One of our first pieces of equipment that we put into our lab was our Agilent HPLC. However, we rapidly realized that we wanted to have more precision and control, which led us to contact Waters. This is where our shift to UPLC (Ultra High Performance Liquid Chromatography) started. Waters makes extremly high end equipment for the pharmaceutical industry, but they are seen much less often in the supplement industry. This is mostly due to cost. They are at the top end of the spectrum in the lab industry, but they are also priced that way as well. We have been luckly to collaborate with Waters with lower pricing, and help them to see the potential in supporting the supplement industry. Their machines help drive our advanced quality control and supplement innovation. These instruments are positioned at the forefront of analytical science, capable of detecting, separating, and quantifying even extremely low concentrations of active compounds with unmatched accuracy.

This level of detail allows us to go beyond verifying purity and potency for routine analysis. We use our UPLC systems as a discovery platform, isolating promising bioactives and mapping their chemical profiles, which informs ongoing research and product development. When we want to research new ingredients, these machines enable us to single out specific compounds, determine their concentrations, and evaluate their biological relevance with confidence. They also allow us to discover things we would not see with HPLC, like coeluting peaks. We have run into this multiple times now, where the base level USP method done on HPLC is overstating the concentration of certain bioactive compounds, simply because the HPLC is not actually separating two very similar peaks from each other. We found this with our groundbreaking turkesterone results, where beta-ecdysterone was coeluting with turkesterone, making people think there was turkesterone there when there was not. We have recently found similar things with certain novel withanolides, and with our expanded ganoderic acid method. Our Waters UPLC machines allow us the ultimate in separaration and precision.

UPLC-MS analysis also streamlines the creation of proprietary reference standards. By accurately identifying and quantifying bioactives that may not be widely available elsewhere, we set benchmarks that raise the industry’s standards. This technology plays a foundational role in the evolution of our products, supporting both everyday quality control and the advancement of new supplement formulas. In addition, our UPLC machines use 10X less solvent than HPLC machines. This not only cuts down on the cost of running the system, but results in a much more green and environmentally-friendly lab.

These tools reflect our commitment to full transparency, scientific rigor, and continuous improvement in every stage of supplement development, from raw material selection to final product validation.

The foundation of truly science-driven supplement development starts with isolating and validating the key bioactives that deliver real benefits. At Nootropics Depot, our lab is equipped to go beyond basic ingredient verification. Using advanced prepratory chromatography and specialized freeze drying techniques, we not only identify the active compounds within botanicals and raw materials, but also create new reference standards used by the industry. One of the main limiting factors we have faced over the years is lack of validated reference materials for the specific bioactives we want to standardize to. This is a limitation all labs around the world face. Without reference standards, you cannot accurately assay for that bioactive. We were not satisfied with waiting around for places like Chromadex or Sigma Aldrich to get around to the specific standards we wanted, so we built our own reference standard creation lab.

Our first piece of equipment was our Buchi flash chromatography and prep HPLC machine. This is what allowed us to first isolate erinacine A years ago, and create reference standards to bring our Erinamax. This machine is very much like an HPLC, but instead of just measuring and discarding things that come out of the column, this system separates and isolates things into different vials as they come out of the column. This allows us to use the chromatographic power of an LC column, and create standards from the things we isolate. However, one of the biggest limiting factors of this machine is that you can only separate compounds from each other using various liquid solvents. This isn't always ideal, as certain compounds have similar solubilities to each other. This means it can be difficult to separate certain bioactives from each other. The other limiting factor is that it only contains a UV detector. This means that for a compound to be "seen" by the machine, it has to contain a chromophore, or a conjugated pi system. For bioactives that do not contain chromophores, the system essentially runs blind. This is where our new machine enters the equation!

Our newest machine in the reference standard lab is the Sepiatec SFC-50 supercritical CO2 prep system. This machine uses supercritical CO2 to separate compounds from each other. It also allows for cosolvent use, meaning you can use liquid solvents like ethanol with supercritical CO2 to separate compounds. This is especially useful for compounds like hericenes and hericenones, that have a fatty acid sidechain. This new machine allows us to get proper separation from the various hericenes and hericenones, which has allowed us to develop the world's most accurate lion's mane fruiting body assay method. This method is actually awaiting peer review in the journal of AOAC as we speak. The other adantage this new machine has is that it doesn't just have a UV detector. It has an additional mass spec detector added on. This allows the machine to see both bioactives that have chromophores and ones that don't. It also allows us to mroe easily determine novel bioactives, because we will have the mass of the molecule to make it easier to determine the structure. This machine has massively sped up our development of new bioactive discovery and reference standard creation.

Building proprietary reference standard libraries allows us to quantify unique compounds, compare across batches, and raise the bar for scientific transparency. This data-driven approach ensures every bottle is formulated around measurable, targeted bioactives, not unverified claims, supporting Nootropics Depot’s commitment to precision, transparency, and industry leadership in supplement science.

Nootropics Depot is pioneering supplement research at the cellular and neuronal level, setting a new standard for scientific rigor in the industry. Our live cell culture lab uses advanced platforms, like the Sartorius Incucyte SX-5 system, to culture human cells and expose them to novel bioactive compounds. This allows us to directly observe and qualify how ingredients affect cell viability, antioxidant status, and most importantly, nerve growth and regeneration. This is an especially powerful tool when you pair it with our reference standard creation lab. This allows us to identify and isolate novel bioactives in plants and mushrooms, then take those compounds right over to our cell culture lab for assay. We can then do both toxicity assays on these bioactives, to determine they are safe, and do biological assays on different human cells to determine the mechanism that these compounds have. It also allows us to simultaneously assay multiple novel bioactives at once, to determine which ones are the most active. This helps both speed up our novel compound discovery, and to kickstart our research on both bioavailability and bioactivity.

By continuously imaging and analyzing live cells over time, our research team obtains real biological data that no traditional supplement company can match. We do not rely on theoretical claims or animal models alone. We generate human-specific results in real laboratory conditions. For example, our studies track how various compounds promote the growth of human neurons or help cells resist stress and damage. Erinacine A is one compound we have taken particular interest in. This isn't just because we think it is cool. We have actual cell data showing that it is the most potent eriancine for nerve growth factor. With each experiment, we gain new insights into the molecular pathways and real-world impact of our ingredients. This allows us to fully understand how each unqiue bioactive is working in the body, and to direct us to the next bioactive that we should be looking for. Stay tuned for new lion's mane products standardized to different erinacines... with corresponding bioactive analysis!

This process is at the forefront of supplement development; merging pharmaceutical research techniques with natural product development. Our cell culture and bioactive research efforts help inform every stage of formulation; from ingredient selection to final product validation. It also provides ongoing evidence for both safety and efficacy of our ingreidents. Customers and practitioners can see not only what goes into our products, but exactly how and why those compounds work for human health. This is also only the beginning, as we have big plans for doing more work with in vivo research with C. elegans, and clinical trials in humans. Stay tuned!

Live cell imaging tracks cell health over time using live cell tracking and specialized dyes that respond to cellular activities such as ROS production, cell division, and cell death. Shown here are HEIC-6 cells treated with Erinacine A, a compound that we have demonstrated to be non-toxic in human cultures, and known for its neuroprotective effects. Stay tuned for our culturing of human brain cells, and the corresponding data showing Erinamax increases neuronal activity and neuronal growth!