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In-House Analytical Laboratory

Nootropics Depot Logo

IN-HOUSE ANALYTICAL LABORATORY

  • 📋 INTRODUCTION
  • 🥼 360° VIRTUAL TOUR
  • 🔬 ISOMETRIC INFOGRAPHIC
  • 🧪 WHY TEST?
  • 🤔 HOW IT WORKS
  • 📘 QUALITY SYSTEMS
  • 📓 QUALITY CONTROL
  • 📊 COMPETITIVE ANALYSIS
  • 🙋 FAQ

WELCOME TO THE NOOTROPICS DEPOT IN-HOUSE ANALYTICAL LABORATORY

Welcome to the Nootropics Depot in-house analytical laboratory! Many of our customers are aware of the insane level to which we go to ensure products are analyzed properly every time, but many have not seen it with their own eyes. We at Nootropics Depot are not only trying to advance the lab and quality control standards of the industry, but are also trying to educate consumers about the science behind the products they love. Use the tabs above to take a 360° virtual tour of our lab and learn why proper analytical testing in both the nootropic and supplement spaces should be an important factor in your purchasing decisions.

WHERE YOU BUY FROM AND WHO YOU CHOOSE TO SUPPORT MATTERS

Over the course of the last several years, Nootropics Depot has made significant financial investments in the construction of our in-house analytical laboratory, its machines and instruments, and staffing highly-qualified scientists to ensure that you receive a quality product each and every time you order with us. We respect and value the great level of trust that has been placed in us, and it is due to the unwavering patronage of our customers, that the Nootropics Depot in-house analytical laboratory is proud to be able to feature:

    • Over $5 Million Dollars Invested In Its Creation
    • 14 Full-Time Laboratory Scientists
    • 4 Dedicated Quality Control Personnel
    • In-House Analytical Method Development
    • Membership in AOAC International (Association Of Official Analytical Chemists)
    • 7 Third-Party Laboratory Partners

WHY SHARE THIS INFORMATION?

On the surface, it may appear as though sharing this information and opening the doors to our in-house analytical laboratory for all to see may put us at a strategic disadvantage. After all, doesn't this mean a business competitor could simply copy what we have built? Technically, yes. However, we are also fully aware of the roadblocks and difficulties that we have faced over the last several years to even be able to get to a point where we can present this information and virtual lab tour to you. Not showing what we have built with the support of our customers and demonstrating what it is possible to accomplish when intentions are genuine would be a disservice to the entire project. Our analytical laboratory can quantify many things, but one it cannot is the amount of resolve and determination that has been required for a company like ours to have accomplished this goal.

Moreover, as we have always stated, Nootropics Depot and its sister companies were founded with the intention of redefining the analytical testing standards for the entire nootropic and supplement industry. That mission has never changed for us. This presentation and the existence of our virtual lab tour is transparent validation of that fact, but we also recognize that it does not necessarily mean that our end-goal of industry-wide change has been accomplished. So, if a business competitor were to accept the challenges and hardships of emulating or re-creating what we have built, we welcome it because we see it as the beginning of the change that is so desperately needed in our industry. In the meantime, our promise to you -- loyal customers and new customers alike -- is that when you purchase an authentic Nootropics Depot product, you can rest assured that you will receive a quality product each and every time you order it from us.

We hope you enjoy this tour. On behalf of our entire staff, thank you for your unwavering patronage.

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TAKE A VIRTUAL TOUR OF THE NOOTROPICS DEPOT IN-HOUSE ANALYTICAL LABORATORY

Welcome to our in-house analytical laboratory virtual tour. This tour, which is viewable with full 360° movement; features audio narration, 40+ points of interest, and 11 total scenes that provides you with a comprehensive view of our entire in-house laboratory. See the virtual tour navigation tips below to learn how to make the most of your user experience.

VIRTUAL TOUR NAVIGATION TIPS

1. TURN ON AUDIO NARRATION: Use the cogwheel located in the upper right-hand corner of the virtual tour to enable audio narration.

2. CLICK POINTS OF INTEREST: Once enabled, click on points of interest to listen to audio narration and read more. There are 40+ points of interest to explore in this virtual tour!

3. EXPLORE IN 360°: With your cursor hovering over a scene; click, hold, and move your mouse to explore any scene with full 360° movement.

 

4. VIEW ALL SCENES: Click the left and right arrows located in the bottom center of the virtual tour to navigate all 11 scenes.

 

VIRTUAL TOUR NAVIGATION TIPS

1. TURN ON AUDIO NARRATION: Use the cogwheel located in the upper right-hand corner of the virtual tour to enable audio narration.

Points of Interest

2. TAP POINTS OF INTEREST: Click on points of interest to listen to audio narration and read more. There are 40+ points of interest to explore in this virtual tour!

3. EXPLORE IN 360°: Using your finger; tap any scene and hold to rotate your view with full 360° movement.

Arrows

4. EXPLORE ALL SCENES: Click the left and right arrows located in the bottom center of the virtual tour to navigate all 11 scenes.

5. LOAD TIMES: Please note that this virtual tour may take a few moments to load. Rapidly clicking through individual scenes may degrade overall performance. We appreciate your patience.

 

6. EXPAND YOUR VIEW: While this virtual tour is viewable on mobile devices, wider displays such as a desktop monitor or laptop may provide a more preferable experience for some users.

LOAD TIMES: Please note that this virtual tour may take a few moments to load. Rapidly clicking through individual scenes may degrade overall performance. We appreciate your patience.

 

 

LOAD TIMES: Please note that this virtual tour may take a few moments to load. Rapidly clicking through individual scenes may degrade overall performance. We appreciate your patience.

SEE AN ISOMETRIC VIEW OF OUR IN-HOUSE ANALYTICAL LABORATORY LAYOUT

WHY SHOULD YOUR NOOTROPICS & SUPPLEMENTS BE TESTED?

In order to know if a quality product has been created, it must be tested. At Nootropics Depot, every incoming batch is tested for identity, purity, and for the presence of dangerous and toxic compounds such as heavy metals. Nootropics Depot has been regularly expanding its lab since launching the company in 2013. We are always adding new equipment, personnel, and capabilities to advance our ability to scientifically analyze products, and to assist in the research and development of new nootropics and supplements. Our efforts will never be complete. We will always be working towards expanding and improving our lab. Your support of Nootropics Depot helps to ensure we can keep pushing our mission forward. New capabilities include the acquisition of an Ultra Performance Liquid Chromatograph (UPLC) and an Ultra Performance Liquid Chromatographic Mass Spectrometer (UPLC-MS) provided by the industry leading Waters, a Headspace Gas Chromatograph (HS-GC) built by Thermo Fisher, and a cutting edge High Performance Thin Layer Chromatograph (HPTLC) provided by the founders of the technology Camag. These upgrades give the analytical chemists at Nootropics Depot the ability to test all incoming batches quickly and efficiently at a lower cost than ever before.

WHAT YOU SHOULD KNOW ABOUT CURRENT INDUSTRY STANDARDS

Most nootropic and dietary supplement companies have a system in place whereby they do not test every batch. This is called “skip lot” testing, or “qualification” testing. At Nootropics Depot, we do not perform any skip lot or qualification testing. Every batch of raw material gets tested for every test, every time. While this adds a lot of work, cost, and time to the laboratory and quality control departments, a much higher level of certainty of product quality can be obtained using this method. This system ensures that our products are always properly identified, quantified, and have met all quality metrics for safety, for every lot produced. It also gives us a higher level of control over our supply chain, because our suppliers know we are testing every batch, so they cannot sneak a failing batch into the supply line. Because of this rigorous level of testing, we reject many more batches than our suppliers, because we only accept the products that meet our high safety and efficacy standards.

HOW TESTING WORKS

Identity testing can be performed in a variety of ways, such as Fourier Transform Infrared Spectroscopy (FTIR), proton Nuclear Magnetic Resonance Spectroscopy (1H NMR), or in the case of mushrooms and botanicals, High Performance Thin Layer Chromatography (HPTLC). For products that are a single type of molecule and single type of crystal structure, such as a pure glycine product, FTIR can be the appropriate choice. For other products for which it is more important to be certain of their structure, or which exhibit high degrees of polymorphism such as some nootropics, 1H NMR is a better option. Lastly, for mushrooms and botanicals, of which the vast majority of the material is cellulose, HPTLC is a better option, as FTIR only measures the cellulose of the plant material, and the identifying compounds of the botanical material are obscured from measurement.

In the menu below you will see the word machine in some places and instrument in other places. This was not accidental. In analytical chemistry, an instrument makes a measurement while a machine performs an operation. The rotary evaporator (rotovap) is a machine, a hammer is a tool, and an HPLC is an instrument. They all perform their functions, but this is how our analytical chemists speak, very precise, and exactingly accurate. Use the menu below to explore and learn more.

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HPLC - High Performance Liquid Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR HPLC

The two main purposes of this instrument in our lab are to determine the purity of isolated compounds, such as Caffeine, and to quantify the exact amount of phytochemicals present in our plant extracts, known as an assay test, such as the EGCG content in our Green Tea Extract. Assays are how we know the purity of our single ingredients or the amounts of actives present in our extracts.

When we test a sample with this instrument, it separates all of the molecules into peaks using column chromatography. The columns in an HPLC separates the compounds by changing the speed of the analytes as they pass through the column, resulting in differing chemicals exiting the column at different times. Once separated into individual peaks, we can measure the total amount of each compound present in the sample. It measures them by shining light through the sample and plotting the absorption with a UV or diode array detector. Proper HPLC analysis of a sample requires the use of reference materials, such as analytical standards. We use these reference materials to calibrate our HPLC instrument and the methods associated with it to ensure the assay results are scientifically accurate.

HPTLC - High Performance Thin-Layer Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR HPTLC

The main purpose of this instrument is to determine which biological species we are dealing with, and going even deeper, which part of the species was used. Essentially our HPTLC allows us to identify which species and botanical part were used in making the extract we are analyzing. This allows us to not only know if a sample is made from Panax ginseng or Panax quinquefolius, but also if it was made from the roots or the leaves. HPTLC analysis gives an incredible amount of insight into the botanical extracts we work with. We also occasionally use this instrument to quantify the exact amount of phytochemicals present in our plant extracts, such as Eurycomanone in our Tongkat Ali extracts. This is another assay technique that we employ in specific scenarios.

HPTLC also uses chromatography to separate the compounds in a sample. However, it does not use a column like HPLC. It uses a flat TLC plate instead. Think of the HPTLC plate as an HPLC column that has been flattened. The mechanisms are similar, but the shape and process differ. The different molecules separate from each other on the plate through what is called “capillary action.” This means rather than exiting the column at different times like in an HPLC, the different molecules separate from each other by their location on the TLC plate and form bands. These bands can then be seen by fluorescing various wavelengths of light in a similar fashion to the UV detector on an HPLC. The “fingerprint” these bands make allow us to identify the species and plant part contained in the sample, then the level of fluorescence can be compared to a reference standard to accurately quantify individual molecules in the sample. There is really no better tool for the regular QC analysis of botanical extracts than HPTLC.

UPLC - Ultra Performance Liquid Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR UPLC

This instrument is very similar to our HPLC, but it utilizes much higher pressure, smaller diameter columns, smaller particles, and less solvent during separation. UPLC analysis allows for results in a much shorter period of time with less chemical waste. For some methods we can use ten times less solvent running a sample on the UPLC instead of the HPLC. This makes the UPLC more environmentally friendly. It results in significantly faster turnaround of samples, due to the faster run times. Some methods can be 20-60 minute run times on the HPLC, but only 3-10 minutes on the UPLC. It can also be especially useful for difficult to test compounds that require high levels of selectivity and sensitivity, such as bacopa and kava. It performs the same function as an HPLC, allowing for the assay of chemical compounds and content analysis of botanical extracts, but does it quicker, more efficiently, and in a more selective fashion.

The UPLC fundamentally works the same as an HPLC, using column chromatography to separate different molecules from each other, then detecting them using a UV detector. An HPLC will run around 6,000 PSI, whereas our Waters UPLC will run at 15,000 PSI. This allows for the columns to be smaller in diameter, which not only reduces time and solvent use, but can result in more clear separation and definition of peaks. While UPLC is the modern version of HPLC, it still requires high quality analytical reference materials for calibration and accuracy of assay results.

UPLC-MS - Ultra Performance Liquid Chromatography Mass Spectrometry

WHAT YOU SHOULD KNOW ABOUT OUR UPLC-MS

Mass spectrometric techniques give us additional information about a sample in ways that spectroscopic techniques do not. We use this technique to develop new products, test competitor’s products, and validate materials internally. Mass spectroscopic techniques are the best ways to analyze compounds that UPLC and HPLC cannot, while also providing an orthogonal data set for additional quantitative or qualitative certainty.

Where a UV detector on an HPLC or UPLC detects the fluorescence of a sample, a mass spec detects the fragmentation patters after molecules are hit with electrons. A UV detector needs the molecule to have a chromophore to be able to “see” it. However, every molecule has a mass. Bombarding a molecule with electrons and measuring how it fragments gives us even more information on exactly what molecule that is. Our Waters UPLC-MS goes beyond what normal LCMS machines can see, as it keeps the PDA, or photodiode array detector, inline with the mass spec. This means we can get a full spectrum analysis of a sample at various wavelengths in addition to the mass fragmentation data. The mass detector works in synergy with the optical detector to give unmatched insight into the contents of a sample.

HS-GC - Headspace Gas Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR HS-GC

Gas chromatography relies on gaseous mobile phase and solid or liquid stationary phases to separate molecules, similar to Liquid Chromatography in HPLC. With this instrument, we test for the presence of Residual Solvents that are the result of the synthesis process or extraction process for botanicals. When a compound is synthesized, or a botanical is extracted, solvents are used in the process. These solvents are then removed during the purification process. However, sometimes they are not completely removed. Our headspace GC allows us to test for the presence of these solvent residues, and to quanitfy them to ensure they are below our specification levels.

How this process works is a sample is dissolved in water, heated in a small oven, and the gas that evolves is automatically injected into the instrument. It then gets separated based on its boiling point, and eventually burns up in the Flame Ionization Detector, or FID. Essentially, we are exposing the sample to a hydrogen flame, then detecting the ions formed during combustion. From this process a chromatogram is generated. When calibrated properly, the amount of each residual solvent present can be identified and quantified to extreme precision. This instrument measures solvents in the parts per billion range (ppb) and is accurately and precisely calibrated. Needless to say, this instrument and method is vigorously validated using known reference standards, which is how we can get the precision.

UV-Vis - Ultraviolet Visible Spectroscopy

WHAT YOU SHOULD KNOW ABOUT OUR UV-VIS

This instrument relies on a chemical property called absorbance, whereby some molecules can absorb light of specific wavelengths in a matter in accordance with Beer’s Law. This law allows a chemist to prepare a liquid sample and place it into the instrument, specify the wavelength to be used, and the instrument measures the amount of light into and out of the sample. If the identity of the molecules being tested are known, the amount can be calculated in accordance with Beer’s Law. We use this instrument to test for the quantity of beta-glucans in our mushroom and yeast products, as well as for our assay values for products like epicatechin and hesperidin. UV-Vis was used for identification and assay of botanical extracts in the past, but HPTLC has taken its place for most scenarios.

UV-Vis does not use chromatography like HPLC. So while the premise is similar to the UV detector on a HPLC and HPTLC, the UV-Vis is measuring the absorbance of light in a whole sample, rather than measuring individual compounds in a sample in an HPLC peak or an HPTLC band. For individual pure compounds, like pure hesperidin or 7,8-dihydroxyflavone, that is perfectly fine. No separation is needed in a sample that only contains a single compound. However, for complex mixtures like milk thistle it can overstate the values. This is why the proper methodology needs to be chosen for a specific instance. For our mushroom beta-glucan testing, we do enzymatic preparation of the sample using Megazyme methods prior to testing on UV-Vis. This uses specific enzymes to hydrolyze a mushroom sample, which can then be assayed for beta-glucan content. UV-Vis is still a useful tool in our lab today.

FTIR - Fourier Transform Infrared Spectroscopy

WHAT YOU SHOULD KNOW ABOUT OUR FTIR

FTIR is a spectroscopic technique that measures the interaction of a sample with infrared light. More specifically, FTIR analysis measures how much infrared light goes in and out of the sample. We mainly use FTIR analysis for identification of the materials we are working with. FTIR requires very little sample preparation, and the analysis is very fast. This makes FTIR a fantastic instrument for identifying products before more costly analytical testing occurs.

Our ThermoFisher Scientific IS50 FTIR has both transmission and reflectance capabilities, along with a near IR module attachment. Almost all of our analyses on the FTIR are done using ATR, or attenuated total reflectance. This shines infrared light through a diamond into a sample, and then provides a spectrum of what is reflected back out. FTIR-ATR is a really good methodology for quickly determining the identity of a sample. It can also tell you if there are any other carbon-based compounds in the sample. While it cannot be used as an assay technique, it can be used as an indicator of what other compounds might be in a sample. Paired with other orthogonal techniques, FTIR can give you great insight into the chemical composition of a sample.

Polarimeter

WHAT YOU SHOULD KNOW ABOUT OUR POLARIMETER

A polarimeter, like UV-VIS and FTIR is a spectroscopic instrument, which means that it detects how light interacts with a molecule. A Polarimeter is used to measure the rotation of plane polarized light. This allows a polarimeter to detect the optical rotation of a molecule, but only if it has a Chiral center or is a chiral molecule. Our Polarimeter is important for the analysis of products that can have different optical rotations, such as Theanine, which can both exist as L-Theanine and D-Theanine. For our Theanine product, we only want L-Theanine, and thus we can utilize our Polarimeter to make sure that we have pure L-Theanine that has no D-Theanine in it.

How polarimeters work is by passing monochromatic light through a polarizing plate. This creates a polarized beam that is then shined through a sample. The light then passes through another polarizer known as the analyzer, which automatically rotates. When the analyzer rotates such that all the light passes through the sample, then rotates so that none of the light passes through, it can measure the angle between those two points. This gives you the optical rotation. When a molecule can be in both dextrorotatory or levorotatory forms, like in the case of theanine, the optical rotation can tell you just how much of each isomer is in a sample.

Melting Point Apparatus

WHAT YOU SHOULD KNOW ABOUT OUR MELTING POINT APPARATUS

This instrument is used to determine at which point a pure compound melts. It does this by slowly heating up the sample, and detecting the temperature at which it turns into a liquid. For well studied pure compounds, the melting point of the material is well known. This can be indicative as to the purity of the sample, and gives us a quick and reliable way in which to gauge the purity of a compound before more time consuming analysis occurs. Often times measuring the melting point is a manually done method. However, ours is an automated melting point apparatus. This allows us finer control over the variables, and also videos the melting and plots the graph of how the substance melts quantitatively. This allows us a much more accurate view into just how a substance melts. This is another orthogonal technique that gives us extra information about a sample, and combines with other methodologies to give us a more comprehensive picture about the identity and purity of a sample.

LOD - Loss On Drying

WHAT YOU SHOULD KNOW ABOUT LOSS ON DRYING

This instrument is essentially an analytical balance with a small oven on top. A sample is poured onto the balance and the oven turned on. As the sample heats, volatiles evaporate, and the material dries. After measurement is complete the percent of mass lost can be determined. This is usually assumed to be only water, but some samples are volatile or melt, and the loss on drying measurement can be confounded. LOD analysis can help us identify if a material was properly stored and not exposed to moisture. This is important for products like Alpha-GPC 99% which has to be stored as dry as possible. Loss on drying is also used in the calculation of some assays. You will often see two measurement on an HPLC: “as is” and “dry basis.” How you calculate the assay on a dry basis is by measuring the loss on drying. If you have a sample that is coming in at a 95% assay, and you have a 5% loss on drying, you can determine if it is just water making up the difference, or if there are actual impurities in the sample. Orthogonal techniques once again come into play. If you pair loss on drying with headspace GC and HPLC, you can be more confident as to what compounds are left in your sample.

Water Activity

WHAT YOU SHOULD KNOW ABOUT WATER ACTIVITY

This is a technique used to measure the energy of water in a material. In order for microbial growth to occur, water must be present at a certain amount, and the water cannot be bound in the sample. Below specific water activity levels, no microbial growth can occur, as not enough unbound water is present for growth. This is a very important and useful instrument for us, because it can tell us whether or not a product is at risk of microbial contamination. Due to the importance of this test, it is performed on every batch of all of our products, both raw materials and finished goods. Any products that fail the water activity test or are suspect to microbial growth are subjected to full microbial testing according to USP standards. Certain products do not work properly with water activity analysis, like Nigella sativa. In those instances, we directly analyze using traditional microbial growth culture analysis.

Lyophilizer

WHAT YOU SHOULD KNOW ABOUT OUR LYOPHILIZER

Lyophilizers are used to remove water and other volatile compounds from a sample via vacuum, which in turn lowers the temperature, and freezes the material. We use this machine for preserving reference materials that we produce in our laboratory. During reference material characterization, small amounts of the material are thawed and used for testing. After characterization is complete, enough preserved remaining material exists to be used as a reference material for methods such as UPLC, which require calibration with reference materials. Lyophilizers can also be used to freeze dry products, like peptides.

Preparatory HPLC

WHAT YOU SHOULD KNOW ABOUT OUR PREPARATORY HPLC

This instrument is a cousin of the analytical HPLC. While the purpose of analytical HPLC is to separate and measure the amount of molecules present in a sample, the purpose of preparatory HPLC is to separate and collect each type of molecule present in a sample to concentrate them. The material collected can then be used for research and development, creation of reference materials in conjunction with a lyophilizer, or re-separated for better understanding of the components of the mixture as it was originally collected. Because the material input is collected back after the use of this instrument, this instrument is a non-destructive technique, unlike most other analytical techniques.

Preparatory HPLCs works identically to normal HPLCs, by using column chromatography to separate molecules in a sample from each other. They even use a UV detector like a normal HPLC does. However, rather than the UV measurement being the end goal like in a normal HPLC, a prep HPLC’s end goal is to separate and concentrate individual molecules for later use. This is how we created our own erinacine-A reference standard. We used prep HPLC to separate the erinacine-A from a sample of lion’s mane, then accumulated and concentrated it till there was enough to be able to use as a reference standard for our HPLC and UPLC assays.

Rotary Evaporator

WHAT YOU SHOULD KNOW ABOUT OUR ROTARY EVAPORATOR

A Rotovap is used to rotate and evaporate a sample under vacuum so that excess solvent can be removed. We use this machine for research and reference material creation in conjunction with our preparatory HPLC instrument. The output of a preparatory HPLC experiment typically has lots of excess solvent present. This machine can remove excess solvents at low temperatures, by using a combination of a strong vacuum which lowers boiling points of solvents and increasing surface area by rotating the sample. This is important for removing solvents from samples that are sensitive to heat, as each parameter can be precisely controlled and monitored during the course of an experiment. Rotovaps can also be used in chemical synthesis, to remove the solvents used in the various synthesis steps.

Microgram Balance

WHAT YOU SHOULD KNOW ABOUT OUR MICROGRAM BALANCE

This balance is extremely accurate and sensitive, and can measure down to 10 micrograms with high precision. At these sensitivity levels, the microgram balance can actually measure the breath of the analyst working with the balance! Measurements can also vary due to changes in temperature and pressure in the laboratory. Due to this, our microgram balance features a sophisticated internal calibration scheme to recalibrate itself every time there is a temperature or pressure change in the laboratory. We use this balance for measuring reference materials used to calibrate instruments like our UPLC. If we did not have this microgram balance, we would waste a lot more expensive reference standards. This is because we would need to use the entire standard each time we needed to run a calibration on an assay. Since we can use this extremely accurate scale to measure out small amounts of our reference standard, we reduce waste and cost associated with the use of reference standards in the lab, in addition to giving us more accuracy and precision in our measurements.

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HPLC - High Performance Liquid Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR HPLC

The two main purposes of this instrument in our lab are to determine the purity of isolated compounds, such as Caffeine, and to quantify the exact amount of phytochemicals present in our plant extracts, known as an assay test, such as the EGCG content in our Green Tea Extract. Assays are how we know the purity of our single ingredients or the amounts of actives present in our extracts.

When we test a sample with this instrument, it separates all of the molecules into peaks using column chromatography. The columns in an HPLC separates the compounds by changing the speed of the analytes as they pass through the column, resulting in differing chemicals exiting the column at different times. Once separated into individual peaks, we can measure the total amount of each compound present in the sample. It measures them by shining light through the sample and plotting the absorption with a UV or diode array detector. Proper HPLC analysis of a sample requires the use of reference materials, such as analytical standards. We use these reference materials to calibrate our HPLC instrument and the methods associated with it to ensure the assay results are scientifically accurate.

HPTLC - High Performance Thin-Layer Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR HPTLC

The main purpose of this instrument is to determine which biological species we are dealing with, and going even deeper, which part of the species was used. Essentially our HPTLC allows us to identify which species and botanical part were used in making the extract we are analyzing. This allows us to not only know if a sample is made from Panax ginseng or Panax quinquefolius, but also if it was made from the roots or the leaves. HPTLC analysis gives an incredible amount of insight into the botanical extracts we work with. We also occasionally use this instrument to quantify the exact amount of phytochemicals present in our plant extracts, such as Eurycomanone in our Tongkat Ali extracts. This is another assay technique that we employ in specific scenarios.

HPTLC also uses chromatography to separate the compounds in a sample. However, it does not use a column like HPLC. It uses a flat TLC plate instead. Think of the HPTLC plate as an HPLC column that has been flattened. The mechanisms are similar, but the shape and process differ. The different molecules separate from each other on the plate through what is called “capillary action.” This means rather than exiting the column at different times like in an HPLC, the different molecules separate from each other by their location on the TLC plate and form bands. These bands can then be seen by fluorescing various wavelengths of light in a similar fashion to the UV detector on an HPLC. The “fingerprint” these bands make allow us to identify the species and plant part contained in the sample, then the level of fluorescence can be compared to a reference standard to accurately quantify individual molecules in the sample. There is really no better tool for the regular QC analysis of botanical extracts than HPTLC.

UPLC - Ultra Performance Liquid Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR UPLC

This instrument is very similar to our HPLC, but it utilizes much higher pressure, smaller diameter columns, smaller particles, and less solvent during separation. UPLC analysis allows for results in a much shorter period of time with less chemical waste. For some methods we can use ten times less solvent running a sample on the UPLC instead of the HPLC. This makes the UPLC more environmentally friendly. It results in significantly faster turnaround of samples, due to the faster run times. Some methods can be 20-60 minute run times on the HPLC, but only 3-10 minutes on the UPLC. It can also be especially useful for difficult to test compounds that require high levels of selectivity and sensitivity, such as bacopa and kava. It performs the same function as an HPLC, allowing for the assay of chemical compounds and content analysis of botanical extracts, but does it quicker, more efficiently, and in a more selective fashion.

The UPLC fundamentally works the same as an HPLC, using column chromatography to separate different molecules from each other, then detecting them using a UV detector. An HPLC will run around 6,000 PSI, whereas our Waters UPLC will run at 15,000 PSI. This allows for the columns to be smaller in diameter, which not only reduces time and solvent use, but can result in more clear separation and definition of peaks. While UPLC is the modern version of HPLC, it still requires high quality analytical reference materials for calibration and accuracy of assay results.

UPLC-MS - Ultra Performance Liquid Chromatography Mass Spectrometry

WHAT YOU SHOULD KNOW ABOUT OUR UPLC-MS

Mass spectrometric techniques give us additional information about a sample in ways that spectroscopic techniques do not. We use this technique to develop new products, test competitor’s products, and validate materials internally. Mass spectroscopic techniques are the best ways to analyze compounds that UPLC and HPLC cannot, while also providing an orthogonal data set for additional quantitative or qualitative certainty.

Where a UV detector on an HPLC or UPLC detects the fluorescence of a sample, a mass spec detects the fragmentation patters after molecules are hit with electrons. A UV detector needs the molecule to have a chromophore to be able to “see” it. However, every molecule has a mass. Bombarding a molecule with electrons and measuring how it fragments gives us even more information on exactly what molecule that is. Our Waters UPLC-MS goes beyond what normal LCMS machines can see, as it keeps the PDA, or photodiode array detector, inline with the mass spec. This means we can get a full spectrum analysis of a sample at various wavelengths in addition to the mass fragmentation data. The mass detector works in synergy with the optical detector to give unmatched insight into the contents of a sample.

HS-GC - Headspace Gas Chromatography

WHAT YOU SHOULD KNOW ABOUT OUR HS-GC

Gas chromatography relies on gaseous mobile phase and solid or liquid stationary phases to separate molecules, similar to Liquid Chromatography in HPLC. With this instrument, we test for the presence of Residual Solvents that are the result of the synthesis process or extraction process for botanicals. When a compound is synthesized, or a botanical is extracted, solvents are used in the process. These solvents are then removed during the purification process. However, sometimes they are not completely removed. Our headspace GC allows us to test for the presence of these solvent residues, and to quanitfy them to ensure they are below our specification levels.

How this process works is a sample is dissolved in water, heated in a small oven, and the gas that evolves is automatically injected into the instrument. It then gets separated based on its boiling point, and eventually burns up in the Flame Ionization Detector, or FID. Essentially, we are exposing the sample to a hydrogen flame, then detecting the ions formed during combustion. From this process a chromatogram is generated. When calibrated properly, the amount of each residual solvent present can be identified and quantified to extreme precision. This instrument measures solvents in the parts per billion range (ppb) and is accurately and precisely calibrated. Needless to say, this instrument and method is vigorously validated using known reference standards, which is how we can get the precision.

UV-Vis - Ultraviolet Visible Spectroscopy

WHAT YOU SHOULD KNOW ABOUT OUR UV-VIS

This instrument relies on a chemical property called absorbance, whereby some molecules can absorb light of specific wavelengths in a matter in accordance with Beer’s Law. This law allows a chemist to prepare a liquid sample and place it into the instrument, specify the wavelength to be used, and the instrument measures the amount of light into and out of the sample. If the identity of the molecules being tested are known, the amount can be calculated in accordance with Beer’s Law. We use this instrument to test for the quantity of beta-glucans in our mushroom and yeast products, as well as for our assay values for products like epicatechin and hesperidin. UV-Vis was used for identification and assay of botanical extracts in the past, but HPTLC has taken its place for most scenarios.

UV-Vis does not use chromatography like HPLC. So while the premise is similar to the UV detector on a HPLC and HPTLC, the UV-Vis is measuring the absorbance of light in a whole sample, rather than measuring individual compounds in a sample in an HPLC peak or an HPTLC band. For individual pure compounds, like pure hesperidin or 7,8-dihydroxyflavone, that is perfectly fine. No separation is needed in a sample that only contains a single compound. However, for complex mixtures like milk thistle it can overstate the values. This is why the proper methodology needs to be chosen for a specific instance. For our mushroom beta-glucan testing, we do enzymatic preparation of the sample using Megazyme methods prior to testing on UV-Vis. This uses specific enzymes to hydrolyze a mushroom sample, which can then be assayed for beta-glucan content. UV-Vis is still a useful tool in our lab today.

FTIR - Fourier Transform Infrared Spectroscopy

WHAT YOU SHOULD KNOW ABOUT OUR FTIR

FTIR is a spectroscopic technique that measures the interaction of a sample with infrared light. More specifically, FTIR analysis measures how much infrared light goes in and out of the sample. We mainly use FTIR analysis for identification of the materials we are working with. FTIR requires very little sample preparation, and the analysis is very fast. This makes FTIR a fantastic instrument for identifying products before more costly analytical testing occurs.

Our ThermoFisher Scientific IS50 FTIR has both transmission and reflectance capabilities, along with a near IR module attachment. Almost all of our analyses on the FTIR are done using ATR, or attenuated total reflectance. This shines infrared light through a diamond into a sample, and then provides a spectrum of what is reflected back out. FTIR-ATR is a really good methodology for quickly determining the identity of a sample. It can also tell you if there are any other carbon-based compounds in the sample. While it cannot be used as an assay technique, it can be used as an indicator of what other compounds might be in a sample. Paired with other orthogonal techniques, FTIR can give you great insight into the chemical composition of a sample.

Polarimeter

WHAT YOU SHOULD KNOW ABOUT OUR POLARIMETER

A polarimeter, like UV-VIS and FTIR is a spectroscopic instrument, which means that it detects how light interacts with a molecule. A Polarimeter is used to measure the rotation of plane polarized light. This allows a polarimeter to detect the optical rotation of a molecule, but only if it has a Chiral center or is a chiral molecule. Our Polarimeter is important for the analysis of products that can have different optical rotations, such as Theanine, which can both exist as L-Theanine and D-Theanine. For our Theanine product, we only want L-Theanine, and thus we can utilize our Polarimeter to make sure that we have pure L-Theanine that has no D-Theanine in it.

How polarimeters work is by passing monochromatic light through a polarizing plate. This creates a polarized beam that is then shined through a sample. The light then passes through another polarizer known as the analyzer, which automatically rotates. When the analyzer rotates such that all the light passes through the sample, then rotates so that none of the light passes through, it can measure the angle between those two points. This gives you the optical rotation. When a molecule can be in both dextrorotatory or levorotatory forms, like in the case of theanine, the optical rotation can tell you just how much of each isomer is in a sample.

Melting Point Apparatus

WHAT YOU SHOULD KNOW ABOUT OUR MELTING POINT APPARATUS

This instrument is used to determine at which point a pure compound melts. It does this by slowly heating up the sample, and detecting the temperature at which it turns into a liquid. For well studied pure compounds, the melting point of the material is well known. This can be indicative as to the purity of the sample, and gives us a quick and reliable way in which to gauge the purity of a compound before more time consuming analysis occurs. Often times measuring the melting point is a manually done method. However, ours is an automated melting point apparatus. This allows us finer control over the variables, and also videos the melting and plots the graph of how the substance melts quantitatively. This allows us a much more accurate view into just how a substance melts. This is another orthogonal technique that gives us extra information about a sample, and combines with other methodologies to give us a more comprehensive picture about the identity and purity of a sample.

LOD - Loss On Drying

WHAT YOU SHOULD KNOW ABOUT LOSS ON DRYING

This instrument is essentially an analytical balance with a small oven on top. A sample is poured onto the balance and the oven turned on. As the sample heats, volatiles evaporate, and the material dries. After measurement is complete the percent of mass lost can be determined. This is usually assumed to be only water, but some samples are volatile or melt, and the loss on drying measurement can be confounded. LOD analysis can help us identify if a material was properly stored and not exposed to moisture. This is important for products like Alpha-GPC 99% which has to be stored as dry as possible. Loss on drying is also used in the calculation of some assays. You will often see two measurement on an HPLC: “as is” and “dry basis.” How you calculate the assay on a dry basis is by measuring the loss on drying. If you have a sample that is coming in at a 95% assay, and you have a 5% loss on drying, you can determine if it is just water making up the difference, or if there are actual impurities in the sample. Orthogonal techniques once again come into play. If you pair loss on drying with headspace GC and HPLC, you can be more confident as to what compounds are left in your sample.

Water Activity

WHAT YOU SHOULD KNOW ABOUT WATER ACTIVITY

This is a technique used to measure the energy of water in a material. In order for microbial growth to occur, water must be present at a certain amount, and the water cannot be bound in the sample. Below specific water activity levels, no microbial growth can occur, as not enough unbound water is present for growth. This is a very important and useful instrument for us, because it can tell us whether or not a product is at risk of microbial contamination. Due to the importance of this test, it is performed on every batch of all of our products, both raw materials and finished goods. Any products that fail the water activity test or are suspect to microbial growth are subjected to full microbial testing according to USP standards. Certain products do not work properly with water activity analysis, like Nigella sativa. In those instances, we directly analyze using traditional microbial growth culture analysis.

Lyophilizer

WHAT YOU SHOULD KNOW ABOUT OUR LYOPHILIZER

Lyophilizers are used to remove water and other volatile compounds from a sample via vacuum, which in turn lowers the temperature, and freezes the material. We use this machine for preserving reference materials that we produce in our laboratory. During reference material characterization, small amounts of the material are thawed and used for testing. After characterization is complete, enough preserved remaining material exists to be used as a reference material for methods such as UPLC, which require calibration with reference materials. Lyophilizers can also be used to freeze dry products, like peptides.

Preparatory HPLC

WHAT YOU SHOULD KNOW ABOUT OUR PREPARATORY HPLC

This instrument is a cousin of the analytical HPLC. While the purpose of analytical HPLC is to separate and measure the amount of molecules present in a sample, the purpose of preparatory HPLC is to separate and collect each type of molecule present in a sample to concentrate them. The material collected can then be used for research and development, creation of reference materials in conjunction with a lyophilizer, or re-separated for better understanding of the components of the mixture as it was originally collected. Because the material input is collected back after the use of this instrument, this instrument is a non-destructive technique, unlike most other analytical techniques.

Preparatory HPLCs works identically to normal HPLCs, by using column chromatography to separate molecules in a sample from each other. They even use a UV detector like a normal HPLC does. However, rather than the UV measurement being the end goal like in a normal HPLC, a prep HPLC’s end goal is to separate and concentrate individual molecules for later use. This is how we created our own erinacine-A reference standard. We used prep HPLC to separate the erinacine-A from a sample of lion’s mane, then accumulated and concentrated it till there was enough to be able to use as a reference standard for our HPLC and UPLC assays.

Rotary Evaporator

WHAT YOU SHOULD KNOW ABOUT OUR ROTARY EVAPORATOR

A Rotovap is used to rotate and evaporate a sample under vacuum so that excess solvent can be removed. We use this machine for research and reference material creation in conjunction with our preparatory HPLC instrument. The output of a preparatory HPLC experiment typically has lots of excess solvent present. This machine can remove excess solvents at low temperatures, by using a combination of a strong vacuum which lowers boiling points of solvents and increasing surface area by rotating the sample. This is important for removing solvents from samples that are sensitive to heat, as each parameter can be precisely controlled and monitored during the course of an experiment. Rotovaps can also be used in chemical synthesis, to remove the solvents used in the various synthesis steps.

Microgram Balance

WHAT YOU SHOULD KNOW ABOUT OUR MICROGRAM BALANCE

This balance is extremely accurate and sensitive, and can measure down to 10 micrograms with high precision. At these sensitivity levels, the microgram balance can actually measure the breath of the analyst working with the balance! Measurements can also vary due to changes in temperature and pressure in the laboratory. Due to this, our microgram balance features a sophisticated internal calibration scheme to recalibrate itself every time there is a temperature or pressure change in the laboratory. We use this balance for measuring reference materials used to calibrate instruments like our UPLC. If we did not have this microgram balance, we would waste a lot more expensive reference standards. This is because we would need to use the entire standard each time we needed to run a calibration on an assay. Since we can use this extremely accurate scale to measure out small amounts of our reference standard, we reduce waste and cost associated with the use of reference standards in the lab, in addition to giving us more accuracy and precision in our measurements.

WHAT IS A QUALITY SYSTEM?

A high-quality final product is the end result of a well-structured, implemented, and monitored quality system. At Nootropics Depot, we strive to implement the most rigorous of quality standards to both our production and quality control functions, operating the production facility under the International Standards Organization (ISO) 9001 and National Safe Foods (NSF) quality standards, while the laboratory part of the quality control system operates under the ISO 17025 standard. The federal current Good Manufacturing Processes (cGMPs) for dietary supplements (21 CFR 111) are only the starting point our quality system and Nootropics Depot is currently in the process of becoming certified by National Safe Foods and Safe Quality Foods, and other accreditation bodies for the ISO 17025 standard. It is within this system can the quality, consistency, and regulatory compliance be assured for all products sold by Nootropics Depot.

WHAT IS QUALITY CONTROL?

Quality Control is a department working within a quality system whose purpose is to ensure that all quality objectives of a product, process, and system are met. At Nootropics Depot, the quality control department includes the analytical laboratory, analysts, technicians, and analytical chemists. It is this department that is directly responsible for the testing and approval of all raw materials and finished products before they are sold to a customer. The quality control department, working closely with the Research and Development team and Nootropics Depot executive management, is responsible for creating specifications, approving labels and claims, and developing and validating analytical, physical, and organoleptic testing methods. The quality control department is responsible for all quality record keeping as well, so that full traceability for every lot created can be traced back all the way to the original raw material supplier. Furthermore, the quality control department performs all analytical, microbial, and physical testing and data analysis, and creates final Certificates of Analysis (CoA) for issuance to customers. If there is a question regarding test results, data integrity, or test method validation and application, the quality control department is the department responsible!

COMPETITIVE ANALYSIS

Nootropics Depot routinely conducts competitive analysis of various nootropic and supplement vendors. Over time, we will be adding additional ingredients to the menu below.

TONGKAT ALI EXTRACTS

BUY TONGKAT ALI EXTRACTS FROM NOOTROPICS DEPOT

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WHAT YOU NEED TO KNOW ABOUT TONGKAT ALI SUPPLEMENT QUALITY

There are a lot of companies out there who have recently started to release Tongkat Ali supplements. However, many of these companies are fairly new to the supplement industry, and are aggressively marketing their Tongkat Ali products as “testosterone boosters”. What ends up happening when a supplement picks up a lot of interest in a short amount of time like this is that quality tends to slip. This is especially true when the companies selling these Tongkat Ali supplements are rarely doing any analytical testing, and just blindly trusting their overseas suppliers. This is why we decided to invest a lot of time and energy into making a high quality Tongkat Ali supplement, while also developing highly accurate testing methods to determine that what we were sourcing was the best Tongkat Ali currently available. In fact, the Tongkat Ali extracts we wanted did not exist. Thus, we built a relationship with a partner in Asia that does the extraction to our specifications, and sources the raw roots from very specific Malaysian farms.

After more than a year of research, sourcing, method development, analytical testing, and beta-testing we finally have two very high quality Tongkat Ali supplements! One of the Tongkat Ali supplements is standardized to 2% Eurycomanone. This extract is intended for general daily use, and is a good introduction to Tongkat Ali. Our other Tongkat Ali extract is standardized to 10% Eurycomanone, making it one of the most potent Tongkat Ali supplements currently available! The 10% extract is intended for experienced athletes, or individuals looking for more pronounced and acute effects. You may think the 2% is the less potent one, but it is still more potent than almost any other Tongkat Ali extract out there. You will see that below in our testing summary.

NOOTROPICS DEPOT TONGKAT ALI VS. THE COMPETITION

Many Tongkat Ali extracts do not even standardize for the main bioactive compound, Eurycomanone. In fact, most Tongkat Ali extracts are marketed as 100:1 or 200:1 extracts. This means that it took 100-200 pounds of root material to make just a single pound of Tongkat Ali extract. You would expect these extracts to then be very high in Eurycomanone, but after some analytical testing of the “100:1” and “200:1” extracts, we found that their Eurycomanone content is actually quite low. We recently tested 8 competitors; many of which claimed extract ratios of either 100:1 or 200:1, with one competitor who claimed their product was standardized to 2% Eurycomanone. Check out the table and image below for our testing results:
VENDOR EXTRACT RATIO EURYCOMANONE SPECIFICATION TESTING RESULTS (EURYCOMANONE)

Nootropics Depot

Not Defined

10.00%

10.4%

Nootropics Depot

Not Defined

2.00%

2.05%

Competitor 1

100:1

No Specification

0.52%

Competitor 2

Not Defined

2.00%

0.43%

Competitor 3

100:1

No Specification

0.24%

Competitor 4

100:1

No Specification

0.23%

Competitor 5

Not Defined

No Specification

0.10%

Competitor 6

200:1

No Specification

*Not Detected

Competitor 7

Not Defined

No Specification

*Not Detected

Competitor 8

200:1

No Specification

*Not Detected

*Method Detection Limit 0.03%

As you can see from the table above, the Eurycomanone content of the 100:1 extracts were generally quite low, with the highest one, competitor number 1, only reaching 0.53 % Eurycomanone. This means that in the pool of the 100:1 extracts we tested, the highest testing 100:1 extract was still about 4 times less potent than our 2% Eurycomanone! So, what about the 200:1 extracts? Surely those should be coming pretty close to our 2% Tongkat Ali extracts, right? The opposite appears to be true, as the 200:1 extracts appear to take a turn for the worse! They give you the illusion that they are going to be extremely potent, based on their claimed extract ratio, but they practically do not contain any Eurycomanone! None of the 200:1 extracts that we tested were able to hit our detection limit of 0.03% Eurycomanone on HPTLC. This means that if they contain any eurycomanone at all, it is below the ability of our machine to accurately measure them.

After talking to some of our most trusted suppliers, it became clear that these very high extract ratios are clearly fake. Multiple suppliers admitted to us that the 100:1 and 200:1 extract ratios are completely made up. They are fake! US vendors started labeling their extracts as 100:1 and 200:1, but they were nowhere near that. The 100:1 extracts are, perhaps, a 10:1 extract at most but this is likely already pushing it. They are most likely lower than that. We have no idea what’s going on with the 200:1 extracts, but the ones we tested do not even appear to be extracted at all, and may just be plain root material. The problem with these extract ratios is that it is practically impossible to verify whether a Tongkat Ali extract is truly a 100:1 extract, or if it is really just a 10:1 or 4:1 extract. Due to this, we decided to move away from these extract ratios, and instead focus on something that we could accurately test for: the Eurycomanone content.

There are a handful of vendors who are now also standardizing for Eurycomanone content as well, so we decided to test one that claimed to match the specification for our own 2% Eurycomanone extract. This vendor is listed as competitor number 2 in the table above. As you can see in the table, this extract did not come anywhere close to containing the 2% Eurycomanone they are claiming. Thus, even if a vendor is claiming a Eurycomanone percentage, this does not necessarily mean this number is correct. This is because most vendors blindly trust their suppliers and do not conduct any analytical testing themselves. So we have false and misleading extract ratios, and we have products coming in way lower than their label claims. Unfortunately, this is the state of the Tongkat Ali market at the moment.

After seeing all of these tests, we decided we needed to bring a quality Tongkat Ali supplement to the Nootropics community! As you can see from the table above, our Tongkat Ali extracts test very accurately. In the case of the 10% Eurycomanone extract, we are even over by 0.4%! Better yet, we perform a whole lot more analysis on our Tongkat Ali including:

⦁ Species and plant part identity testing by HP-TLC
⦁ Loss on drying by moisture balance
⦁ Heavy metal testing by ICPMS
⦁ Microbial proliferation by water activity

Check out our specification sheet for the 2% Eurycomanone extract below to get a good idea about the tight specifications we follow:

Nootropics Depot Tongkat Ali Spec Sheet

If any raw material comes in out of these specifications, then we reject it. No exceptions! This means that you will always get a clean and efficacious product when you get your Tongkat Ali from Nootropics Depot!

Unfortunately, it appears that the same cannot be said for many of our competitors. However, we remain hopeful that our efforts to shed light on these issues will help move the entire industry in a better direction. Our goal is not to corner the market. Instead, we are advocating for the supplement industry to adhere to proper quality standards like we are, so that we all have access to effective and safe supplements that are not fake, misleading, or improperly tested. We believe that we all have the right to high quality supplements, and that we should never have to worry about whether or not what we are taking is real or fake. We do our part in taking this worry out of the equation for our customers, and hope the rest of the supplement industry will strive to achieve this similar goal!

A DEEP DIVE INTO HPLC TESTING

After reading the above section, you may be wondering how we determined the Eurycomanone percentages, and if we really did the testing for those competitors. For the sake of transparency, which we believe is incredibly important in the supplement industry, we will now go over the testing methodology used, and the results of the analytical tests. It is complex, but also very interesting to dive into!

For Tongkat Ali, we are using High-performance thin-layer chromatography (HPTLC) testing. HPTLC is quite similar to HPLC, however, instead of using columns to separate compounds, HPTLC uses specialized plates called TLC plates. At its most basic, we have developed methods to dissolve all of the compounds in the Tongkat Ali samples into a solvent. We then spray this solvent onto a plate made of specialized silica gel. The compounds then travel up the TLC plate by means of capillary action. The separation occurring on the TLC plate is very similar to the separation happening in an HPLC column, like a C18. C18 stands for octadecylsilane, or 18 carbons bound to silicon dioxide. These silica-bound carbons stick out into the column and separate molecules from each other as they are pumped by at high pressures. In a TLC plate, you are essentially flattening out the column, and having the separation occur under normal air pressure, rather than being pumped at high pressure through a column. This capillary action is what spreads the different molecules across the plate, and separates them based on their polarity, which allows us to both identify and quantify each of the bands on the TLC plate.

If you want to see capillary action for yourself, find yourself some food coloring and put it in water. Now, take a piece of paper towel, and dip it into the food coloring solution, and slowly but surely you will see the colored liquid moving up into the paper towel. In the context of HPTLC, this capillary action separates the different compounds in Tongkat Ali by their polarity. Based on their polarity, different compounds in Tongkat Ali will travel up the TLC plates at different rates. This variation in how the various compounds in Tongkat Ali travel up the TLC plate is what separates the compounds from each other. This means that they will be deposited at different positions on the TLC plates. This may sound a little bit vague right now, but it is about to make a whole lot more sense because we can visualize it!

Once our TLC plate is ready, we can actually visualize where the compounds are on the plate. We do this by exposing the plates to UV and visible light, typically ranging from 190nm to 900nm. In our case, we used two wavelengths: one at 254nm and the second at 366nm. When we hit the plates with these wavelengths, the compounds on the plate either fluoresce or prevent fluorescence, allowing us to visually see where they are on the plate. This is very similar to the UV detector on an HPLC. So you can see how an HPTLC and HPLC are doing the same thing, just using different methods to do it. It’s about chromatographic separation of molecules, then detection using a UV detector. Let's take a look at the plate visualized with 254nm first:

All of the dark spots you see are where the compounds ended up due to the capillary action. The scale on the vertical axis are Rf values. An Rf value is the ratio of distance the compound travels on the Rf plate to the distance travelled by the solvent the compound is dissolved in. The Rf value then falls between 0 and 1. To simplify it, imagine you and three friends are doing a one mile running race with a time limit of 9 minutes. At the end of 9 minutes, your experienced running friend made it all the way to the one mile mark, whereas your other friends were scattered between 0.1 miles and 0.5 miles. One of your friends twisted his ankle at the 0.1 mile mark and didn’t make it much further. Another friend was pretty slow and could only make it to the 0.5 mile mark. You are also an experienced runner and tied with your friend at the 1 mile mark. These mile marks can be seen as Rf values, and these Rf values are being visualized on the TLC plate above.

If you check out the columns labeled 1 on the left, you can see those are our reference standards. The first column on the left is pure Eurycomanone from Chromadex, with a known concentration. The one on the right is the botanical reference material standard, which is also from Chromadex. Since the amount of eurycomanone in the leftmost lane is a known concentration, we can use that as our reference for the other samples. This is how we can quantify Eurycomanone in other samples accurately. As you can see, through capillary action, Eurycomanone made it up to Rf 0.65. When we visualize our TLC plate at 254nm, the entire plate fluoresces, hence the bright green color. At 254nm, Eurycomanone however does NOT fluoresce, and thus it shows up as a dark band. You can see this dark band in most of the samples we tested, indicating the presence of Eurycomanone. As you can see in our own 2% and 10% Eurycomanone extracts, the band with Rf value 0.65 are quite dark, and you can see that the 10% Eurycomanone Tongkat Ali extract is slightly darker than the 2% Eurycomanone Tongkat Ali extract. These bands are how we identify specific compounds in the extracts. We’ll get to how we quantify the exact amounts of Eurycomanone via HPTLC in just a second, but let's look at the plate that was exposed to a wavelength of 366nm first:

At 366nm, we see the reverse of the TLC plate that was visualized at 254nm. On this plate, only the compounds of interest fluoresce, and thus they show up as colorful bands on a dark background. As you can see, there is a band that fluoresces right around Rf value 0.65 on this plate as well. However, since we are using a different wavelength of light, and given the fact that Eurycomanone prevents the fluorescence, this is not actually Eurycomanone. You can see how the Eurycomanone reference standard in the leftmost lane doesn’t fluoresce at this wavelength. This is a sure indication that the band we are seeing in the Rf 0.65 region on the 366nm plate is something other than Eurycomanone. According to our research, this is likely a flavone that has yet to be elucidated in Tongkat Ali. This is based on the fact that it is fluorescing at 366nm and has an Rf value of 0.65. The interesting thing here is that two different compounds, due to similar polarities, both ended up at around Rf value 0.65. However, if we only visualized the plate at 254nm, then we would have only seen Eurycomanone and not the other flavone compound.

If we revisit our 1 mile race example that we discussed earlier, you and your experienced running friend both made it to the one mile mark in 9 minutes. This is easy to visualize, as we can visually see two different people at the 1 mile mark. In the case of HPTLC it is a little bit more complex, but by using different wavelengths of light, we can visualize two different compounds with the same Rf values. This is because different molecules will respond differently to various wavelengths of light. This is why it is oftentimes important to run a HPTLC test at different wavelengths, rather than just running it at one wavelength of light, as we may miss some potentially interesting compounds! So in this analogy, viewing race positions from one angle might allow an observer to see you standing at the one mile mark of the race, where looking at the positions from another angle allows them to see your experienced friend at the same one mile mark. Both you and your friend are standing at the same mark, just like Eurycomanone and this flavone are at the same Rf of 0.65 on the plate. Viewing them differently allows you to see the two things at the same place. So how can we empirically show this? We have to get further into the data. Take a look what we see when we zoom into the range of Rf 0.645.

You can see that we see a couple distinct peaks close to the Rf 0.65 region when we get further into the data. The peak that the red arrow is pointing at is Eurycomanone. The peak that the blue arrow is pointing to is the suspected flavone compound. Why do we think the peak by the blue arrow is a flavone? Benzene usually absorbs around 280nm, which is about where that peak is located. Flavones contain multiple benzene rings. So we are assuming that there is a flavone that is yet to be elucidated in Tongkat Ali that just happens to fall close to the same Rf value as Eurycomanone. If we only visually looked at the plates, we would not be able to tell the difference between the bands. However, our HPTLC software allows us to really get an accurate view of each band, and make determinations based on that data. That data explains why we see a band around the Rf 0.65 mark in both the 254nm and 366nm plates, even though Eurycomanone itself only shows up on the 254nm plate. You can see how the devil is in the details here. If you do not do a thorough job with your method development and validations, you can misinterpret or miss crucial factors in identifying and quantifying specific compounds.

Another interesting thing to note here is that on the 366nm TLC plate, we see a stronger fluorescence at around Rf value 0.65 for the 2% Eurycomanone Tongkat Ali extract than we see for the 10% Eurycomanone Tongkat Ali extract. In fact, there appears to be more fluorescence overall at 366nm for the 2% Eurycomanone Tongkat Ali extract than for the 10% Eurycomanone Tongkat Ali extract. The most likely explanation for this is that the 2% Eurycomanone Tongkat Ali extract is a more “full spectrum” extract. This means that it contains a larger percentage of the other compounds in Tongkat Ali besides Eurycomanone. Oftentimes when we are aiming for a high standardization of one specific compound, in this case Eurycomanone, it is at the expense of the other compounds in a plant. This means that the 10% Eurycomanone Tongkat Ali extract likely has a more focused effect that is more representative of pure Eurycomaone, whereas the 2% Eurycomanone Tongkat Ali extract is likely more representative of the whole root material. Overall, this results in the character of effects being different between the 2% and 10% Eurycomanone Tongkat Ali extracts. It is not simply the case that the 10% Eurycomanone Tongkat Ali extract is just 5 times more potent than the 2% Eurycomanone Tongkat Ali extract. Our goal was to maintain the ratios of the raw root as much as we could, and we have done a very good job of that. The 10% extract keeps many of the compounds you would find in the raw root. However, more potent extracts are inevitably going to concentrate some things, and lower others. This means that you will always have differing ratios of ancillary compounds between extracts that range in potency from each other. If we look at our extracts compared to a raw root botanical reference material, you can see what we are referring to.

Lane 7 is the botanical reference material of raw Tongkat Ali root from Chromadex. Lanes 8-11 are increasing concentrations of our 10% extract. Lanes 12-15 are increasing concentrations of our 2% extract. As you can see, we do a very good job of keeping the various compounds in the raw root intact. However, you can see that the bands in the 2% fluoresce differently, and are at different ratios/concentrations than the 10%. As we concentrate Eurycomanone, we inevitably change the ratios of the final product. This is also why we find more of that flavone compound at Rf 0.65 in our 2% extract, even though it has less Eurycomanone. This is one way to visualize why our 2% extract might feel different than our 10% extract, even if you tried to just increase the dose. You can see how we can use our analytical tools to get a very detailed view into our products, and exactly which compounds are in them.

It’s pretty clear that after seeing all this, getting exact numbers is definitely a complex science. Determining exact percentages of a given compound like Eurycomanone based solely on visual analysis is obviously not doable. Thus, these plates only allow us to visualize qualitatively that Eurycomanone is present, but it does not allow us to quantify exactly how much Eurycomanone is present. Remember how we mentioned the identification/quantification issue earlier? Luckily HPTLC generates a lot more data via its UV detector than just visual bands on a plate. As we mentioned before, this is very similar to how HPLC works. It’s all about chromatographic separation, identification, then quantification. Through computer software we can analyze the height of the peaks that are generated by the UV detector at specified Rf values. The peaks are analyzed for Absorbance Units (AU), and this is what allows us to quantify exact percentages. Fair warning, we are about to discuss a fair amount of math! Below is an example of this for sample SSF-266 which corresponds to competitor number 1 in our table above.

As you can see in the chromatogram above, peak number 4 is Eurycomanone. This matches the Rf value found in the Eurycomanone reference standard. We can then take this AU value and plug it into a calibration curve.

This calibration curves gets created by plotting the concentration of the reference standard against its AU value, then regressing the data. This can then be used to accurately quantitate subsequent samples. If your methods and measurements are valid, they will fall on the line connecting the various reference standard measurements. By using the calibration curve above, we can calculate the amount of Eurycomanone present in each sample. Check out the table below for our results for sample SSF-266 (competitor 1):

For sample SSF-266, our HPTLC test determined that it contains 53.70 micrograms of Eurycomanone per mL of solution. However, this number is not representative of the powder material, only of the sample as prepared, which is diluted for measurement purposes. With this in mind, we have to take into account the amount of dilution that took place while preparing the sample. This particular sample was created by dissolving 0.5116 grams of Tongkat Ali extract into 50 mL of our solvent. Using these data, we can calculate a dilution factor:

50 mL 0.5116 g = 97.733

Now we can determine how much Eurycomanone a pure, undiluted sample would contain:

53.70 μg/mL x 97.733 = 5248.262 μg/mL

We now need to convert this value into a percentage by mass. To do this, we can assume that 1 mL is equal to 1 gram, as is standard for diluting solids into liquids. With this in mind, we can convert our microgram per milliliter value into a gram per gram value. Below is the math used to get to this point:


5248.101 μg/mL 1000 μg/mg = 5.248101 mg/g
5.248101 mg/g 1000 = 0.005248101 g/g

Now that we have the concentration expressed in a Eurycomanone per total sample mass amount, we can easily convert it into a percentage by mass:

0.005248101 g/g x 100 = 0.5248101%

After running through these calculations, we can determine with a very high level of accuracy that sample SSF-266 contained 0.5248101% Eurycomanone, which rounds to 0.52%. While that may have gone over many people’s heads, we felt it was necessary to really get into the details behind the numbers. The amount of method development and validation work we do in the background would surprise many people. We want our numbers to be accurate. We want people to trust that when we make a claim, that claim is backed up by valid scientific proof. We feel that inaccurate or misleading numbers are worse than no numbers at all. Consumers trust science. However, when science is used improperly, or worse… to purposely mislead, it erodes people’s trust in valid scientific evidence. That is extremely dangerous. This is why we always ensure we are doing everything scientifically necessary to validate our chemistry, measurements, and calculations. You can see from our calibration curve that all our measurements fall within expected parameters, and our method results in a linear response at varying concentrations. This is a positive confirmation of assay performance, and shows that all our results are within a valid analytical range.

 

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FREQUENTLY ASKED QUESTIONS

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Don't see your question answered here? Help us build this FAQ by submitting your question via the form at the bottom of this page.

Do you test your products for heavy metals? If so, what specifications does Nootropics Depot adhere to?

ANSWER: Yes, we test all of our products for heavy metals. Not only do we test every product, we also test every single lot. We do not do skip lot testing, as is the standard in much of the industry.

Our heavy metals specifications are generally .5ppm and below to comply with California Prop 65. California has the most stringent heavy metal regulations in the country. Some specs are a little higher due to supplier specifications but still are within the Prop 65 limits for the amount of product that is in a capsule or serving.

Can I request a Certificate of Analysis without a lot number or purchase? 

ANSWER: Our Certificates of Analysis are created for a specific lot number. Unfortunately, until a purchase is made, we will not know the specific lot number for the item you are purchasing or inquiring about. However, you can check out the specification sheets on the product page for your specific product. We adhere to these specifications and do not deviate from them. Any raw material that does not meet our specifications is rejected and does not end up on the shelves as a finished product.

When you purchase, please contact us with your lot number and we will be happy to provide the Certificate of Analysis for your particular lot.

Does Nootropics Depot have any lab certifications?

ANSWER: Nootropics Depot is FDA, cGMP (current Good Manufacturing Practices), USP and HACCP compliant. In addition, our company has an ISO-5 compliant in-house analytical testing lab. Our lab is a registered lab with USP and Sigma Aldrich, whom we use for our reference standards. Nootropics Depot is NSF compliant, and is working toward full NSF certification very soon.

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The Nootropics Depot in-house analytical laboratory proudly utilizes state-of-the-art machines and instruments from the following industry-leading manufacturers and more.

The Nootropics Depot in-house analytical laboratory proudly utilizes state-of-the-art machines and instruments from the following industry-leading manufacturers and more.

The Nootropics Depot in-house analytical laboratory proudly utilizes state-of-the-art machines and instruments from the following industry-leading manufacturers and more.

Agilent
Anton Paar
Buchi Switzerland
Camag
Thermo Fisher Scientific
Waters
Anton Paar
Buchi Switzerland
Camag
Thermo Fisher Scientific
Waters

Proud Member Of AOAC International (Association Of Official Analytical Chemists)