Dietary Supplement Adulteration Testing
In the US, dietary supplements (DS) are not required to undergo the same stringent testing, standardization and regulation as pharmaceutical drugs, leaving opportunities for DS companies to make misleading claims, mislabel packages and use contaminated ingredients. Adulteration of DS has long been a concern of consumers and regulatory bodies due to the lack of required testing. With the advent of new developments in DS standards, regulations and instrumentation, the market for LC/MS for adulteration testing of DS is growing. LC/MS is among the most common techniques used to test DS for adulteration.
The NIH defines DS as products that are meant to supplement the diet; contain at least one dietary ingredient (i.e., vitamins, herbs, minerals, amino acids); are intended to be taken orally; and are clearly labeled as being DS on their packaging. According to the FDA, as of 2015, the US DS industry totaled approximately $35 billion, with approximately 150 million Americans taking DS every year.
The regulation of DS has undergone many changes. In 1994, the Dietary Supplement Health and Education Act (DSHEA) was established, permitting the FDA to implement GMP for DS, which the Act classified as foods. The DSHEA described adulteration in a DS as a substance that presents a health risk under both the recommended use on the DS label and in ordinary usage; contains a new ingredient for which there is insufficient information about its assurance of not causing any health risks; poses a public health risk; and meets a conventional food adulteration standard in the Food, Drug and Cosmetic Act. In 2007, the FDA officially published the GMP rule with a phased implementation from 2008 to 2010. The GMP rule requires DS manufacturers to confirm that each batch of DS meets identity, purity, strength, composition and contamination limits.
Like the FDA, the US Pharmacopoeia (USP) also works to monitor DS and serves as the official compendia for DS in the DSHEA. A DS must correspond to a DS monograph in the USP–National Formulary in order for it to comply with federal standards. In 2001, the USP created the voluntary Dietary Supplement Verification Program, giving DS companies the opportunity to mark their products as “USP Verified” if they contain the proper amounts of safe ingredients that are advertised on the packaging. To date, over 400 million DS have the “USP Verified” mark on their labels. In 2009, the USP launched the Dietary Supplement Compendium (DSC), a comprehensive collection of monographs, regulatory guidance and other reference tools for DS and DS ingredient manufacturers, published every three years. Currently, the USP has 800 monographs and over 200 Reference Standards in the DSC.
Instrument companies also work to advance DS adulteration testing. Waters, for example, collaborates with industry leaders to create analytical technologies for DS adulteration testing. “Waters’ strategy is to partner with industry leaders to facilitate and help the standard setting with appropriate and suitably advanced analytical technologies,” stated Kate Yu, PhD, senior manager for Business Operations, Pharmaceutical Business, at Waters. The company created joint open labs and works with regulatory-affiliated labs to educate and train inspectors and end-users. “We’ve established the University of Mississippi–Waters Natural Products Training Center (opened April 2016 in Oxford, Mississippi) and … we’ve formed scientific collaborations with key players from industries, such as Dr. Maged Shreif from the American Herbal Products Association, Dr. Rudi Bauer from the University of Austria, Dr. Jim Harnley from USDA, etc., with the aim of jointly solving key challenges industries are facing,” elaborated Dr. Yu.
Companies such as Thermo Fisher Scientific and Agilent Technologies are also involved in advancing DS adulteration testing, working with the AOAC’s Stakeholder Panel on Dietary Supplements to ensure that customers have the information they need to overcome the challenges associated with DS adulteration testing. “Agilent also collaborates with various leading labs looking at the holistic information that can come from untargeted profiling of dietary supplements and their ingredients using MS,” explained John Lee, Global Food Market manager at Agilent. According to Mr. Lee, Agilent is focused on “the promotion by the dietary supplement community of key efficacious components, either occurring naturally in a commodity or which are legitimately fortified, […] and the way that dietary supplements are promoted—simply because they are derived from a popular and well-respected raw material. Here the key question is one of authenticity rather than efficacy.”
New regulations and standards for DS continue to be introduced. In 2015, the USP implemented the “General Chapter Adulteration of DS with Drugs and Drug Analogs,” a reference tool to aid adulteration detection in DS. Later that year, the FDA created the Office of Dietary Supplements (ODS). According to FDA spokesperson Marianna Naum, PhD, “ [The Office] works with FDA labs to ensure we have appropriate, scientifically valid methodology in place for FDA’s testing of dietary ingredients and supplements. These methodologies are necessary when FDA samples and tests products for potential regulatory action.” The Office works with certain third-party programs to establish methodologies for DS ingredients, and also to implement methodologies and testing for regulatory purposes to ensure product quality. “As our methods are publicly available, they are certainly appropriate for the industry to use in applicable situations,” noted Dr. Naum. “According to the FDA’s DS GMPs, the industry is responsible for ensuring its methodology is appropriate and scientifically valid for its purpose. It is not required to use any particular method for analysis.”
Even with the recent efforts made in DS regulations and standardization, the FDA is still working on improving the participation of DS companies. “At this point, all-size firms have had the requirement of being compliant with GMPs for more than five years,” said Dr. Naum. “Testing is a large part of a GMP system and we still see a lack of testing records during facility inspections, which is concerning. This is part of the reason the FDA is invested in ensuring methodologies are developed and in use, in both the FDA’s and the industry’s labs.”
Likewise, the USP told IBO it has not seen a significant increase in DS testing inquiries following the implementation of Chapter . “Generally, we have little feedback from the industry when the specified procedures work as expected,” said USP’s Anton Bzhelyansky, Scientific Liaison for Botanicals. “I have not received any questions from the industry so far. It is not uncommon for the contract laboratories to implement their own procedures which may differ from the ones General Chapter specifies. No inquiries from individual DS companies either.”
Creating methods and standards for DS is a challenge for organizations like the FDA and USP, since much of the burden for proving product integrity falls onto them. The FDA is required to prove that a DS ingredient is harmful instead of the manufacturer having to prove that the ingredient is safe. As for the USP’s challenges, Mr. Bzhelyansky explained that with pharmaceutical testing, there is a basic set of knowledge guidelines that are required for product analysis, such as knowledge of the analyte; placebo; amount of the analyte’s content per unit dose; content uniformity; lot-to-lot reproducibility; and the components of the sample. With adulteration testing of DS, however, these guidelines are not so useful. “One by one, the basic tenets of pharmaceutical analysis are proven to be inadequate and inapplicable to analysis of adulterated [DS] products,” Mr. Bzhelyansky stated. “In essence, the analysis scheme is closer to a forensic exercise, not typical pharmacopoeial work.” With DS adulteration testing, the subject of analysis is not the actual DS, but the adulterant. As Mr. Bzhelyansky stated, “How does one construct an analysis when so little is known about the subject of the analysis, i.e., the adulterated product?”
Instrument companies must address the challenges for their customers that are testing for adulteration in DS. Using MS with chromatography is a useful way to fingerprint and distinguish botanical ingredients in DS. “Both LC/MS and GC/MS are important in such work and increasingly the use of high-resolution Q-TOF has proved particularly powerful in developing such methods. ICP-MS is also a useful tool,” stated Mr. Lee.
(U)HPLC and triple quadrupole LC/MS are used for targeted analysis. But for non-targeted analysis, (U)HPLC and High Resolution MS (HRMS) (such as Q-TOF MS) are the most effective and commonly used, according to Dr. Yu. “Overall, the biggest challenge in adulteration testing in the field of DS is to identify key chemical markers related to both the authentic samples and to the potential adulterants from complex samples,” said Dr. Yu. She stated that sample preparation also poses a challenge, as does maximizing separation power with the proper balance of analytical run time and peak separation capacity. “Typically, the longer the separation time, the better the separation capacity,” noted Dr. Yu.
Thermo Fisher Scientific’s Khalil Divan, PhD, senior director for Food and Beverage, also cited complex sample matrices as a challenge in adulteration testing. “The perfect platform to be able to detect any potential contaminant or adulterant would be able to detect and quantify the known and unknown analyte in the same run,” he said.
With triple quadrupole LC/MS, Ms. Yu states that the biggest challenges are matrix interference and isomer analysis. “As triple quad LC/MS is unit mass resolution, it can only differentiate compounds/markers with parent mass or MRM transition at least one amu apart,” said Ms. Yu. “For complex samples, coelution is not uncommon, isomers are also not uncommon—chemists have to rely on chromatographic separation to differentiate the isomers and clean up the matrix.” She states that for DS adulteration testing, HRMS is preferred over triple quad LC/MS, if possible.
In the future, integrated workflows may play a significant role in developing methods. “A workflow incorporating (U)HPLC/HRMS with a strong informatics solution is becoming increasing common for method development,” Dr. Yu said. “Once target markers are identified, a fast, mobile device is highly desirable especially for initial screening purpose. For example, direct MS analysis with ambient ionization techniques, such as ASAP and DART.” Advances in instrument sensitivity for detecting adulterants is also an important factor. “Even though, today, triple quadrupole MS is still the gold standard of LC/MS quantification, moving forward, with the fast advancement of the HRMS technology, being able to perform qualitative and quantitative analysis on HRMS will also be a clear trend,” Dr. Yu noted.
Agilent also views MS techniques, including LC/MS, as significant for DS testing in the future, though Mr. Lee believes instrument cost also plays a role in choosing testing methodologies. “While more and more customers appreciate the sensitivity, specificity and the ability to perform untargeted profiling using MS, other labs are satisfied working with existing and validated methods based on LC, which clearly also represents a low investment level and can be implemented with a lower training requirement (both with respect to MS usage and the statistical skills that are required for profiling investigations),” he said.
USP Chapter suggests alternate methods to LC/MS that can also affect testing costs. “[The Chapter] emphasizes the advantages of other, orthogonal approaches, which could be less expensive and laborious, and still yield screening results equivalent, and occasionally, superior to those achievable by LC/MS,” said Mr. Bzhelyansky. “Use of orthogonal techniques is encouraged because while it may be possible to ‘trick’ one test, it is much harder to achieve this with two or more orthogonal ones.”
The DS industry has steadily become more regulated, not only in the US but also globally. In 2014, for example, China’s FDA passed DS regulations. Waters is participating in the implementation of DS adulteration testing in China with its Pharmacopeia–Waters Joint Open Lab, which was opened in Beijing in 2015. Agilent has seen an increase in DS testing since the Nutritional Supplement regulations for DS was passed. “The pressure to ensure that safety, identity and authenticity of Chinese exports is certainly driving increased testing by both LC and mass spec,” said Mr. Lee. “The needs for DS testing in China are mainly from Institute for Drug Control labs in the China FDA system. Some [China} CDC and China Exit-Entry Inspection and Quarantine Bureau labs are also qualified for such testing.” He noted that the majority of these applications use triple quadrupole LC/MS and LC/Q-TOF instrumentation. Thermo Fisher Scientific also noted interest from China for DS testing. “We have been requested to analyze several traditional Chinese medicines and to reproduce Chinese Pharmacop methods using the Thermo Scientific Vanquish platforms and mass detection,” said Dr. Divan.
With new advancements in DS adulteration testing, instrument companies like Waters, Thermo Fisher Scientific and Agilent can participate in creating methods and standards to contribute to hastening the regulatory process, as well as continue to develop instruments to simplify and enhance the accuracy of the testing process. “This is where contaminant testing is headed,” Dr. Divan said. “From highly targeted to non-targeted methods that are able to provide much more comprehensive information about a sample.”