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
“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 illegal levels of contaminants in your food or supplement, verification is how trust is ensured.
While trust is the ultimate goal, verification comes first.
#verifythentrust
First published on LinkedIn, April 2016
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.
Why 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:
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…
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.
Validation 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:
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.
The 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.
We are QC Testing Consultants for Supplements and Foods.
NaturPro offers independent quality control analysis and testing for foods and dietary supplements.
Why Testing is Important: Supplement and natural product testing with a compliant laboratory is required by FDA for foods, dietary supplements and ingredients to ensure they meet standards for safety, quality and effectiveness.
For example, identity, potency and purity are quality control parameters needed to legally sell a natural product as an ingredient, food or dietary supplement.
Have food or supplement testing questions? Contact Us..
NaturPro Scientific is driven to achieve the right answers for our clients.
We are not a lab. And this independence means that we are free to pick from the top scientific experts for each type of test or analysis.
As a result, we are truly able to work on behalf of our clients, to ensure that correct methods are used and reliable results are obtained.
3 main benefits of food and dietary supplement QC testing consultants:
We know the shortcuts that testing labs can take, and how to prevent that from happening to you, to ensure testing gives the most scientific, accurate and defensible results possible.
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
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.
Today’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. 
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.)
Exciting 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
The longtime policy of the US Patent and Trademark Office (USPTO) to prohibit patenting of natural products is controversial because it has strong arguments both for and against. Now, the patentability of natural products has come under new scrutiny recently, as the USPTO recently offered a new guidance document regarding how natural products patent applications should be examined in response to recent Supreme Court decisions addressing the patentability of genes.
On one side, the lack of patentability for natural products allows for greater access to natural products in the form of foods and dietary supplements. The flip side is that the significant investments needed to adequately research and develop many natural products into what the medical establishment considers “evidence-based” therapeutic products are not protected without strong patents. The consumer choice could be viewed as either a completely high-quality bottle of plant extract with fantastic clinical research and validation costing hundreds of dollars and available only by prescription—or the model that we have for most natural products today: accessible and generally high quality, but not quite at the level of pharma.
This dichotomy has led to the pharma and supplement/food industries existing, in a sense, on different planets. And since patents are the critical requirement for large R&D investments, natural products often get left in the dust. Although natural product molecules form the underlying structural skeleton for the overwhelming majority of drugs, adding even a seemingly innocuous carbon group to a natural compound creates something that would never be found in nature, and could never be considered a food or supplement—but is fully patentable.
How can we bridge the gap between “evidence-based” therapies and high-quality, accessible products from natural sources? This remains as the billion-dollar question, one whose answer will hopefully be addressed by future innovations resulting from the new patent law.
According to the new USPTO guidance, patentable inventions based on natural products are those that are “significantly different” from natural products, principles and phenomena. How to interpret “significantly different” gets very complicated and is outside the scope of this post, but is described in some detail in the guidance. Here are some key examples given:
While the USPTO guidance is still in a public comment period, there are many on the natural products drug discovery side who believe that the new rules will hurt development efforts. But there are others who believe that the new guidance will force inventors to be truly innovative and apply new technologies and processes to creating natural products, while continuing to allow Americans access to our trusted herbs at a reasonable price. This onion has yet to be fully peeled, but it will be interesting to see how this story develops due to its potential impact on our access to effective healthcare.
By: Blake Ebersole
This article was originally published in Natural Products Insider in March 2015.
The human pursuit of spices has helped to create the world (and America) as we know it today. Hundreds of years ago, merchants from Europe traveled by land and sea to transport exotic and expensive plants such as cinnamon, rosemary, nutmeg and turmeric from Asia. But when the Ottoman Empire restricted Europe’s spice routes to Asia in the 1400s, explorers such as Christopher Columbus looked for alternate routes to India and instead stumbled on our glorious land. It’s not a far stretch to thank cinnamon for our providence.
Spices hold a special place in human existence that we are just starting to understand. Sure, they are prized to provide bold and unique flavors, aromas and colors to otherwise bland foods. But many don’t know the hidden story: before the invention of refrigeration, spices’ underlying bioactivity, in the form of potent and diverse antioxidant and antimicrobial food-preserving properties, helped to prevent sickness and contagion caused by food spoilage. Thus, spices carried a magical aura for those who demanded them, and at the same time, they provided a livelihood for many generations of farmers, harvesters and suppliers.
Today, our interest in spices has shifted to the scientific study of their health benefits, to see if they can help us live healthier lives. On a molecular level, the chemical properties that make spices great flavorings, colorings and food preservatives are closely linked to the properties which help to promote human health. Polyphenols, carotenoids and terpenoids are all highly bioactive and health-supporting classes of compounds common to many spices, and are the focus of thousands of medical research studies.
Consuming enough of these active compounds to make a difference in our health can be tough through food alone. The mantra of many is that a diet with a diversity of spices can help us live longer, but no one is suggesting that fried chicken made with 14 of them is a health food (yet!). And while variety may be the “spice of life,” research suggests a variety of spices added to food can lead to a tendency to overeat.1 Likewise, consumer health media recommendations to sprinkle some cinnamon on toast or add a pinch of turmeric powder to curry may be naïve to some key underlying practical and scientific caveats such as compliance, dose response and opposing effects.
For instance, a clinically significant effective dose of cinnamon powder often recommended for managing blood sugar is a teaspoon or more—quite a “cinnamon challenge” for the palate and the stomach. Impurities that can be found in cinnamon powder, such as added sulfites and naturally occurring coumarin can tip the opposing-effects equation in the wrong direction, especially when doses are in baking measurements. On the other hand, science has validated the efficacy of concentrated, purified extracts, both from Chinese cinnamon (cassia) as well as “true” cinnamon (Cinnamomum verum syn. zeylanicum). Both the “whole food” and the scientific approaches have merits, but the second seems to garner increasingly more credibility among top medical experts.
In another example, four-week supplementation with the amount of straight turmeric powder contained in a strong curry (2.8 g) did not improve oxidative stress, inflammation or global metabolic profile in overweight women.2 But in another study just published on a purified, brain-optimized form of curcumin, just 80 mg of the potent turmeric active consumed daily for four weeks led to significant improvements in measures of short-term memory, attention and mental energy.3
On the other side of getting enough of the active compound absorbed to make a difference is the argument for moderation. Again, we seek to know what the relationship is between the amount of dose and the health benefit observed, and no two natural compounds are exactly alike in this way. The scientific results can be hard to predict. For example, in cell culture experiments where one biological mechanism is isolated, it is common and desirable to see the response increase as the dose increases. But for human trials, more does not always mean better. In one example, daily low dose (750 mg) of rosemary marginally improved cognitive function in elderly adults, but the higher 6-gram dose did not.4
For some spices, their aroma and impact on the brain through our nose is the main source of impact on health. A fair number of well-designed studies have shown positive results with herb and spice aromatherapy on various cognitive-related measures. One study found lavender or rosemary aromatherapy improved relaxation and test scores in nervous nursing students.5 However, rosemary consumed in a capsule form—while wearing a nose clip to block effects of the aroma—did not induce consistent short-term improvements in cognitive function in young adults.6
Topical applications of spices have been used in traditional medicine like Ayurveda for hundreds of years, with turmeric being well proven and used by allopathic physicians for its wound-healing capabilities. The bioactivities of spices that preserve food also promote health in ways that are well known mechanistically, but in a clinical-sense are just now emerging. For example, in a 2014 study, an ointment containing cinnamon was effective at reducing pain after childbirth.7 In another study, a topical application of black pepper essential oil improved vein visibility for IV insertion better than the standard of care.8 This study did not measure whether sneezing increased, although the essential oil used in the study would probably have improved dinner too.
The potential of spices in human health and wellness is vast, and with sound science, more is learned every day about how and why spices can be beneficial.
References:
1. Jones JB et al. “A randomized trial on the effects of flavorings on the health benefits of daily peanut consumption.” Am J Clin Nutr. 2014 Mar;99(3):490-6. DOI: 10.3945/ajcn.113.069401.
2. Nieman DC et al. “Influence of red pepper spice and turmeric on inflammation and oxidative stress biomarkers in overweight females: a metabolomics approach.” Plant Foods Hum Nutr. 2012 Dec;67(4):415-21. DOI: 10.1007/s11130-012-0325-x.
3. Cox KH, Pipingas A, Scholey AB. “Investigation of the effects of solid lipid curcumin on cognition and mood in a healthy older population.” J Psychopharmacol. 2014 Oct 2. PII: 0269881114552744.
4. Pengelly A et al. “Short-term study on the effects of rosemary on cognitive function in an elderly population.” J Med Food. 2012 Jan;15(1):10-7. DOI: 10.1089/jmf.2011.0005..
5. McCaffrey R, Thomas DJ, Kinzelman AO. “The effects of lavender and rosemary essential oils on test-taking anxiety among graduate nursing students.” Holist Nurs Pract. 2009 Mar-Apr;23(2):88-93. DOI: 10.1097/HNP.0b013e3181a110aa.
6. Lindheimer JB, Loy BD, O’Connor PJ. “Short-term effects of black pepper (Piper nigrum) and rosemary (Rosmarinus officinalis and Rosmarinus eriocalyx) on sustained attention and on energy and fatigue mood states in young adults with low energy.” J Med Food. 2013 Aug;16(8):765-71. DOI: 10.1089/jmf.2012.0216.
7. Mohammadi A et al. “Effects of cinnamon on perineal pain and healing of episiotomy: a randomized placebo-controlled trial.” J Integr Med. 2014 Jul;12(4):359-66. DOI: 10.1016/S2095-4964(14)60025-X.
8. Kristiniak S et al. “Black pepper essential oil to enhance intravenous catheter insertion in patients with poor vein visibility: a controlled study.” J Altern Complement Med. 2012 Nov;18(11):1003-7. DOI: 10.1089/acm.2012.0106.
By: Blake Ebersole
This article was first published in Natural Products Insider, December 2014