A Review
of Recent Conferences Focusing on Botanical Ingredient Adulteration
UNPA Analytical Summit in Salt
Lake City, Utah
The United Natural Products Alliance (UNPA)
organized an analytical summit on November 5-6, 2015, in Salt Lake City, Utah,
in response to increased herb and dietary supplement industry concerns about
the identity and authenticity of botanical dietary ingredients and supplements
resulting from the investigations by the New York Attorney General (NY AG). The
goals of the summit were to update participants on the latest developments with
regard to the NY AG’s actions and on the outcome of the meeting of the National
Association of Attorneys General (NAAG) held on November 2-4, 2015, in St.
Louis, Missouri, and to discuss the current status of analytical technologies
to authenticate dietary ingredients and to 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 then gave a brief
review of events following the initial investigation into the authenticity of
dietary supplements by the NY AG in February 2015. He reported on the meeting
of the NAAG, held on November 2, 2015, where the state AGs were informed on
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). Also speaking at the event was Rend Al-Mondhiry from the
Council for Responsible Nutrition (a leading supplement industry trade
association).
Mr. Israelsen then made a few remarks on the seven papers published in
the scientific periodical Drug Testing and Analysis,1-7
most of which were highlighting the dangers of consuming dietary supplements
and suggesting 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 USFDA,1 were
embraced by a majority of the UNPA summit attendees according to an instant
poll during the summit, which was conducted by using a handheld device handed
out to each of the attendees.
For example, in the November 5, 2015 poll, 81% of the respondents agreed
to 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), presented the
experience of GNC in 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 to go
through a lengthy litigation process, which may have ultimately caused more
damage to consumer confidence.
The larger part of their talk was focused
on GNC’s current strategies to move forward, in particular the establishment of
four working groups to enhance 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 what the advantages are to have an
in-house quality control laboratory, and what it takes to build one. He listed
the better control over the analytical work, faster turnaround time, lower
costs in the long term, and the ability to be on the cutting edge of compliance
as positive aspects of an in-house laboratory. 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. He suggested that costs to build an in-house quality control
laboratory could be between 8-10% of the annual company sales, and the ongoing
investment, between 4-6% of yearly 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 a particular emphasis on
space to expand over time), plumbing and electrical infrastructure, and
requirements for the waste stream. 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 confounded 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 was, suggesting that it should be used mainly to
identify original plant material for which taxonomic characteristics are
missing. She emphasized that there were 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 had 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 to the participants 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 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 on
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 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, National Center for Natural Products
Research, University of Mississippi, Oxford, 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, 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 their raw materials and finished
products, which comprises 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. Drug Test Anal.
November 2, 2015; [epub ahead of
print]. doi: 10.1002/dta.1892.
2.
Johnston DI, Chang A, Viray M, et al. Hepatotoxicity associated with the dietary supplement OxyELITE
Pro™ — Hawaii, 2013. Drug Test Anal. November 2, 2015; [epub ahead of
print]. 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. Drug Test Anal. November 2, 2015; [epub ahead of
print]. doi: 10.1002/dta.1899.
4.
Dodge T. Consumers' perceptions of the
Dietary Supplement Health and Education Act: implications and recommendations. Drug Test Anal. November
2, 2015; [epub ahead of print]. 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. Drug Test Anal. November 2, 2015; [epub ahead of
print]. doi: 10.1002/dta.1905.
6.
Rasmussen N, Keizers PHJ. History full circle: ‘Novel’ sympathomimetics
in supplements. Drug Test Anal. November 2, 2015; [epub ahead of
print]. doi: 10.1002/dta.1852.
7.
Marcus DM. Dietary supplements: What's
in a name? What's in the bottle? Drug
Test Anal.
November 2, 2015; [epub ahead of
print]. 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, 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 2, Regulations on Botanicals, was organized
in Tunis, Tunisia, from October 5-7, 2015. The third Module, entitled 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, Dr. Vittorio Silano (chair of the European
Food Safety Authority Panel on Food Contact Materials, Enzymes, Flavourings and
Processing Aids), and Prof. Anastassios Troganis (University of Ioannina,
Greece), a natural products chemist specializing in spectroscopic techniques
for structure elucidation. The American Botanical Council participated in the
last two modules.
Modules IV and V started with a
presentation by Prof. Chlodwig Franz (University of Veterinary Medicine in
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. He was followed by Prof. Laura Di Renzo (University of Rome Tor
Vergata, Rome, Italy), who presented on traditional use of botanicals and the
Hazard Analysis and Critical Control Point (HACCP) process for botanicals. Next
was Dr. Joris Geelen (Food Law
Consultant and former expert on botanicals within the Belgian Government), who
gave a talk on the regulatory framework of food supplements and their
notification system in the European Union 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
USA. 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 the American Botanical Council,
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 on 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, where samples are aligned in space based on the similarity
of their principal components (principal component analysis [PCA]); soft
modeling techniques, where a model is built using a number of known materials
(e.g., vouchered samples of black cohosh [Actaea racemosa,
Ranunculaceae]), and unknown samples are evaluated with regard to their
resemblance to the established model; and hard modeling, where 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 the 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
to secure global police function which provides tools permitting 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 represent 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, 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
US $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, frequency of reports of adulteration, and
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, Dr. Little suggested that
DNA is present in ca. 90% of all dietary supplements (90% includes every
species detected in targeted analyses: the labeled species and/or close
relatives; e-mail communication, December 21, 2015), but that the
presence is highly dependent on the starting material and processing steps. He
told the audience that smaller DNA fragments usually survive the processing,
but said that high heat can destroy DNA, certain types of filtration can remove
DNA, and that 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, and that it rarely
provides accurate species identification, since distinction among closely
related species is difficult, often impossible, and that 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 that 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 the other closely related species.
He finished his talk by
presenting the results of the work by 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, based on these
supplements containing 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. Professor Pauli presented the overarching concept of Botanical Integrity, which
includes the determination of the identity, composition, and pharmacological
effects of a botanical ingredient. He made an eloquent explanation about the
difference between identity and authenticity; the former referring to the
correct species, while the latter also includes geographic origin and
processing consistent with the sample known to have a specific health effect.
Dr. Simmler gave details on 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 out 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 made 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 the fitness for purposes 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.” (e-mail communication, 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.