectroscopy technologies, reconciling the fact that processing of standardized extracts may diminish target compounds used for species identification. The AFCC isolated fraction in the previous Schizandra extract HPTLC example can also be characterized by LCMS and NMR to identify the structure and mass. Further routine testing can be done using this standard on a triple quad or other MS system to rapidly test subsequent shipments to confirm identity or to identify the marker species in a pre-mix.

Practical Solutions

CRO Outsourcing: The industry has the option to invest in various technologies that can be used by skilled staff internally to quickly assess ingredient identity or outsource the testing work to a contract laboratory, which would require a laboratory proficiency demonstration by check samples or an audit as well as the characteristic delay time for shipping a sample, putting it into queue and awaiting results. However, caution is advised for firms proceeding with outsourcing; qualifying questions to determine the level of expertise in identity are often necessary. The firm will need to determine if the CRO possesses the proper equipment, whether analysts have adequate training and qualifications, learn how instruments are calibrated, and determine whether the incoming standard is characterized properly. Note that even though materials may be sent out for testing by a third party lab, the manufacturer is still responsible for the work being done.

The article, Choosing an Analytical Lab, developed in cooperation with the American Herbal Products Associations (AHPA) Analytical Laboratories Committee, is an excellent resource for businesses considering contract laboratory services.

In-House Lab: Consideration for ROI, including hiring, training, equipment, space, safety, and other factors, should be considered when choosing to create an in-house lab. Other factors such as intellectual property protection could also be a consideration.

Business Benefits of Implementing AFCC

Independent of whether evaluations are performed by an in-house or third party lab, via FTIR, HPTLC or mass spectroscopy, the One Pass Flash" technique eliminates other time-intensive sample prep work and reduces costly column replacements for a LC/MS system.

 By applying AFCC to the FRL arsenal of technologies to perform automated sample prep, businesses can recognize many benefits impacting operations, including:

* Reduce sample prep time by up to 90 percent

  • Samples are simplified and ready for qualitative examination or quantitative analysis typically in minutes instead of hours. This is like having an extra full-time technician in the laboratory.

* Faster sample analysis times

  • Samples can now be properly analyzed in one to two days versus 10 to 15 business days, which is more typical for exotic or complex mixtures. For companies bringing in multiple pre-mixed products to encapsulate (ready to encapsulate or RTE), this could mean tens of thousands of dollars per year saved on each product SKU.

* Detecting/isolating sample components quickly

  • Isolating authentic reference standards by AFCC saves thousands of dollars yearly and, in some cases, it is the only means to obtain the compound unless one is willing to contract a standard company to purify it, which will usually cost as much as buying an AFCC unitand that is just one standard. So one is ahead of the game after an initial compound is isolated and characterized when working on novel products. Plus, by performing the work in-house businesses IP can be better protected.

  • In addition, instead of attempting a complex sample separation with a long 60 to 90 minute compromised HPLC gradient, a series of simple HPTLC separations or simple HPLC marker specific compendia methods can be used to save a great deal of R&D time and avoid extra validation work.

* Lower consumable costs (savings depends on sample throughput, but could be thousands per year)

  • AFCC systems use small, inexpensive disposable cartridges preventing the early demise of LC columns which are very expensive and critical to throughput.

* Easy to learn and use

  • Lab personnel quickly come up to speed on the operations and develop simple methods to address a variety of complex botanicals and product formulations.

  • Replaces other methods such as preparative TLC, open column chromatography and preparative HPLC.

  • The embedded wizards included in next-generation AFCC literally walk you through each step and help assure you have the right amount of solvent, proper cartridge and that other parameters are satisfied before you start the experiment. This can help you get the job done right the first time every time.

Under the new cGMP regulations, all companies must assure their products are compliant. Further, FDA is actively inspecting facilities at an ever increasing rate, meaning not if" but when" the agency will inspect your facility. By properly implementing the correct modern technologies and utilizing trained staff, with consideration given to cost and ROI, companies can comply with the cGMP regulations and greatly mitigate the risk of an FDA Warning Letter and potential financial liability/loss of client trust. Choosing the right partners is an essential step in developing or evolving your companys laboratory operations. When you have the right equipment, right staffing, strong partners, and a commitment to excellence, your company moves from being a high risk entity to an entity anchored in a solid foundation of quality.

For more information on appropriate techniques for the verification of the identity of and meeting specifications for popular botanicals, and in particular how AFCC can help with botanicals/botanical extracts/complex mixtures, click here to register for a free webinar presented by AHPA on June 12, 2013 from 2 to 3:30 p.m. EDT.

James Neal-Kababick is the director of Flora Research Laboratories and Kathryn Lawrence is with Grace.

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