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Testing Lab and Method Qualification


There are thousands of variations of analytical methods used for natural products.  Not all of them are fit for purpose, failing to meet a central requirement for dietary supplement cGMP’s.

Identity methods for botanical products are especially problematic, as demonstrated by the mass confusion set off by the NY A.G.

And manufacturers and contract labs are stuck in the middle of a supply chain that can lack an understanding of the source or processing method, which are important factors for labs to determine appropriate methods.

The slide deck at the link below is from a 2016 talk at SupplySide West that presented challenges and best practices for method selection and validation against established guidance. A process for laboratory qualification, method development and ‘red flags’ are included in these slides.

Access the slides here

NaturPro Supports AOAC Official Method for Ethanol in Kombucha


NaturPro Scientific presented method validation data leading to a unanimous vote for adoption of first action Official Method status at the 130th Annual Meeting of AOAC International, September 18-21, 2016.

The Official Method status is thought to be the first method to demonstrate scientific validity for ethanol in kombucha under peer review, and is the result of a truth-in-labeling initiative supported by industry.

Kombucha is a fermented tea marketed for probiotic properties, which is expected to continue 25% yearly growth, to nearly $2 billion by the year 2020.

The validation data has been submitted to the Journal of AOAC for publication.

A preliminary validation study, including data from multiple laboratories, is available here. 

NDI, GRAS and Supplement Safety Assessment


The objective of NDI and GRAS  for supplements and foods is to provide a baseline evaluation of the safety aspects of an ingredient.

FDA issued draft guidance on the NDI and final guidance on the updated GRAS requirement in August 2016. The guidances are likely to require a significant amount of information related to safety and quality of dietary ingredients to be compiled and evaluated by scientific experts.

Four Steps to Compliance:

1. Determine the most likely regulatory status of your ingredient:  ODI, NDI, GRAS or other based on a preliminary review of regulatory status and toxicology data.
2. Compile a master file of all your safety and quality documents supporting the new CFR 117 and the new FDA guidances. Most master files are more than a hundred pages long, including references.
3. Have the master file reviewed for gaps according to the regulatory status. Perform a risk assessment to safety, quality or brand presented from the analysis.
4. Chart a plan of action to meet the requirements.
Toxicology and safety studies are expensive to conduct, so you need to know if your ingredients need to go through the new GRAS Notification process, require a NDI Notification, and also carry any specific risk for adulteration or contamination. Take care of these elements, and you can be fairly sure that you will not be blind sided by regulators, consumers, or class action attorneys.

A pre-assessment is typically conducted quickly to determine the appropriate strategy and level of risk.  Contact us to determine the best strategy for you.

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For more detail, here is a framework of basic requirements for a safety assessment

  1. Clinical, Medicinal and Food Use 
    1. In country to market
    2. Global
  2. Regulatory Status
    1. ODI or NDI (if ingredient used for supplement)
    2. GRAS
    3. Other
  3. Toxicity Summary
    1. History of human consumption in foods/supplements including dosage amount and composition
    2. LD50/acute toxicity/chronic/subchronic toxicity studies
    3. Bioavailability and ADME
    4. Clinical trials
    5. Other (genotoxicity, carcinogenicity, reproductive toxicity etc)
    6. Case reports, AER and Drug Interaction Review
  4. Dietary Supplement Manufacturing Risk Review (or CMC, Chemistry/Manufacturing/Control)

    1. Chemical and Nutritional Characterization
      1. Literature review
      2. Specification, Certificate of Analysis, ID and contaminants
    2. Manufacturing Facility GMP evaluation (self-assessment and audits)
    3. Potential adulterants and their controls

Contact us for more information.

Identity Testing for Dietary Supplements

by NaturPro in Quality Comments: 0

The mass confusion around identity testing has brought more awareness to the issue, but the industry is still lacking clear guidance on how to deal with some of the practical issues.

How to Properly Conduct Identity Testing for Dietary Supplements

Verifying identity is like a lot of sciences drawn from multiple disciplines: it’s not usually plug-n-chug, and it requires replication of results to achieve meaningful conclusions. Luckily for science, there are valid ways to determine ID. They just take some thought, and in many cases, more than one test.

The thought behind ID requires some understanding of the context of the material being identified. Species expected, plant part, physical form, likelihood and type of adulterants, degree of processing and concentration all dictate what type of method should be used.

Believe it or not, identity testing is easy for a lot of botanical raw materials. Whole or cut herbs, fruits and roots are often easily distinguished by their quality, and by physical features such as size, shape, color and odor. However, we lose this ability when we take a whole leaf, dry it and grind it into a powder whose parts cannot be identified by microscope. At this point, we have to switch to chemical testing to determine what ancient terminology should be used, using classifications created hundreds of years ago by people for plants.

Luckily, whatever form of ID methods we choose, we still need to compare to a ‘reference’ or a sample of the plant as it exists in nature. So it’s really important to be sure the reference material is authentic and representative of the target species in the population, because it forms the entire basis for the test. The method itself should be based on clear and objective pass-fail criteria that includes at least one positive match to the species expected, plus in many cases for higher risk materials, at least one negative match to rule out a possible adulterant.

The documentation is as important as the activity. If it’s not in writing then it didn’t happen, says the auditor. One shortcut often taken for verifying ID is to write ‘conforms’ on the COA. But what, exactly, does the sample conform with? A memory of what the sample looks and smells like? A powder that has been verified to contain some of the same chemical markers as the expected species, which you are ‘pretty sure’ is 100% of the expected species? Or to a specific authenticated reference material? Because shortcuts on the reference are so often taken, it is best to list the reference material number used in the Certificate of Analysis for the sample, under ID Method. Accompanying the COA and available on request should be a test report which lists all methods, reference materials and their validations and verifications.

How to know your reference material is authentic? The best is to acquire whole fertile plant parts from a source that has authenticated the identity of the sample using a trained botanist or herbalist. For chemical testing, a lower grade of botanical references (milled powder, extract or liquid forms) are often used, but their validity as reference material are considered inferior or secondary in the absence of authentication and chain of custody to the whole plant part.

The difference between trusting the source of reference materials versus verifying it can make all the difference. For example, a bottle of olive oil purchased from the grocery store may be used as a reference standard without verifying the identity with testing. However, olive oil is known to be adulterated with other oils, so our trust in the label should not be sufficient to verify its identity. We need more proof, in the form of independent test reports using valid methods, that the olive oil is 100% from olives and is not adulterated, before we can consider it a valid reference material.

Botanical references, even authentic ones, are not ‘ready-made’ to reference all types of samples from the same species and plant part. Again, this is an area that requires some thought by laboratory technicians. For example, when a botanical is extracted or heated, chemical changes occur. To prevent the processing method from falsely impacting the match, a reference must undergo the same process as the sample, or else the chemical differences from heating falsely appear as differences between the species. So, if your sample was extracted in hot 70% ethanol for five hours, then the lab should prepare the reference in the same way.

Now, for the top 5 ID testing lessons they don’t teach in school:

  1. Much of ID testing relies on a sufficient understanding of the material being tested. Communicate to the lab as much info as possible, such as specification and manufacturing process, so that they can make sure to prepare the reference standards correctly and select the correct method.
  2. No ID method covers all potential adulterations or mis-identifications. And ID covers contamination if it may be considered harmful. Make sure you know what the risks are for your materials. Risk assessment for adulteration is necessarily a team effort across the supply chain, not something that ‘someone else should be doing’.
  3. Authentic references and their preparation are as important (or more) than the method used. Always check the sources and make sure they are authenticated and representative of the species and identity.
  4. Testing the same sample with different methods substantially improves the meaningfulness of any one test. High risk materials with known adulterants often need a test for a positive match to the species and a separate test for a negative match to the adulterant.
  5. Many adulterants, especially newly developed ones, are not caught with typical ID methods, which is why strong specifications, and supplier verification is so important.

The issue of ID is not going away, and it’s a big issue in food now too (see sawdust in cheese, sushi that’s not sushi, etc). The wave of attention on food and supplement ID issues has not yet begun to crest, but now at least we know how to swim.

This article was originally published by Natural Products Insider in August 2016.

Single Laboratory Validation of Ethanol in Kombucha Tea

by NaturPro in Uncategorized Comments: 0

Single Laboratory Validation of Ethanol in Kombucha Tea by Gas Chromatography with Flame Ionization Detection

The objective of this study was to ensure the validity of test results of ethanol in kombucha tea by performing single laboratory validation (SLV) of a method using gas chromatography with flame ionization detection (GC-FID).

Downloads:

SLV Study of Ethanol in Kombucha

Research News: SLV Study of Ethanol in Kombucha

AOAC SMPR

 

Why Verify, Then Trust?

by NaturPro in Uncategorized Comments: 0

“Verification” is the 2016 buzzword for food and supplements, due to the sequence of food safety crises that arguably started with salmonella in peanut butter in the early 2000s.  Recently, FSMA and the “Identity Crisis” for botanical ingredients in supplements have renewed the requirement for verification of quality and safety practices in the supply chain: raw materials, manufacturing practices and test methods being three big areas of focus.

“Trust But Verify” is attributed to President Reagan and later FDA and quality assurance folks. Although it is a well meaning mantra, doesn’t it make verification seem optional?  Shouldn’t we verify BEFORE trusting?

We do know that trust disappears soon after a failure to verify becomes apparent. From Salmonella in peanut butter, to misidentified plant extracts, to pesticides in unregulated medicinal plants — verification is how trust is ensured.

So while trust is the ultimate goal, verification comes first.

#verifythentrust

First published on LinkedIn, April 2016

2-Minute Tip: 6 Ways Ingredients Communicate Value

by NaturPro in Uncategorized Comments: 0

Product development is an increasingly painful process, taking weeks and months to sort through and evaluate ingredients.

That’s because the evaluation process involves cutting through the marketing fluff and understanding (and communicating) the core value of your product.  This makes it a difficult and time-consuming task for your customers.

Marshmallow fluff GinnyWhy should your customer pick your product or ingredient over all the others?  Because they are able to communicate it’s value.

Effective customer education  is one great way to help customers navigate the pitfalls of the product development process, and keep your product top of mind.  The results often include higher customer conversion and less wasted activity.

 Here’s a 2-Minute Tip listing a few things to be sure to include in your customer education materials:

2-Minute Tip: Six Ways Ingredients Communicate Value

 

 

What do supplement testing and Star Wars have in common?

by NaturPro in Uncategorized Comments: 0

Star Wars and science fiction fans know that technology is a double-edged sword. On one hand, advances in science offer us fantastic powers to solve difficult problems (space travel, light sabers). On the other hand, the potential for catastrophe is also greater. With better technology comes a greater responsibility to prevent its misuse.

Early botanical scientists understood both the power and limitations of science to describe a complex natural world. Carl Linnaeus, who developed the original system to classify plants and animals, recognized that all organisms are not discrete species necessarily, but exist on a continuous spectrum of life.

Five ways NaturPro helps to ensure scientific validity

star-wars-episode-7-production-release-date

Today, scientists in academia work to identify and quantify the diverse array of chemical constituents in botanical products, while industry works to ensure a safe, effective and consistent product. At our disposal are alphabet soups of various analytical technologies that offer increasingly better detection of constituents, even down to the picogram, which relative to a gram can be visualized as a drop of water in a thousand swimming pools.

But with picoscale resolution comes a lot of noise (one trillion per gram, to be exact) and even more responsibility to reliably separate a signal from it. Even at the parts-per-million (ppm) level—equivalent to a cup of water in a swimming pool—we often observe unexplainable results that defy logic.

How our “UnLab” approach controls for shoddy methods and unexplainable results… 

For example, only today’s best and most expensive instruments, such as multiple mass spectrometers linked to a chromatograph, such as LC/MS/MS (also known as tandem-MS, which means two mass spectrometers are hooked to each other; the first MS removes a lot of the “junk” that can interfere with the result from the second MS), are able to account for matrix effects that occur when testing complex mixtures. The reason complex mixtures are so difficult to examine is they contain so many different compounds, and therefore the chances are relatively high that one of these is observed at the same retention time (or peak) on the chromatogram as the compound a scientist is trying to quantify. Also, because the sample is being injected into super-heated, high-pressure instruments, there are often chemical reactions create new interfering compounds. Matrix effects can falsely change results in a significant way that cannot be resolved without further work. Results should always be questioned and replicated, and ultimately, investments in the development of methods are required to generate confidence.

FDA Supplement FactsValidation of matrix-specific methods across multiple laboratories address these challenges, however few methods have been validated to the extent required to be confident in the results. An example from the nutrition field: the inherent challenges in quantification of vitamin D (a pure compound and age-old vitamin, no less!)

Both the best and worst thing about good science is that with each answer comes another question. There is always more work to be done to achieve the greater goal: reproducible results. Needless to say, rigorous analysis of complex mixtures such as botanical products is often not straightforward. Unfortunately, the aims of science often oppose the aims of high-throughput lab testing.
How do you know whether a lab is focused on getting the right results? Here are some criteria to help decide whether or not to work with an independent laboratory:

  • Is it transparent? Does it share methods, chromatograms, observations, historical data and control charts?
  • Does it perform validation? Does it verify methods using appropriate controls such as calibration curves and spike recovery? What steps are taken when it initially sets up a method?
  • Does it have a process for dealing with out-of-specification results, and will it share that process? Does it have an internal recordkeeping system that tracks method precision and alerts them when a method or system is out of calibration?
  • Does it run internal control samples? Does it run samples in triplicate or duplicate at least, and does it report statistical analysis on the certificate of analysis (CoA), such as standard deviation from multiple runs?
  • How does it validate the purity of reference standards? When it gets a new batch of reference standard, does it run it against an internal control sample? How often does it make fresh reference standard solution?
  • Is it a proactive communicator, for example how often does it advise on the best methods to use, and alert their customers on new developments in methods?

 

Not all testing needs to be expensive or high-tech, but every method needs to be rigorous enough to provide results that are reproducible in another lab. For example, thin layer chromatography (TLC) is not high-tech, but it can be valid to determine botanical identity with the right mix of expertise, a rigorous and validated set of reference standards, and enough trial and error to develop the method and be confident in reproducibility of results. High-performance liquid chromatography (HPLC) is great when actual validation of the method and reference standards have been certified for their purity.

MicroscopeThe true test of scientific validity is when multiple labs running different methods achieve the same result, especially when they are blinded as to the expected result.

Despite all of the challenges in quality control (QC) testing of botanicals, the world is changing, and our industry is rapidly improving. With scientific validity mandated by supplement GMPs (good manufacturing practices), and increasing demands for transparency and validity from all stakeholders, everyone is upping their game. Good science, not science fiction, provides reproducible results we can all be confident in.

Learn about reproducible results through our UnLab…


By: Blake Ebersole

This article appears with revisions, and was originally published in the March 2014 issue of Natural Products Insider.

Eight Steps to Developing Research Relationships

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Developing relationships with scientists is much like any other; the first step is in understanding scientists’ challenges and needs. Sensitivity to the ways of the scientific research world, especially academia, is one of the best ways to get the most out of your research investment.

As for what else a supplement manufacturer needs to do:

Show an interest in the science. Like anyone, scientists can sense if you’re more interested in doing great science or just the marketing benefits from it. Offer solutions that boost both scientific and business objectives. Add to the debate and question assumptions.

Try to discover something new. There are thousands of questions to be answered and thousands of different study designs. To be industry-relevant, adopt “standard” methods used widely—but allow some space for new discoveries. Also, test some new hypothesized bioactivity or clinical effect.  One-hundred percent “me-too” science just isn’t very interesting to scientists or consumers. Plus, new findings are more likely to go viral.

Decide on a budget and be realistic. Most research costs money, unless you can get into a study funded by someone like the NIH. But government funding is decreasing every year, while grant applications have multiplied exponentially. Performing strong research often requires expensive labor and materials, and the coordination of many different shared resources.

Offer unrestricted grants for basic research. Research seeking to understand mechanisms of action often best developed step-by-step, making long-term planning difficult. Unrestricted grants that don’t guarantee a specific study plan allow you to support critical shared resources, and they prevent you from painting yourself into a corner at the beginning of your scientific journey.

Agree to milestones for projects, but anticipate delays. University-based, public-funded research requires the alignment of many parts, so some projects hit snags. Plan in advance to prevent potential troubles with approval, recruitment, testing, or finances. Add a “delay buffer” to your timeline for a more realistic expectation.

Decide whether to publish research results and, if so, where. Agree early on who owns the data and who has final decision on whether to publish results. Deciding this early on is a good idea because it sets the standard for the rigor of study design. It’s not necessary to always publish in a patent application or journal. Consider the fact that by publishing, you are likely helping both humankind and your competition. Decide which one outweighs the other.

Presentations at research conferences are sometimes a good idea because you can “publish” data that is somewhat peer-reviewed, and isn’t widely available to the public.

Scrutinize everything. Analyze all methods, data, and reports closely; question them to the best of your ability. Form an internal peer review panel of experts from related disciplines. Be sure to give yourself and other sufficient time to review and discuss revisions.

License technology. Many universities have inventions or start-ups that quietly clamor for attention and funding. Look for available technologies that are scalable and offer a new benefit for humankind.

By: Blake Ebersole

First published in Natural Products Insider, December 8, 2015

Keys for Meeting Supplement GMP Testing Requirements

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A core concept across GMPs for many industries is scientific validity, and this is also one of the necessary requirements of the dietary supplement GMPs. For example, the purpose of an ingredient specification is to disclose scientifically valid methods and results for the tests, and these methods and results are used to verify the quality and identity of the material being sold.

Scientific validity means that tests must be suitable for what they are intended to measure. In a rapidly evolving industry, scientific validity is a core principle guiding our efforts to ascertain the identity, safety, and label claims of the material that millions of people take to support their health.


Here’s some ways NaturPro helps to ensure scientific validity


To apply scientific principles to the measurement means that we develop a foundation of confidence in test results that accumulates only through repeated testing of viable hypotheses. During the process, we understand that like with many scientific measurements, sources of error exist which tend to increase with complexity. For example, complex samples containing thousands of chemical constituents (e.g., botanical extracts), and instrumentation methods that have a lot of variables all contribute to our bank of “known unknown” and “unknown unknowns.”

Testing using any single method can be an educated guess as an answer to a different question, especially for labs that may only sporadically test a given matrix with a single type of test.

gel electrophoresisToday’s analytical technology to measure analytes in complex mixtures is way ahead of the not-too-distant past, but now we understand a mitigating factor: that with greater power and resolution comes an increasing number of factors that may cause test results to be inaccurate or imprecise.

For example, it can be difficult to account for systematic error associated with dirty chromatography columns or non-optimal instrument conditions. Inaccurate purity data on reference standards (due to either inaccurate standard purity values, or unaccounted-for degradation during storage) are also a common sources of error — when we are simply trying to figure out the “actual” composition of a material. Another source of error arises from the calculation of the results; for example, moisture can account for a certain amount of the measured weight of both samples and standards, which is often simply estimated, even if it is accounted for.


What more does supplement testing and Star Wars have in common?


Other sources of error in testing can be chalked up to incomplete extraction and isolation during the sample preparation.  The subject of dissolution is an interesting one. For example, it is a common assumption that when a sample “dissolves” during HPLC sample prep, then it is fully “ionized” and thus is not strongly bonded to any solid particles (which then often get caught on the filter and not pass into the detector).

If both standard and sample dissolve to the same degree, no problem!  But (unknown unknown) error due to lower than expected ‘percent recovery’  creeps in when your sample is prepared with heat and time, becoming different compounds and binding differently to the protein-fat-and-sugar matrix of a biological product.  So the analyte that you are trying to extract into another phase is often a lot easier using the pure, unbound. chemical reference standard — leading to a difference in percent recovery.  So chemical reference standards are best complemented in testing with an additional control being the original, authentic botanical reference — yes a whole plant part, taken from the same source as the raw material in question.  Sounds easy, but its actually not for a lot of people. 14963749580_49e4e7ed8a_k

Then compound the sample preparation challenges with the high heat and pressure applied by an analytical instrument like HPLC, where more chemical reactions can happen in the complex sample to degrade what you are measuring, all while your pure reference standard survives nicely to the detector. (Theoretically, this scenario can also happen the other way around, where the matrix stabilizes the analyte better than the standard solution under the HPLC conditions.)

Good-Manufacturing-PracticesExciting stuff, all this mystery, which we eventually find answers to through validation and repetitious testing.  While it’s difficult to predict analytical uncertainty, the point is to control it to the extent possible, hopefully to within 5-10% of your expected result — not bad compared to the 20% tolerance limit required by pharmaceuticals.

The practical question facing suppliers and manufacturers is how to ensure your specification accounts for testing variance?  One solution commonly opted for in the short term is surprisingly simple: add the testing variance to the label or spec requirement, to ensure a high statistical probability that the material won’t fail due to inherent imprecision of the test.

The implications of an imprecise test often means that manufacturers are forced to add an ‘overage’ of material, which essentially makes the cost of the material 10% more expensive for every 10% difference in test results. 

Scientific validity in QC testing for supplement all too often is discussed not on a daily basis, but when the cost of “mistakes” has finally sunk in.  Many a product formulator saw hours and months of work go down the drain due to quality testing failures, and everyone involved in product development can testify to the measurable waste of time and resources that result from testing failures, which can include both the approval of bad material, as well as the rejection of good material.


Five ways NaturPro helps to ensure scientific validity


Here is a short list of some practices that QC units can perform to achieve scientific validity as per GMPs:

–Review your lab’s methods for their suitability for the intended purpose. There are good independent labs out there that will share method information, and answer your questions. Always ask whether the sample is being tested in triplicate and request to receive the individual values.

–Review the documentation on the reference standard, specifically the methods and results of the testing used to determine its purity. When was the standard made, when was its purity last tested, and how was it stored in between?

–Blind your sample so your lab does not know what value to expect.

–Test control samples (samples that do not contain the suspected analyte, OR samples that you previously sent to the same lab).

–Work with labs that can demonstrate having worked to some basic degree to optimize/validate the method.

Sounds like costly work, but not so much when put in perspective of the potential costs. With transparency among customer, supplier, and lab together, a little teamwork goes a long way to reduce the costs and maximize the benefits of quality systems.

By: Blake Ebersole

This article was first published in Natural Products Insider in June 2013