Authenticity of Herbal Supplements: The Analytical Tug-of-War

Is DNA barcoding the best technique to test product authenticity or adulteration? Or chromatography and mass spectrometry?

The New York State attorney general’s office accused four major retailers of selling fraudulent and potentially dangerous herbal supplements and demanded that they remove the products from their shelves, according to the New York Times in a story Tuesday, Feb. 3. 2015.

The news broke after the attorney general’s office sent cease-and-desist letters on Feb. 2 to four chain stores instructing them to stop selling a range of products.

What’s at stake?

Authorities in New York said they had conducted tests on store brands of herbal supplements at four national retailers — GNC, Target, Walgreens, and Walmart — and found that four out of five of the products did not contain any of the herbs on their labels.

The retailers were ordered  to stop selling “adulterated” and “mislabeled” herbal supplements that independent lab tests show do not contain ingredients as stated on the labels, according to Food Safety News.

Bottles of Walmart-brand Echinacea, an herb said to ward off colds, were found to contain no Echinacea at all. GNC-brand bottles of St. John’s wort, said to alleviate depression, held rice, garlic and a tropical houseplant, but not a trace of the herb, reported the Associated Press.

Not only did many of the tablets contain potential allergens not listed in the ingredients; some showed none of the supposed active ingredient at all, reported IFLScience.

How did they test samples?

In the letter sent to GNC, for example, the AG office states, “The supplements tested included Ginkgo Biloba, St. John’s wort, Ginseng, Garlic, Echinacea and Saw Palmetto. By using established DNA barcoding technology, analytic testing disclosed that 5 out of 6 types of dietary supplement products tested were either unrecognizable or a substance other than what they claimed to be, and therefore constitute contaminated or substituted products.”

The New York Times previously reported in 2013 that researchers had selected popular medicinal herbs, and then randomly bought different brands of those products from stores and outlets in Canada and the United States. Of 44 herbal supplements tested, one-third showed no trace of the plant advertised on the bottle — only another plant in its place.

UPDATE 3/30/2015:
GNC Reaches Deal With NY AG Over Herbal Plus Products

DNA Barcoding: Is it the right technique?

Industry advocacy groups from across the dietary supplement and herbal-based products industry are reacting strongly.

The Council for Responsible Nutrition (CRN), which represents the supplement industry, has criticized the New York State attorney general’s office, calling into question whether DNA barcoding technology is an appropriate test for quality tests. DNA barcoding was at the center of the New York Times 2013 article, which based on a BMC Medicine research article.

In addition, the American Herbal Product Association (AHPA) issued a statement that the AG’s office “used an inadequate and unproven analytical method to test herbal supplement products.”

“Using DNA barcoding as the only method for identifying ingredients in popular herbal dietary supplements and ignoring all other well-established and validated methods of herbal analysis is a suspect analytical process at best, and likely to provide results that are inaccurate,” commented AHPA Chief Science Officer Maged Sharaf, Ph.D.

The AHPA pointed out some of the known limitations of DNA barcoding include its inability to identify extracts and highly processed herbal products due to the loss or denaturation of DNA material during processing. DNA barcoding may also provide misleading results by disproportionally amplifying minor components in a product, which could erroneously imply its higher content in the tested product.

Likewise, the American Botanical Council (ABC) issued a statement saying, “The AG’s study is not based on adequate science and its actions are thus premature. The use of DNA barcoding technology for testing of the identity of botanical dietary supplements is a useful but limited technology.

“DNA testing seldom is able to properly identify chemically complex herbal extracts as little or no DNA is extracted in many commercial extraction processes. Basing its actions on the basis of only one testing technology from only one laboratory, the NY AG results are preliminary and require further substantiation. Additional testing using microscopic analysis and validated chemical methods should be conducted to confirm the initial results upon which the AG is acting,” ABC’s statement said.

ABC also pointed to recently published DNA studies on commercial ginkgo extracts conducted at the New York Botanical Garden and the highly respected University of Mississippi’s National Center for Natural Product Research.

“The AG’s test results do not comport with other recently published research on herbal dietary supplements. Two recent tests from reputable laboratories on commercial ginkgo extracts have found ginkgo in all or almost all of the samples tested,” said ABC’s Chief Science Officer Stefan Gafner, Ph.D.

Of note, not only is the scientific technique used for testing the supplements under scrutiny, so too is the warning brought by the New York attorney general’s office as a law enforcement agency vs. industry regulation and oversight by the U.S. Food and Drug Administration. The New York Times noted that while the FDA has targeted individual supplements in the past, this is the first time that a law enforcement agency has targeted retail and drugstore chains that sell the products for misleading products.

UPDATE 3/12/2015:
Trade Association-Commissioned White Paper Refutes NY AG Botanical Supplement Results

Good Manufacturing Processes rely on chromatography and mass spectrometry

The CRN, in its objection letter noted, “Dietary supplement companies are required by law to adhere to Good Manufacturing Practices (GMPs) which include identity testing for all botanical materials used. Different identification test methods, from simple titration to chromatography and mass spectrometry, are appropriate for different stages of the processing—from the whole plant to the extract, to the finished product.

“Companies identify all incoming materials and keep records of these test results, and those test results could have been shared if companies had been approached in a reasonable manner with a reasonable timeframe. The FDA, which enforces these identity requirements through routine inspections, does not require DNA barcode testing; nor should it, given that this novel approach has not been validated for finished products,” the CRN stated.

The AHPA reiterated that manufacturers of herbal supplements have high standards to meet in terms of GMPs that are enforced by the FDA.

The role of analytical LC and MS in QC testing for authenticity or adulteration

As an analytical technology provider, Waters has seen chromatography and mass spectrometry used on both sides of this fence by leading dietary supplement providers, herbal and natural products manufacturers, and by food and vitamin manufacturers.

LC and MS are used to prove the authenticity and quality of end products and origin of ingredients; and for adulteration tests that show products don’t contain what is claimed on the labels.

Most dietary supplement companies are aware that multiple methods of analysis are often required to verify the identity of herbal products and herbal extracts. In addition to visual and organoleptic evaluation, HPLC and TLC are the most commonly used methods in the industry and rely on comparison of chemical constituents of an extract to that of a known standard.

For complex samples or where additional confirmation is required, companies often turn to mass spectrometry, which provides even greater selectivity and confirmation of identity.

The ACQUITY QDa Mass Detector, introduced by Waters in 2013, offers a unique opportunity for QC labs to add mass as an orthogonal detection mode in their LC/UV analysis. The detector provides simple, straightforward access to mass spectral information and integrates directly with existing Waters HPLC and UPLC instruments under the control of Empower 3 Software.

Dietary supplement companies wanting to provide their customers with greater assurance of ingredient identity can now readily incorporate mass detection into their laboratory workflow, reserving the use of DNA barcoding to uses more appropriate for that particular technology.

Additionally, the chromatography data system Empower 3 Software is used by pharmaceutical and supplement industry laboratories around the world to manage analytical data, to meet compliance and regulatory requirements, and to report on the quality of final products.

Following are some examples of how chromatography, mass spectrometry, and applications such as multivariate statistical analysis and reference libraries for screening herbal components can provide more certainty about product authenticity.

The Good:
Uses of LC and MS in proving product quality

Accelerating Product Development and Release
with a Rapid, High-Precision Vitamin Analysis Method
In this case study, learn how a global health care company uses UPLC/MS/MS for the routine quantiative analysis of vitamins to support the development of new or enhanced products and label confirmation of finished products.

Reducing Vitamin QC Analysis Times by Up to 90% with UPLC
DSM Nutritional Products is a premier global nutritional ingredient manufacturer and supplier to producers of foods, beverages, and dietary supplements. In this case study, learn how UPLC is used to accelerate sample analysis turnaround time, reduce the cost of analyses, and increase overall efficiency in their QC analytical processes that ensure product quality.

Characterizing the Natural Product Goldenseal
Using Solid-Core HPLC Columns and ACQUITY QDa Detection
Natural product analysis and characterization can be a difficult process due to the complex nature of samples such as Goldenseal. By combining state-of-the-art solid-core columns for HPLC separation with orthogonal mass detection using the ACQUITY QDa Detector, a simple separation of five sources of Goldenseal from five manufacturers was performed in five minutes; seven compounds were identified.

Advancing the Characterization and Quality Control of Botanical Extracts with Mass Spectrometry
With a continuing focus on innovation, Bio-Botanica has pioneered numerous advancements in the botanical field over its 40+ year history. Its laboratories rely on analytical technologies to provide comprehensive characterization of botanicals and help ensure final product quality. Learn how the company now routinely uses UPLC-MS/MS to support a wide variety of analytical testing needs including those for product safety, R&D, final product QC, and various customer requests.

Comparison of the Chemical Ingredients of Shuang-huang-lian Oral Drink
from Two Different Manufacturers
This application demonstrates the fast and accurate detection of the differences and similarities of chemical ingredients in Shuang-huang-lian oral drink from two different manufacturers. It shows how a manufacturer can use UPLC, MS, and workflow-driven software to more accurately determine quality control between batches of product.

The Bad:
Determining actual ingredient adulteration with LC and MS

UPLC/MS/MS for the Screening, Confirmation, and Quantification of Drugs Illegally
Added to Herbal/Dietary Supplements for the Enhancement of Male Sexual Performance
We demonstrate an analytical screening method for the detection of both known and unknown erectile dysfunction (ED) drugs and their analogues. The screening method is complemented by a highly sensitive and selective MS/MS confirmation assay.

Identification and Characterization of Key Chemical Constituents for the Authentication
of Hoodia Gordonii Using UPLC/Tof-MS
In this application note, we present a novel UPLC/MS workflow using multivariate statistical analysis (MSA) for the holistic characterization of the chemical constituents from H. gordonii and to authenticate the dietary supplements that claim to contain H. gordonii.

Real-Time Mass Spectrometry of Adulterants in Herbal Slimming Products
Use of tandem quadrupole MS with a Direct Analysis in Real-Time (DART) source enables fast and direct analysis of solids or liquids for simultaneous intact molecular ion and fragmentation information with rapid sample analysis.

The Ugly:
When LC and MS identify health-risking ingredients

Detection and Identification of Synthetic Phosphodiesterase Type-5 Inhibitors
in Adulterated Herbal Supplements using UPLC and MS
Adulteration of herbal dietary supplements with synthetic pharmaceuticals is a growing problem. Reports in published literature that show many of the supposed natural alternatives to approved synthetic phosphodiesterase type 5 (PDE5) inhibitors used to treat erectile dysfunction (ED) are actually adulterated with them. Due to the threat to public health posed by unknowingly ingesting both the known PDE5 inhibitors and their analogues, it is vital that analysts have tools that allow them to characterize these complex samples adequately. In this work, we evaluate the use of the tandem quadrupole mass spectrometry for the detection and identification of PDE5 adulterants in herbal supplements.

Rapid Detection and Identification of Synthetic Phosphodiesterase Type-5 Inhibitors in Counterfeit and Adulterated Products using the Atmospheric Solids Analysis Probe for MS
When hyphenated methods such as LC/MS and GC/MS are used to analyze herbal dietary supplements for illegal counterfeiting with synthetic pharmaceuticals, the need for chromatographic separation and sample extraction often creates a sample processing bottleneck. We demonstrate the use of a unique tool that enables direct sample analysis by mass spectrometry that is useful for rapidly screening large amounts of solid or liquid samples. When paired with TOF-MS, the ASAP probe enables analysts to rapidly identify unknown compounds using exact mass measurement and elemental composition determination with isotope ratio comparison; the turnaround time from sample receipt to structural identification and unknown compound determination is greatly accelerated.

More information

• See applications work that our scientists have performed on Ginkgo Biloba, Ginseng, and Echinacea
• Learn how Waters technologies are used in Food Profiling and Authenticity
• Learn how Waters technologies are used in Quality Control and Quality Assurance laboratories, whether in traditional pharmaceutical industry or by dietary supplements and natural remedy manufacturers
• Vitamin Analysis: New Methods for Improving Data Quality & Laboratory Productivity (on-demand webinar)