Author: Wiater Julia

In the afternoon, there wasn’t too much to do since Dr. Pandi had to go back to Raleigh to do some lab tests in RTP. So, after learning about data analysis with him in the morning, we were instructed to work on our group presentations. Tomorrow, we will give a summary of our lab work and individual project findings not just to Dr. Pandi, but also to Dr. Taylor and the Integrated Pain Solutions staff as a whole! (which is super scary). Both day 7’s afternoon and day 8’s morning will be dedicated to rehearsing and revising our presentation.

After we got to see the bottling process in the morning, we met with Dr. Pandi to go over our finalized research projects about various cannabinoid compounds and receptors. We had two main discussions: the first was a slide-by-slide talk-through of what we researched.

As we went through, he showed us the ways that all of the receptors, enzymes, and cannabinoid compounds work together to create the endocannabinoid system, which was helpful in completing our understanding of our studies.

After we presented our projects to him, he showed us a few ways that we could deepen our findings for the presentation to the staff at Integrated Pain Solutions on Thursday; specifically, he showed us a fantastic website called SciFinder by the American Chemical Society – it allows us to check for the molecular structures of each compound we researched, like looking for its chemical fingerprint! But… we could only use the demo version, because this software costs thousands of dollars per year for use by an institution (because it’s such a valuable tool for researching!)

This final discussion of our projects not only left us with a more complete understanding of the topics at hand, but also gave us insight into how to research more effectively in the future!

To start off our day, we helped Ryan out by labeling and bottling formula 16 for distribution under the brand name RitualX (manufactured by Integrated Pain Solutions). Ryan first formulated the serum, then took it to the bottling room, where he and a bottling machine make quick work of filling up each serum bottle. His setup is below:

After filling each bottle, he has to then label each individual bottle; each serum looks the same as all the others since formulaic differences are mostly in the fragrances and additives, not the core formula. Ryan also acts as the labeling machine for Integrated Pain Solutions, and he affixes a label to and seals hundreds of bottles at a time with his secondary setup:

After seeing the process for making the base balm and serum solutions last week, it was really interesting to see how the base formula is further processed and prepared for sale in the store. We have now seen the complete process by which Integrated Pain Solutions takes crude CBD extract and makes their products!

To finish off our first week at Integrated Pain Solutions, Dr. Sean and Dr. Pandi walked us through the process of interpreting mass spectra to determine the relative concentrations of various cannabinoid compounds. Firstly, the LCMS machine we learned about yesterday produces a page of integrals for each compound to show much of each was in each sample:

From that, Dr. Sean exports the file to excel, where the actual milligrams of each compound in the sample can be more accurately measured, and the concentrations can be calculated:

Finally, he compared the expected rate of travel through the chromatography part of the machine to what it actually was. He then adjusted the expected time accordingly to match what the actual time was. This is a sort of continuous adjustment that they make to the LCMS machine to make sure that it stays accurate over time. From the data we collected today, we confirmed that the process Dr. Pandi carried out on the initial sample didn’t significantly change its chemical composition, as there were still many cannabinoids present in the final solution.

To start off our fifth day at Integrated Pain Solutions, we got to see Dr. Sean process samples of CBD extract from the day before. Yesterday, Dr. Pandi stayed in Raleigh to make these samples, and he walked us through his process… Firstly, he diluted a sample of crude extract, which is the consistency of tar, to make a viscous concentrate of CBD. Then, he performed a series of processing steps to further purify the concentrate to its final form, which was much more fluid and less dirty than the original form. But, to make sure that the chemical composition hadn’t changed too much, the samples were brought back here to Southern Pines for testing.

Dr. Sean dissolved the liquids in ethanol to dilute them and prepare them for the Liquid-Chromatograph Mass Spectrometer (LCMS) machine that we learned about yesterday. This involved a graduated processing procedure, which took the samples from the various processing stages from thick and difficult to work with to homogenous and easily processable solutions. The vials used for analysis are below:

After lunch, we’ll meet back with Dr. Sean and determine if Dr. Pandi’s processing of the original sample significantly altered its chemical makeup!

After continuing our individual research projects at the beginning of the day, we had the opportunity to observe Dr. Sean preparing hemp plant samples for chemical analysis for CBD, THC, and other cannabinoid content. To begin, he soaked a sample of hemp in Isopropanol to draw out and dissolve the cannabinoids into a solution that can be easily tested.

Next, he showed us how to appropriately dilute the samples so that the Liquid Chromatograph Mass Spectrometer (LCMS) machine could accurately analyze them. He performed a 100x dilution of the initial sample using Isopropanol and Acetonitrile, and also brought out control samples to calibrate the machine. He created a standard curve for the concentrations of each cannabinoid in the calibration samples. Then, we went over to the machine, where the samples would be analyzed. His setup is below.

After we learned the basics of pipetting in the morning, we met with Dr. Pandi again, and he introduced us to our individual research projects that we will be doing over the remainder of the week. He assigned us each a specific class of compounds/effects to study: JR will be studying the CB1 cannabinoid receptor, Julia will be studying the CB2 cannabinoid receptor, and Alekh will be studying the specific molecule CBD (Cannabidiol). To complete these projects, Dr. Pandi directed us to the research databases of the American Chemical Society, the American Institute of Biological Sciences, and other academic journals. He also lent us two books on the history, development, and implications of CBD-based pain management solutions. Over the next few days, the three of us will continue to deep-dive into our respective research areas, culminating in a presentation to Dr. Pandi with our findings at the end of this week. This will serve as the theory-based portion of our time at IPS, and will supplement the lab-based experiences we will continue to have.

To start off our day at Integrated Pain Solutions, we decided to apply our acquired knowledge of urine analysis screens for drugs with Calculus. For screening urine samples for drugs, the samples are placed into a machine that ionizes the drugs. Through vibrational frequencies, other molecules in the urine are blow out of the sample, leaving only narcotics. Then using argon gas, the samples are fragmented and recorded by mass spectroscopy. To analyze the concentration of the drug, the mass spectroscopy curve is compared to the control set beforehand through the area under the curve or AUC. Then, the AUC of the sample is compared to the control to determine the % concentration, depicting if the patient has legal amounts of sample. Today, we decided to hand-integrate equations that match a typical curve for the machine to determine our hypothetical % concentration. In our sample, we calculated 79.0% compared to the control, which was a great trial.