HerbalEGram: Volume 13, Issue 1, January 2016

By Stefan Gafner, PhD

UNPA Analytical Summit in Salt Lake City, Utah

The United Natural Products Alliance (UNPA) organized an analytical summit that took place fromNovember 5-6, 2015, in Salt Lake City, Utah, in response to increased concerns about the identity and authenticity of botanical dietary ingredients and supplements. These concerns resulted from the 2015 investigations by New York Attorney General (NY AG) Eric Schneiderman. The goals of the summit were to update members of the herb and dietary supplement industry about the latest developments related to Schneiderman’s actions and about the outcome of the meeting of the National Association of Attorneys General (NAAG) held November 2-4, 2015, in St. Louis, Missouri. The summit also included discussions of the current status of analytical technologies used to authenticate dietary ingredients and detect adulteration, with emphasis on DNA-based methods.

The event opened with a presentation by UNPA president and event host Loren Israelsen, who introduced the goals of the summit and gave a brief review of events following the initial investigation by the NY AG, in February 2015, into the authenticity of dietary supplements. Israelsen reported on the meeting of the NAAG, at which the state AGs were informed about issues related to herbal dietary supplements by industry critics Maia Kats (Center for Science in the Public Interest [CSPI]) and Pieter Cohen, MD (Harvard Medical School). Rend Al-Mondhiry from the Council for Responsible Nutrition (a leading supplement industry trade association) also spoke at that event.

Israelsen then made a few remarks about the seven papers published in the scientific periodical Drug Testing and Analysis,^1-7 most of which highlighted the dangers of consuming dietary supplements and suggested an overhaul of current regulations. Of interest is that some of the suggested regulatory changes proposed in the paper by Akshay Kapoor, MSPH, and Joshua M. Sharfstein, MD, a former official at the US Food and Drug Administration (FDA),¹ were supported by a majority of the UNPA summit attendees according to an instant poll during the summit.

In the poll, 81% of respondents supported having dietary supplement products registered with the FDA prior to marketing, 80% were in favor of monographs that would provide standard manufacturing processes for botanicals, 87% agreed that the FDA should have new authorities to remove/suspend sales of dietary supplements in “serious” cases (i.e., when there is an established unreasonable risk), and 68% were willing to consider the Kapoor and Sharfstein proposals as a starting point for discussions on new regulations.

GNC representatives Jim Sander (chief legal officer) and Guru Ramanathan, PhD (chief innovation officer), talked about GNC’s experience dealing with the NY AG, and detailed the amount of paperwork (incoming raw material specifications, complete manufacturing batch records, testing methodology for identity and authenticity, benefit claims substantiation, allergen testing, contaminant testing, adverse events, etc.) that was provided to the investigators to comply with their requests. GNC explained that they preferred to settle with the NY AG rather than go through a lengthy litigation process, which may have ultimately caused more damage to consumer confidence.

The majority of their talk focused on GNC’s current strategies to move forward, in particular the establishment of four working groups to enhance the quality of botanical dietary supplements throughout the industry. The working groups focus on work with the media, quality seals and facilities certification, raw material Good Manufacturing Practices (GMPs), and the review of the current industry product notification and numbering database.

Also representing the manufacturing industry, Aaron Secrist (director of quality assurance/quality control and research and development at NOW Foods) explained the advantages of having an in-house quality control laboratory (e.g., better control over analytical work, faster turnaround time, lower costs in the long term, and the ability to be on the cutting edge of compliance), and what it takes to build one. The costs to build and maintain a laboratory are decided by the amount of risk a company is willing to take, the financial support that is available, and the type of products that a company sells. Secrist suggested that costs to build an in-house quality control laboratory could be between 8-10% of annual company sales, and that the ongoing investment could be between 4-6% of annual sales.

Another part of Secrist’s presentation was devoted to important considerations when building the in-house laboratory, such as lab safety equipment, space considerations (with particular emphasis on space to expand over time), plumbing and electrical infrastructure, and waste stream requirements. Finally, he emphasized the importance of building the laboratory in an area where highly trained analysts can be recruited, and, more importantly, how to motivate these employees to stay with the company for a long time.

Since genetic testing methods were the main focus of the analytical methods discussions, the two expert talks by Susan J. Murch, PhD (Professor & Canada Research Chair in Natural Products Chemistry, University of British Columbia, Kelowna), and Danica Harbaugh Reynaud, PhD (CEO of AuthenTechnologies), were followed with much interest. Dr. Murch explained that DNA, not to be confused with proteins, is a very large molecule that is principally composed of four nucleic acids: adenine, cytosine, guanine, and thymine. In many genetic technologies, it is the establishment of the sequence of these four nucleic acids that allows identification of a material. She then detailed what DNA barcoding is, suggesting that it should be used mainly to identify original plant material for which taxonomic characteristics are missing. She emphasized a number of potential sources of error (e.g., the selection of the right primer, DNA degradation, or a lack of variability in the chosen sequence), and said that the species identification success rate of the method varies between 80-98% when two or more sequences are used. Dr. Murch concluded by stressing the importance of evaluating if the DNA method is fit for purpose, and that it should only be used if it has been properly validated for the particular matrix of the tested material.

Dr. Harbaugh Reynaud pointed out that there are a number of genetic methods, and that it is important to select the appropriate method for the targeted endpoint (i.e., the botanical material being analyzed). She explained that DNA barcoding is a process, not a technology, and that the process has not been validated with regard to specificity. She made it clear that DNA barcoding can be used with fresh, raw material that is made from only one species, while in all other cases, a different approach is needed. For processed botanical ingredients and finished products, specific primers (primers that will amplify one or a few closely-related species) that can amplify shorter pieces of DNA are needed, and if multiple plant species are present, next generation sequencing should be the method of choice. She then took the audience through the steps for developing and validating a DNA method for identification.

For validation, Dr. Harbaugh Reynaud suggested following the AOAC International guidelines for botanical identification methods. Towards the end, she presented data from her own research showing that universal DNA barcoding was able to authenticate only 10% of crude raw materials, and none of the finished products made with extracted ingredients, compared to 90% and 60%, respectively, when using specific DNA authentication methods. During the research project, she analyzed a number of botanical ingredients, and detected adulteration in two out of nine garlic (Allium sativum, Amaryllidaceae) samples, one out of 13 echinacea (Echinacea spp., Asteraceae) samples, three out of nine St. John’s wort (Hypericum perforatum, Hypericaceae) samples, eight out of 29 ginseng (Panax spp., Araliaceae) samples, and one out of 18 skullcap (Scutellaria lateriflora, Lamiaceae) samples. None of the 22 black cohosh (Actaea racemosa, Ranunculaceae), 12 ginkgo (Ginkgo biloba, Ginkgoaceae), or eight saw palmetto (Serenoa repens, Arecaceae) samples were adulterated.

There were a number of presentations about identification of botanical ingredients and detection of adulteration by chemical means, including those by Botanical Adulterants Program members Roy Upton of the American Herbal Pharmacopoeia (AHP) on challenges, pitfalls, and solutions using botanical taxonomy, and this author on the implications of the NY AG’s investigations on the quality control of botanical ingredients.

The summit ended with the keynote presentation by Ikhlas Khan, PhD (research professor of pharmacognosy and assistant director for the National Center for Natural Products Research at the University of Mississippi). Dr. Khan gave an honest assessment of the issues within the dietary supplement industry, with examples of adulteration (ginkgo, tea tree [Melaleuca alternifolia, Myrtaceae], and hoodia [Hoodia gordonii, Apocynaceae]), the presence of hepatotoxic pyrrolizidine alkaloids in natural products, and the sale of unauthorized ingredients (e.g., 1,3-dimethylbutylamine [DMBA]). He emphasized the need for the dietary supplement industry to implement a “quality by design” approach for raw materials and finished products, which encompasses the knowledge of the seed/genetic source of the plant material; the identity of the specific species; information on the climate or environment where the plant was grown; harvest date; details about how the plant was stored, processed, and extracted; and information about the overall phytochemical composition. Finally, he told the audience that a quality plant material is one that is botanically, chemically, and pharmacologically well-characterized.

The UNPA summit was attended by 140 people, with brand manufacturers, contract manufacturers, and ingredient suppliers making up 65% of the participants. Based on its success, UNPA decided to organize another summit on February 23-24, 2016, with a focus on supply chain qualification.

References

1. Kapoor A, Sharfstein JM. Breaking the gridlock: regulation of dietary supplements in the United States [published online November 2, 2015]. Drug Test Anal. doi: 10.1002/dta.1892.
   
2. Johnston DI, Chang A, Viray M, et al. Hepatotoxicity associated with the dietary supplement OxyELITE Pro™ — Hawaii, 2013 [published online November 2, 2015]. Drug Test Anal. doi: 10.1002/dta.1894.
   
3. Eichner A, Tygart T. Adulterated dietary supplements threaten the health and sporting career of up-and-coming young athletes [published online November 2, 2015]. Drug Test Anal. doi: 10.1002/dta.1899.
   
4. Dodge T. Consumers' perceptions of the Dietary Supplement Health and Education Act: implications and recommendations [published online November 2, 2015]. Drug Test Anal. doi: 10.1002/dta.1857.
   
5. Austin KG, Farina EK, Lieberman HR. Self-reported side-effects associated with use of dietary supplements in an armed forces population [published online November 2, 2015]. Drug Test Anal. doi: 10.1002/dta.1905.
   
6. Rasmussen N, Keizers PHJ. History full circle: ‘Novel’ sympathomimetics in supplements [published online November 2, 2015]. Drug Test Anal. doi: 10.1002/dta.1852.
   
7. Marcus DM. Dietary supplements: What’s in a name? What’s in the bottle? [published online November 2, 2015] Drug Test Anal. doi: 10.1002/dta.1855.

BRAMA Training Modules IV and V in Aqaba, Jordan

Botanical Risk Assessment Training in the Mediterranean Area (BRAMA) is a 40-month project co-financed by the European Neighborhood and Partnership Instrument (ENPI)* as a cross-border cooperation project in the Mediterranean Sea Basin area with four partners: Hylobates Consulting Srl (Rome, Italy), the University of Ioannina (Ioannina, Greece), the National Research Centre (Cairo, Egypt), and Ben-Hayyan Laboratories (Aqaba, Jordan); and one associate partner, Wageningen University (Wageningen, the Netherlands). The main goal of the project, initiated in July 2012 with an overall budget of €1.708 million (more than $1.8 million), is to support the training and professionalism of young researchers, inspectors, and technicians from the Euro-Mediterranean area to improve knowledge of quality control and technologies for raw plant materials and dietary supplements based on plants.

The Program has organized the training sessions in five modules. Module I on Advanced Analytical Methods and GLP (Good Laboratory Practices) was held from June 29-July 1, 2015, in Rome, Italy. Module II, Regulations on Botanicals, was organized in Tunis, Tunisia, from October 5-7, 2015. Module III, titled Safety Assessment of Botanicals Preparations, took place in Cairo, Egypt, from October 19-21, 2015. Aqaba, Jordan, was chosen as the site for the final two modules (Module IV, GMP, GAP, HACCP, and Module V, Planning and Management of Official Controls), which were held from November 16-19, 2015. Speakers included, among others, Prof. Ivonne Rietjens, expert on risk assessment and food toxicology at Wageningen University, the Netherlands; Prof. Jacques Vervoort, also from Wageningen University, who investigates health effects on humans and other organisms directly via food (plant ingredients/botanicals) or via environmental pollution; Vittorio Silano, PhD, chair of the European Food Safety Authority Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids; and Prof. Anastassios Troganis, a natural products chemist from the University of Ioannina who specializes in spectroscopic techniques for structure elucidation. The American Botanical Council (ABC) participated in the last two modules.

Modules IV and V started with a presentation by Prof. Chlodwig Franz (University of Veterinary Medicine, Vienna, Austria) on Good Agricultural Practices (GAPs), an overview on current Good Manufacturing Practices (cGMPs) — as far as applicable for botanicals — and on quality issues related to botanical ingredients. Then, Prof. Laura Di Renzo (University of Rome Tor Vergata, Rome, Italy) presented on the traditional use of botanicals and the Hazard Analysis and Critical Control Point (HACCP) process for botanicals. Next, Joris Geelen (managing director of Food Law Consult and former expert on botanicals within the Belgian Government) gave a talk on the regulatory framework of food supplements and their notification system in the European Union (EU) and in Belgium in particular. This author presented the final talks on issues related to adulteration of botanical preparations and ingredients, and on the regulatory framework and cGMPs in the US. A total of 103 students (researchers, food inspectors, and food safety consultants, among others) from 14 countries, with the largest representations coming from Egypt, Tunisia, and Italy, signed up for the training classes.

*Note: The ENPI CBC Mediterranean Sea Basin Programme is a multilateral Cross-Border Cooperation (CBC) initiative funded by ENPI. It is funded by the European Union under administration of the Directorate General for International Cooperation and Development. The Program objective is to promote a sustainable and harmonious cooperation process at the Mediterranean Basin level by dealing with common challenges and enhancing its endogenous potential. The Program finances cooperation projects as a contribution to the economic, social, environmental, and cultural development of the Mediterranean region. Participating countries include: Cyprus, Egypt, France, Greece, Israel, Italy, Jordan, Lebanon, Malta, Palestine, Portugal, Spain, Syria (participation currently suspended), and Tunisia.

Adulteration and Fraud in Food Ingredients and Dietary Supplements Workshop in Rockville, Maryland

This workshop, co-sponsored by ABC, the Food Protection and Defense Institute, and the United States Pharmacopeia (USP), was held at the USP headquarters in Rockville, Maryland, from December 3-4, 2015. The workshop featured a keynote presentation by James Harnly, PhD (research chemist at the US Department of Agriculture [USDA]), six plenary lectures, and a number of talks held in parallel sessions focusing on either food or dietary ingredient/supplement adulteration.

The keynote address by Dr. Harnly gave an overview of USDA research on the detection of botanical ingredient adulteration where chemical fingerprints are submitted to pattern recognition techniques. He explained the differences among unsupervised chemometric methods, in which samples are aligned in space based on the similarity of their principal components (principal component analysis [PCA]); soft modeling techniques, in which a model is built using a number of known materials (e.g., vouchered samples of black cohosh), and unknown samples are evaluated with regard to their resemblance to the established model; and hard modeling, in which the unknown samples are forced to align within one of the clusters of the known samples in the established model (e.g., a model that was built with black cohosh and a number of known adulterants).

While the availability of new technologies, particularly with regard to improvements in computer software to perform multivariate statistics, has allowed analysts to have a more objective way to compare samples, Dr. Harnly noted that it is important to compare apples to apples. Many of the chemometric models to detect adulteration are based on crude raw materials that were consistently processed to build the model. In reality, dietary supplement ingredients are subject to a variety of processing steps, which often differ from one manufacturer to another. In addition, the finished product usually contains a number of excipients, which also change the chemical profile and thus may lead to erroneous conclusions when compared to the set of samples that the model was built upon. Dr. Harnly concluded by saying that non-targeted analysis will be the future for the detection of botanical ingredient adulteration since, in many cases, the analyst does not know what the adulterant is, and therefore what specific phytochemicals she/he is looking for.

Françoise Dorcier (criminal intelligence officer at Interpol) introduced the program at Interpol (a network which facilitates global police function and provides tools allowing for the identification of crime and criminals), to collaborate with police around the world to counteract food and dietary supplement fraud. Interpol investigates cases of dietary supplement adulteration since these may pose a public health threat and may involve members of criminal gangs. She gave some examples of international collaborations: as part of an Interpol operation, the police department of economic crime in Ho Chi Minh City, Vietnam, confiscated 597 boxes filled with fake dietary supplements that contained only flour. Another operation led to the seizure of 23,530,553 units of illicit, counterfeit, or illegally sold medicines, mostly products marketed for erectile dysfunction; 1% of the products that were seized were sold as dietary supplements.

Shaun Kennedy (associate professor at the Food Policy Research Center at the University of Minnesota) gave an overview on the economics of adulteration. He explained that there are two ways for fraudulent manufacturers to make a profit—either by selling cheap materials as high-value products (for example, when vanilla [Vanilla spp., Orchidaceae] prices soared due to a supply shortage, some suppliers started to add mercury to the vanilla beans in order to increase their weight) or by selling large volumes of adulterated, low-value ingredients, where the low margin can be offset with a high volume. For the latter, he gave the sale of basil (Ocimum spp., Lamiaceae) as oregano (Origanum spp., Lamiaceae) as an example, where the price difference often is in the range of $1/pound, which is enough to make a profit if the sales volume is large enough. He urged manufacturers to be familiar with the supply chain, including, for example, harvest volumes, commodity pricing (including looking at price stability over the years, price comparisons among producing countries, etc.), ingredient production standards, and frequency of reports of adulteration. He also urged performing an economic risk analysis, starting with the most important ingredients.

Botanist Damon Little, PhD (associate curator of bioinformatics at the New York Botanical Garden), gave an overview on how to confirm the identity of a species with DNA-based methods, using devil’s claw (Harpagophytum procumbens, Pedaliaceae) as an example. Based on his research, Little suggested that DNA is present in about 90% of all dietary supplements (90% includes every species detected, including the labeled species and/or close relatives [email, December 21, 2015]), but that the presence is highly dependent on the starting material and processing steps. He said that smaller DNA fragments usually survive the processing, but high heat can destroy DNA, certain types of filtration can remove DNA, and liquid herbal preparations are particularly challenging.

DNA work can be done using either a general approach, attempting to identify all the species present, or a targeted approach, where the presence/absence of a particular species is verified. Dr. Little noted that the general approach suffers from the misconception that DNA is uniformly preserved and available. This approach also rarely provides accurate species identification, since distinction among closely related species is difficult, often impossible, and the diagnostic features examined may not be distinctive enough for a given species. On the other hand, targeted DNA analysis provides accurate sample identification if it is done correctly. However, it cannot detect species outside the target group, and it is difficult to establish the absence of a species in a dietary supplement solely based on the absence of detectable DNA.

Dr. Little emphasized, on a few occasions, that quantitative DNA work is not reliable since the amount of DNA tends to correlate poorly with the amount of ingredient, and certain DNA types amplify more readily than others. There are a number of important aspects to consider when doing DNA-based plant authentications, such as the availability of vouchered samples and database records to estimate inter- and intraspecific variations, the availability of sample materials and database records from closely related species, and whether the target sequence can differentiate the species of interest from all other closely related species.

Dr. Little finished his talk by presenting the results of the work of his student, Genelle Diaz-Silveira, on the distinction between H. procumbens and H. zeyheri using variations in the psbA-trnH sequence. These results were later used by the New York attorney general as the basis to send cease-and-desist letters to 12 companies selling devil’s claw supplements that contained various amounts of H. zeyheri.

The College of Pharmacy at the University of Illinois at Chicago (UIC) was represented by Guido Pauli, PhD, and Charlotte Simmler, PhD. Dr. Pauli presented the overarching concept of “Botanical Integrity,” which includes the determination of the identity, composition, and pharmacological effects of a botanical ingredient. He gave an eloquent explanation about the difference between identity and authenticity (the former refers to the correct species, while the latter also includes geographic origin and processing consistent with the sample). Dr. Simmler gave details about the ongoing work at UIC to identify licorice species (mainly Glycyrrhiza glabra, G. inflata, and G. uralensis, Fabaceae) based on DNA sequencing and ¹H NMR fingerprinting followed by chemometric analysis. The results, which were published in the Journal of Natural Products,¹ showed that of 51 commercial licorice samples, 11 did not indicate the correct species on the label (although six of these 11 samples were licorice hybrids), and another eight samples were mixtures of several species.

As the technical director of the ABC-AHP-NCNPR Botanical Adulterants Program, this author gave a presentation on the scope and activities of the program, focusing on the three Laboratory Guidance Documents (LGDs) published in 2015 on skullcap, bilberry fruit extract, and black cohosh root/rhizome. These LGDs provide summaries and evaluations of 21, 39, and 36 published analytical methods, respectively, available in the scientific literature and are intended as guidance for in-house industry laboratories and third-party analytical labs to help ensure authentication of problematic botanical ingredients (i.e., those in a category that is known to be adulterated).

The “Adulteration and Fraud in Food Ingredients and Dietary Supplements Workshop” was attended by over 120 people. According to Nandakumara Sarma, PhD, director of USP’s Dietary Supplement program, “It was a clear success in terms of the quality of speaker presentations, and participant engagement, and discussions. We received wonderful feedback from the participants,” he wrote (email, December 8, 2015).

Reference

1. Simmler C, Anderson JR, Gauthier L, et al. Metabolite profiling and classification of DNA-authenticated licorice botanicals. J Nat Prod. 2015;78(8):2007-2022.