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Single Laboratory Validation of Ethanol in Kombucha Tea

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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).

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SLV Study of Ethanol in Kombucha

Research News: SLV Study of Ethanol in Kombucha

AOAC SMPR

 

Why Verify, Then Trust?

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“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 cannabis, 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

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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?

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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

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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