Yvonne attends the virtual ACS Fall 2020 National Meeting

My name is Yvonne DePorre is and I’m an organic chemistry PhD from the University of Michigan. After defending my thesis, I worked as an Assistant Lab manager and Senior Scientist of PSI labs, the first cannabis testing facility licensed for adult use in the state of Michigan, where I specialized in pesticide and vitamin E acetate analysis. I am currently a Scientific Advisor at Orion GMP Solutions, and part of my current role is to stay on the cutting edge of scientific publications related to cannabis, so that as a company, we can distill that knowledge and information into addressing our client’s needs.

In my opinion, and with my background in academia, one of the best resources to stay updated on current science is through the American Chemical Society, which I have been a member of for almost 10 years, with the mugs to prove it! Additionally I have had the opportunity to publish scientific papers in its various specialty journals: Organic Letters, Chemical Reviews, Biochemistry, Inorganic Chemistry, and ACS Catalysis. The American Chemical Society, or ACS, is a professional chemistry organization that was founded in 1876. The organization represents cutting edge chemistry, exemplified by its flagship publication Journal of the American Chemical Society, or JACS. The organization hosts 50 other peer-reviewed journals as well, exemplifying the unique findings and talents of its many subdivisions.

Fortunately, even cannabis is not left out of this organization’s territory. Under the Division of Chemical Health and Safety, DCHAS, there is a Cannabis Chemistry subdivision, also known as CANN, which was started much more recently. Prior to the fall ACS meeting in Boston of 2018, I attended the CANN workshop “Health and Safety Training for Cannabis Businesses”, presented by Melissa Wilcox, Jahan Marcu, and Julia Bramante. I would highly recommend this workshop as part of an employee or manager training option or requirement at any cannabis facility, whether it’s growing, production, or testing, this workshop really covers so many great topics and connected me with some of the most helpful and brightest minds in the industry. This year I opted for the full conference experience virtually, and I am very grateful to be supported by Orion GMP Solutions in this endeavor. Here, I will summarize the content of presentations that I viewed, for the purpose to spread chemistry knowledge and foster collaborations across the cannabis community. Enjoy!

“What do we really know about the health risks of cannabidiol (CBD) in products?” by Thomas Lewandowski and James Rice of Gradient

With CBD becoming more commonly infused in food, beverages, and cosmetics, and without FDA-approval, concerns must be raised over the risk of multiple products being used together or products being used at the incorrect dosages or routes of administration. It’s also worth noting that CBD does not directly act on the same receptors as THC, but it does have many complex interactions with the cannabinoid system indirectly, affecting anandamide. Studies related to the FDA-approved CBD drug Epidiolex™ have found that 5 mg/kg as a starting dose, with a top dose of 20 mg/kg is safer considering liver effects in order to reduce seizure frequency by 20-30%. Only 13% of CBD treated patients had liver effects, and these were subjects taking the higher end dosage of CBD in combination with Valproate, another seizure medication. Liver effects were also shown to return to normal after the CBD regimen had ceased, indicating no permanent liver damage in these short-term studies. Animal studies with more aggressive dosages saw increased liver weights in rats and monkeys. More long-term exposure studies are needed for both humans and animals.

CBD has countless claimed health benefits with limited data, but a few of these benefits have actually been more rigorously proven in scientific studies thus far. Treatment for anxiety has actually been well supported by both animal and human studies, although some dosages were higher than what was recommended by the Epidiolex™ studies. There is also evidence for CBD treating psychosis; studies have shown that pre-dosing THC with CBD can lead to less paranoia and memory loss than with THC alone. Additionally, for treating schizophrenia, CBD shows similar effects, with fewer side effects than phenothiazine drugs, boosting anadamide levels, although at much higher dosages compared to the Epidiolex™ study, ranging from a dosage of 120 to 1,500 mg/kg.

They then proceeded to look at possible product combination and misuse scenarios for both children and adults. It was found that if a child were to consume an entire pack of gummies (300 mg CBD), or were to combine enough lotion, chocolate, and tinctures, that would be on the high end of the Epidiolex™ maximum dosage studies, but for adults these same acute dose scenarios would be much safer and not be a cause for concern or toxicity. I also learned that THC is listed under the California Prop 65 warning label, so that will be important for CBD product manufacturers to include this warning if their THC levels are over the allowable threshold. Overall, the presenters stress that with these new products available, manufacturers will need to be aware of possible misuse scenarios, and create their dosages, packaging, and labeling accordingly to address these concerns. Additionally, more research needs to be done on the effects of long term CBD use, as it has shown promise in treating anxiety and psychosis in humans and animals.

“Cannabis Extract Adulterants: Not all Rosin is Good” by Jiries Meehan-Atrash and Robert Strongin of Portland State University

To begin with, solvent-less cannabis extracts that are made by applying physical pressure to extract the cannabinoids are commonly referred to as rosins within the cannabis community, however the rosin that will be discussed in this talk is of a different kind altogether, not referring to this kind of concentrate.

Taking a step back, there was a major outbreak of EVALI (e-cigarette or vaping associated lung injury) in the United States that resulted in 68 deaths between September 2019 and February 2020. One of the first linked culprits found by the CDC vitamin E acetate, which was identified in both seized vape cartridges by New York as well as in lung tissue fluids. EVALI was originally thought to be lipoid pneumonia, but then later was further identified to be a toxicant-induced airway centered pneumonitis. Furthermore, simply vitamin E itself has not been shown to be harmful when inhaled, so this could indicate that a breakdown product of vitamin E acetate could be a potential culprit as well.

At the height of the EVALI outbreak, these researchers came across some unknown materials in vape cartridges obtained from industry partners. Using NMR, they found that one of the viscous liquids was pure vitamin E acetate, and another golden semi-solid liquid contained an unknown mixture of substances. Spiking the sample with MCT oil confirmed its presence, and quantitative NMR confirmed it to be 50% by mass. To figure out the rest of the material, GC-MS found the presence of dehydroabietic acid, which is a sesquiterpene acid found resin acids from pine trees, also known as pine rosin. This material has several uses including soldering, shoes, musical instruments, and even as a food additive in its glycerol ester form. Further analysis with HPLC-ESIMS found even more resin acids, including isopimaric acid and palustric acid. Additionally they found oleamide, a nitrogen-containing compound that has been used in Spice synthetic cannabinoid mixtures and as a sleep aid supplement. Pine rosin was ordered from Sigma Aldrich as an analytical standard, and compared to the cannabis extract adulterant. An overlay of the NMR chromatograms indicated that the cannabis extract adulterant contained pine rosin mixed with MCT oil. 2D NMR techniques were used to make assignments to specific fractions isolated by HPLC. Although oleamide was not visible by NMR in the crude extract, when the specific fraction of it was isolated by HPLC, the NMR spectra was found to overlay with commercially available oleamide.

The presence of pine rosins in cannabis extracts is significant and alarming because when used in soldering it actually can cause occupational asthma. Vaping hash oil, even with only 1% pine rosin, results in exposure over 3500 times the occupational health limit. The researchers propose that forensic labs that test black market products should also do pine rosin testing. They also propose that resin acids should be searched for in the lung tissue of EVALI patients, if this has not yet been done.

The first night of programming’s theme was “Mind the Gap: Specialized Cannabis Formulations” was moderated by Monica Vialpando.

“AI and drug discovery: Search for new psychoactive candidates from cannabis sativa” by Ramesh Jagannathanof International Medical Cannabis Association, Toronto, Ontario, Canada

This presentation was about an application of machine learning to cannabis, and began with a brief history of Cannabis Sativa. Its oldest and first use was in China, and then spread to India, Arabia, Africa, Europe, and the Americas. In the second half of the 19^th century, about 100 scientific articles were published about cannabis in the United States and Europe, along with many drug companies selling cannabis products. Then in 1937, the Federal Bureau of Narcotics passed the Marihuana Tax Act Law, which restricted cannabis use, and eventually led to it being taken out of the US pharmacopeia in the 1940’s. Currently, cannabis use has started to become legal in some areas, beginning in the 2000’s and continuing today.

This project used AI, or artificial intelligence, and applied it to drug development. The goal was to explore the psychoactive status of some of the less common cannabinoids, other than THC, and also to explore the psychoactive metabolites of the smoke. Interactions with the blood brain barrier are also of concern along with receptors in the brain. The machine learning study began with a total of 468 metabolite structures, not just from cannabis, but from other plants as well. Factors considered were the physiochemical descriptors of the structure along with the clinical parameters of the compound. Several iterations of machine learning algorithms narrowed the candidates down to 330, then to 165 and later 112. After running a 3D scaffold homology with THC, 54 candidates were left. Substances that had a high homology with THC included 8-hydroxy-THC and delta-8-THC-acid. Moderate homology was found with 10-oxo-delta-6a-THC and isocannabitrol. Low homology was found with CBD and also CBD-V. The future directions of this research are to test the highly homologous structures for psychoactivity in-vivo, followed by Phase 1 animal level testing. The details of this machine learning study were published in an ACS Omega article.[1]

“Product marketing: Bridge the gap between formulation science and consumer” by Stefanie Maletichof MedPharm, Denver, Colorado, United States 

Stefanie Maletich has BS from Regis University, and worked in analytical testing back in 2015. Since 2017, she has been a part of MedPharm, a vertically integrated facility that is both GMP and GAP-certified. It also holds the first and only cannabis research license in the state of Colorado. The talk began with a discussion of compliance, basically, don’t make a product that you can’t sell in your area, and pay attention to the differences between medical and retail cannabis rules and limits, as well as relevant FDA guidelines. Regular cannabis products cannot advertise to treat or cure diseases. In terms of demographics, you must consider your target audience, for example, how often will they use the product? What is their experience level with cannabis? How much are they willing to spend? What kind of routes of administration or different product types do they prefer? Are you looking to compete with existing products or to fill a gap in the marketplace? What kind of flavors are they going to prefer, and are these cannabis flavors or not? Overall, it is a balance of appealing to a target customer along with being appealing to general customers too. Other things to consider are how your company adds value to the product’s value proposition. For example, is your company GMP or GAP certified? Are you working with experts in scientific formulation?

When considering the final packaging, factors to be considered are the price of said packaging materials, the type of packaging depending on the method of administration, and also making sure to be in compliance with local regulations. Also, it must be clear on the packaging at what temperature a product should be stored along with its shelf life; these can be accomplished by stability studies. Obtaining user feedback is also important, although the feasibility of this may depend on local regulations. If possible, users can report back how they consumed the product, its onset and duration, what effects they felt, along with any additional positive or negative feedback. Pricing of your product takes many factors into account, including the cost of raw materials and ingredients, packaging, and labor. Overall, Stefanie suggests that product marketing groups put themselves in the customer’s shoes, and be willing to compromise and explore alternatives to meet demands, but definitely don’t bend the rules to sell more product, complicate the message of your product, or sell your product for as cheap as possible, as competitors may easily take you out.

“Does “cannabis-derived” mean to the public what it does to a chemist?” by Brad Douglassof The Werc Shop, Monrovia, California, United States 

Brad Douglas, PhD begins by mentioning the 2018 Farm Bill, and how that set a limit of 0.3% THC or less on industrial hemp. The biosynthetic pathways that create CBC-a, THC-a, and CBD-a are discussed. These three cannabinoids are made directly from their respective synthase enzymes. In most hemp, THC-a synthase is not active, that’s why levels are generally low. That does not, however, mean that it will not be made at all, even if the synthase is completely inactive because THC can be made without THC-a synthase, but through other pathways of CBD cannabinoid degradation. Additionally delta-8-THC is not directly synthesized by a cannabis synthase enzyme, but made instead by light and oxidation. Recently, there have been certain vape products out there that are reportedly derived from hemp but made of mostly delta-8-THC. Furthermore, they can contain up to 10% delta-9-THC. This new vape source’s lack of consistency or legality indicates that this is probably not a safe hemp product.

Cannabinoid esters have been found in cannabis plants: when the acid group of the cannabinoid reacts with a terpenoid alcohol, an ester is formed. Cannabinoids have been chemically converted by acetylation of their hydroxyl groups. This is considered a synthetic derivative. THC has been hydrogenated, and this is known as delta-9-HHC. The metabolism of THC in the body results in hydroxylation and oxidation. Overall, it is worth noting that all of these cannabinoids and synthetic derivatives can be created from CBD hemp with synthetic manipulations. Therefore, academia and industry need to work together more and establish standards for derivatives of hemp cannabinoids. He suggests a Decision Tree Approach as a starting point to tackle this problem. In other words, the degree of division from cannabis can be classified for these derivative molecules.

“How recent federal court decisions and USPTO proceedings are shaping future cannabis intellectual property rights” by Katherine Rubinoof Caldwell Intellectual Property Law, Boston, Massachusetts, United States 

Katherine Rubino, from Caldwell IP Law, knows about cannabis IP rights. She is a former compounding pharmacist, making topical creams with CBD, and a patent attorney, protecting cannabis inventions. She’s also the chair elect of the CHAL subdivision of the ACS. She began by explaining three main types of patents: utility patents, which are the most common in chemistry processes, plant patents, which is the protection of the actual plant, which began in 1930, and design patents, which can protect the ornamental appearance of something, how it looks basically, and is common in fashion and clothing industries. However, plant patents can be difficult to prove of their infringement. In 2010, the first cannabis plant patent was issued to “Ecuadorian Sativa”. More recently, there is one called “Rainbow Gummeez” which has a unique mix of cannabinoids and terpenes. It was interesting to learn that the top filer of cannabis patents is not actually any company, but the category of sole inventors. Biopharmaceutical companies and academic institutions are also big filers of cannabis patents, but sole inventors make up the top category. The most common category of cannabis patents is medical, in essence, to treat specific ailments such as neurological, pain, psychiatric, cancer, immune, ophthalmic, sexual, respiratory, or nausea, to name a few. The second most common category is compositions of a product, followed by processing patents and device patents. She stressed the importance of well-written patents, patents can be invalidated if they are claimed to be obvious, but a more thorough patent will address this angle with strongly written claims. In conclusion, she says that the cannabis patent landscape is rapidly evolving and maturing.

“Role of applied molecular microbiology in increasing cannabis product quality” by Khyrrah Shepardof College of Agriculture and Food Science, Florida Agricultural and Mechanical University, Lauderdale Lakes, Florida, United States and College of Biochemistry and Cell Science, University of Florida, Gainesville, Florida, United States

Kyrrah Shepard is actually one of the first analytical microbiology presenters at a CANN symposium, which is typically more chemistry-focused. She highlights that “Biology is the driving force of this industry” and also that interest in cannabis microbiology has served as an indication and coincided with an overall spike in investors’ interest in the cannabis industry. Microbiology is so important because immune-compromised individuals may use cannabis products, so how can we provide more protection for such individuals now that cannabis use is more widespread? Microbiological testing can make the use of consumables in a testing lab skyrocket, but there actually can be ways of melding methods together to reduce consumable usage. Labs also need to be in control of their data as well as the bio-hazardous substances they create. It takes both cost and time for a company to be able to make safe products.

“Novel LC-MS/MS method with dual ESI and APCI ion source for analysis of California regulated pesticides and mycotoxins in medium-chain triglyceride (MCT) oil cannabis tinctures” by Avinash Dalmia¹, Charles Johnson², Saba Hariri¹, Jacob Jalali¹, Joey Kingstad², Erasmus cudjoe¹, Feng Qin¹
1. PerkinElmer, Hamden, Connecticut, United States. 2. Napro Research, Sacramento, California, United States 

Avinash Dalmia from Perkin Elmer discusses a method for detecting and quantifying 66 pesticides and 5 mycotoxins in MCT oil tinctures. It uses LC-MS/MS with a dual APCI and ESI source. They started the project planning that all compounds could be done with just ESI, but then it turned out that some the more nonpolar ones that are normally ionized by GC-MS, could not be done by ESI, which is a liquid phase ionization technique that commonly works for more polar molecules. In contrast, APCI is a gas phase ionization technique, but it’s still done on the same LC-MS/MS system instead of a GC, it’s just a different source part. They were working with the California list, and were able to meet all action limits. Some of the ones that needed APCI were PCNB, chlorfenapyr, chlordane, and methyl parathion. They discussed the mechanism of PCNB ionization. Typically, compounds lose a hydrogen atom, but PCNB has none available, so it actually fragments by losing chlorine and gaining oxygen. They proved this math by the mass fragments. In terms of sample prep, they do a solvent extraction only. They use many internal standards throughout the method in order to account for matrix suppression. Specifically, the pesticide bifenthrin without an internal standard showed quite poor recovery but was completely fixed with the appropriate internal standard. Mycotoxins were analyzed by this same method, but not much detail was given about the specific analytes.

“Analytical advances for the assessment of cannabis-derived products in the European scenario” by Michele Protti, Marco Cirrincione, and Laura Mercolini of the Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum – University of Bologna, Bologna, Italy 

Laura Mercolini discusses the European scenario of cannabis consumption, specifically in the European Union. She runs a research group at the University of Bologna, Italy. In the European Union, cannabis is the most used illicit drug, 7.4% of people have used cannabis in the past year there, with cannabis being the most commonly drug seized (three quarters of the time) of drug types that are seized. Cannabis resin can come from Morocco and is trafficked in large volumes. The overall potency of cannabis has increased in recent years, in line with the trends seen in the Unites States, as well as the prevalence of edible cannabis and new vaporization devices. In Italy, commercial hemp must be under 0.6% THC, and in cannabis products, should be under 0.5% THC. CBD oils from hemp that are sold as wellness products must have a THC level under 0.2%. Sensitive analytical methods are needed to reach and accurately quantify at this level. Edibles are also analytically challenging, with a complex matrix.

In a matrix of hemp seeds, they optimized a microwave-assisted extraction procedure, and compared it to other extractions such as refluxing, and looked at major and minor cannabinoid content through standard analytical approaches. HRMS gave them a crucial insight about the different cultivars of cannabis. The group has an ACS Medicinal Chemistry Letters paper about this research.[2] The group also has bio-sampling analysis techniques with dried blood spots, detecting blood levels of THC in a finger prick test. Overall, she emphasizes that cannabis being tested and sold is new in both the United States and Europe, and there is still a lot of research to do because hemp is a difficult matrix, and CBD and legal hemp is an emerging market in Italy.

“Improving throughput and overcoming the challenges associated with pesticide & heavy metal analysis of cannabis & hemp extract and edible samples” by Toby Astill of Perkin Elmer, Victoria, British Columbia, Canada and Kevin Smithof Napro Research, Sacramento, California, United States

With cannabis, as a global commodity, how can we improve the throughput of pesticide and heavy metals testing? The United States has twelve legal recreational states, and hemp is legal in all states. Cannabis has a complex chemistry and is heterogeneous. Seed-to-sale means tracking from cultivation to harvest to extract to purification, combining or formulating with other raw ingredients, then tested, giving the final product. Products must be safe for consumption, and contamination is a real issue. There are no guidelines from federal organizations, and from this they see things such as a huge variation in sample preparations techniques from lab to lab. Looking at the California regulations as an example, it a testing program requires many different platforms for the different tests. When they were using an 8-step extraction procedure, they found that 61% of the time was spent on sample prep, and also that pipetting was differing from person to person. They automated the sample prep procedure with the Janus 420 Liquid Handler, programming it to do the sample dilutions instead rather than with hand pipettes. It reportedly gives reproducibility, fewer errors, and improves compliance. It goes all the way down in dilutions to making the HPLC vial. It can be used with both any LC and GC mass spec instruments, regardless of vendor.

On the news it has been shown a high incidence of heavy metal contamination increasing lately, specifically in Michigan and California vape products. They remind that one of the required metals for testing, mercury, is volatile, so a sealed vessel must be used for digestion, and the digest should be clear and transparent following the digestion. The Emerald Test validated these two methods that they discussed. Their entire workflow is traced electronically, and they emphasize the improvement they gained with the Liquid Autohandler, shortening sample preparation time bottlenecks.

“Evaluation of a liquid chromatography and photo-diode array detection method for the screening of 11 cannabinoids in hemp plant and oil samples as part of the NIST cannabis quality assurance program (CannaQAP)” by Walter Wilson¹, Maryam Abdur-Rahman¹, Melissa Phillips¹, Catherine Rimmer²
1. Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States. 2. Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, Maryland, United States

NIST provides tools for improving chemical measurements across disciplines; this includes calibration solutions, traceability, and quality assurance programs. This presentation by Walter B. Wilson of NIST goes into detail about a new hemp plant and oil NIST quality assurance program that THC and CBD testing labs can sign up for right now until August 31, 2020. First a need was identified for this test, namely that it is important to be able to detect and quantify THC that’s around 0.3%, because of hemp regulations. The study was designed by NIST because of this current need for consistency in this area, so labs are now signing up for this test. The hemp and oil samples will be distributed in September; the labs will then analyze the samples and give the data back to NIST in October. NIST will then analyze the composite data and give feedback to the participants. Different types of labs that may choose to participate in this include regulators, product developers, forensic labs, and contract testing labs.

Quality assurance programs are not the same thing as a proficiency test. QAPs are better to use for newer methods, while proficiency tests are designed for more established methods. The goal of the quality assurance program is to help cannabis and forensic labs demonstrate and compare their abilities. The NIST program has two exercises: the first being a commercial hemp oil sample, and the second part of the test is a cannabis/hemp plant that contains 0-2% THC from the University of Kentucky. Some suggestions they had were to use the lower portion of the calibration plot to quantify cannabinoids at low levels, resulting in a better fit than when using the whole plot, but be careful there are still sufficient data points. Samples must be diluted carefully and calibration ranges observed. They also recommend using an AOAC extraction procedure for the hemp oil, and run dilutions at both 10 and 100 fold. Some of the CBD samples may contain just 0.15% THC, so there is not a lot of room for error. In the end, users will be able to compare their measurements and uncertainties to those of NIST. Future exercises will include more complex matrices and test for contaminants as well.

“Discovering the un-tapped channels of ISO accreditation: How laboratories add value to industry” by Shannon Swantek of Enlightened Quality Analytics, Santa Cruz, California, United States

Shannon Swantek was on the advisory committee for pesticide testing and sampling rules, getting Oregon labs ready for regulations in their state a few years back. After Oregon, she moved on to the Massachusetts department of Public Health. Currently, she’s in California and is supporting labs and licensees with Enlightened Quality Analytics, LLC. She says that we have come a long way from the “wild, wild west” and that we are now finally holding ourselves to the same standards as other mature industries. State-licensed cannabis-testing labs have to be ISO 17025 accredited, this accreditation actually spans across different industries. Within this type of accreditation, it must assume that each industry has its own standards to follow and that the lab is following those standards specific to its industry.

ISO 17025 is actually very focused on the needs of the clients, and a major part of an ISO 17025 certification is evaluating whether testing labs are able to meet the needs of their clients. These needs definitely do not include requesting anything that would compromise valid data. These needs, which Shannon says are not taken advantage of enough, are in place so that the clients can ensure that lab is doing everything correctly to deliver data that’s going to help the customer with their goals. The testing lab must have its own set of standards that they follow, including procedures for when these standards are compromised. Clients must be notified if their data was compromised. There are also procedures for when a client has a complaint and the corrective actions that must take place because of it. Clients can request for data including blanks, duplicates, and spiked samples. Clients can audit labs, but if entrance to the lab is not possible, data packages can be requested.

ISO 17025 reduces the need for over-regulation because quality is driven internally. Additionally, clients may have their own ISO accreditation, which only allows them to work with other ISO-accredited labs. The most common kind of cannabis testing is “point of sale” testing, when it is tested basically right before it goes on the shelves. This does not capture any risk management of the product, though. Additional kinds of testing such as shelf life, testing of source material, and contamination control give the client more control over their process and final product. Clients also need to be able to identify risks, so testing over a long period of time will show any deviations in product data trends. Values below the action limits could be of interest to clients, so they should request them if needed. In the federal landscape, producers and processors will have to do much more testing than what is required currently. Also, reference methods are becoming more common in the industry, being worked on continuously by different groups. Overall, clients may not actually be aware that they have all of these entitlements when working with an ISO 17025 lab, so Shannon really thinks that client services and open communication should be used more instead of lab shopping.

“Cannabis analysis: The intersection of scientific integrity, accountability, and analytical technology” by Susan Audino of Audino & Associates, LLC, Orient, Ohio, United States 

Susan draws some parallels between the cannabis testing industry and testing for covid-19. In both areas, we have seen examples of how a false positive or a false negative result can have consequences. Additionally, decisions are being made on both the state and federal levels about how to react to or control covid-19, which is similar to how both states and the federal government work to regulate cannabis and hemp.

Scientific integrity is very important and has been defined by the USDA. The cannabis industry is not fundamentally different from other industries, but it does have its own unique challenges – a desire for speed as well as regulations that don’t always make sense. ISO 17025 does not always guarantee for the best science because people can take shortcuts. For example, calculation of a % recovery, spiking should be done before extraction, but sometimes the spiking happens after extraction, and this does not actually convey true recovery. Also, expiration dates on vials only apply before the ampules are broken. Once the ampule is broken, it is up to the lab to determine an appropriate expiration date for the opened standard. These are just a few examples of what Susan has seen.

“USP Considerations for quality attributes of cannabis inflorescence for medical purposes” by Nandakumara Sarma of United States Pharmacopeia, Rockville, Maryland, United States

Nandu Sarma is part of Dietary Supplements and Herbal Medicines at the United States Pharmacopeia. The USP improves global health through public standards. Convention members work together with USP and alongside committees and panels of advisory experts. USP standards are public and used in over 140 countries. Everyone benefits from increased quality control. They recently published a paper in the Journal of Natural Products.[3] This paper looks at three different types of cannabis: THC-dominant, CBD-dominant, and intermediate strains that contain considerable levels of both THC and CBD. They describe the microscopic features of this cannabis, and perform HPTLC, that is, high-pressure thin layer chromatography, to identify 11 cannabinoids compared to their analytical standards. The USP provided some storage and labeling rules, for example, the dominant terpenes should be labeled. The product should also have an indication as to whether it was remediated in order to reduce the microbial load. A USP panel of experts wrote this paper. On its way is similar work on the quality attributes for hemp.

“Formulation strategies required to design fit-for-purpose oral cannabis products” by Monica Vialpando¹, ², Carolyn Burek³, ²
1. Vialpando, LLC., Baltimore, Maryland, United States. 2. Cannabis Chemistry Subdivision, DC, District of Columbia, United States. 3. Department of Biochemistry and Molecular Biology, Colorado State University, New York, New York, United States

Monica Vialpando, PhD is the founder and CEO of Via Innovations, and gave a huge thanks to CANN. Kicking off the session, “Cannabis, Soil to Oil: How Cannabis Savita L. Products go from the Bench top to the Marketplace”, she began with some fundamental strategies for cannabis-infused oral products, for example the categories of beverages, gummies, and baked goods, just to name a few. For these kinds of products, oral bioavailability is very important, and there are major challenges in achieving sufficient bioavailability in the product for the consumer. The first being that the drug must have enough solubility to dissolve, and then secondarily, the hostile environment of the GI tract’s pre-systemic metabolism, combined with factors affecting the permeability and extent of intestinal absorption.

Cannabinoids and terpenoids have shown poor aqueous solubility. Dissolution is defined as the kinetics of a drug passing into solution, and this is affected by the dissolving particle’s available surface area. The three main strategies for increasing solubility are to reduce the particle size, to add a solubility excipient such as lipids, surfactants or cyclodextrin, or lastly, manipulation of the solid form, considering that an amorphous solid will be more readily dissolved than a highly ordered crystal form. She provides the example of cotton candy vs. rock candy. They’re both made of the same ingredients, sugar, but the cotton candy is going to dissolve more readily.

It’s not uncommon to combine different strategies for solubilizing, for example nano-emulsions are most commonly used in beverages. It’s reportedly easier to make a nano-emulsion from an isolate compared to using full spectrum material for nano-emulsions. Formulators must be careful when using them in complex beverages, though, because of pH stability. Some nano-emulsion ingredients could be stable in their concentrated form, but then prove to be unstable in the finished product. Reportedly, a nanoemulsion beverage ended up failing for benzene because sodium benzoate reacted with vitamin C, making benzoic acid, which degreded into benzene and carbon dioxide. The beverage had to be reformulated, and this shows why testing is so important. In conclusion, Monica says to practice caution and understand the fundamental science principles when formulating edible cannabis products, because they are going to end up being consumed by people.

“Ready-to-run eMethods to aid rapid startup of your laboratory” by Jennifer McCulley¹, Elizabeth Almasi¹, Anthony Macherone¹, ²
1. Agilent Technologies, Wilmington, Delaware, United States. 2. School of Medicine, The John Hopkins University, Baltimore, Maryland, United States 

Jennifer McCauley, a Program Manager at Agilent, began with an overview of the current landscape of the cannabis testing lab industry. Currently there are many testing considerations that must be taken into account including potency, pesticides, heavy metals, residual solvents, terpenes, and also microbial testing. In addition, the many matrices involved can include beverages, pet products, personal care items, cannabis flowers, concentrates, tinctures, and edible goods. Furthermore, each state has its own set of regulations that they follow. Setting up a testing lab is like climbing a tall mountain, however, e-methods can do some of the initial hard work for you, getting you half way up the mountain to help you climb the mountain. E-methods are downloaded directly and contain the method and extensive support information. It’s an electronically delivered set of information that also contains application notes and consumable part numbers. For example there is an e-method for a 20 minute GC triple quad pesticide screen. It comes with all the right GC and MS instrument configurations, and it’s designed for accuracy and robustness. It’s also editable, you can add or delete analytes. It also comes with steps for sample preparation and support information. It includes best practices and was made by Agilent scientists. Steps for method maintenance and tips for avoiding sample overload are included. Another one available is the 10-minute potency method; it’s an LC with a variable wavelength detector. With these e-methods, you can shorten your start up time; get to running more samples sooner and making a profit sooner.

“Stability in cannabis product development” by Andrea Baillo, Cheri Pearson, and Sarah Lundfelt of Research & Development at Green Thumb Industries, Centreville, Maryland, United States

Andrea Baillo, PhD begins with a little background on cannabis: it’s just one drug but has been associated with over 100 conditions and/or benefits. Manufacturers need to be cautious, and regulators show a lack of conformity because there are different action limits in different states. In terms of pharmaceutical product stability, it is critical that a product is made correctly, stored correctly, and that it’s shelf-stable. This requires stress testing, accelerated shelf life testing, and long term testing; it’s a continuous improvement process. The main factors to consider are the stability of the active ingredients, the container that will be used for packaging, and also the heat and moisture conditions at which the raw materials are shipped and stored. You should also look for any possible interactions between active ingredients and inactive ingredients. There are many types of stability: therapeutic, physical, chemical, microbiological, and toxicological. Shelf life studies are done at ambient conditions, a normal room temperature and regular humidity, but during accelerated shelf life studies, the sample is kept at 40°C at 75% relative humidity. In these hotter more humid conditions, 7 days of time equates to one month of ageing at room temperature. Single source data comes from just one batch or lot over time, while multisource data comes from multiple batches or lots over time.

Vape stability was studied over a period of 2.5 years. After 1 year, there was less than 10% relative percent difference in the THC value, and after two years, the RPD was roughly 10%. However, after 2.5 years, the RPD is up to 25%. For the terpenes, there was already a 20% RPD after just 3 months, but that loss actually mostly plateaued over the next 2 years, retaining the major terpenes. Something interesting that they found was that nerolidol was not found in the original carts, but seen after 3 months. In accelerated conditions, chromium leached from the carts over time, as well as lead, but there were no yeast or mold after 2.5 years for the carts.

They also looked at the stability of flower in clear glass packaging. There was a small decline in THCa, but with no comparable increase in THC after 8 weeks. The terpene beta-myrcene was also shown to be not quite stable in the long term, but the yeast & mold and water activity were stable. In summary, there is a need for standardization of state testing programs and a need to emphasize more research into stability and product formulations. There are benefits to understanding stability. To have control over a process, you have to monitor stability throughout the process. You can’t assume the stability of a product stays the same if you’re changed any of its hardware or packaging. The stability tests need to be re-done to test how any changes could affect shelf life or storage conditions.

“Truly green plastic: Bio-based, biodegradable polymers from cannabis plant waste” by Tarek Moharram, Najwa Zebian, and Dmitry Bryzgalovof Moharram Ventures Inc., London, Ontario, Canada

Tarek Moharram is a lawyer and project manager. The initial purpose or need for a biodegradable polymer comes from the inability of dog waste bags to break down under normal conditions, and also being that there are too few processing locations for the bags. They brought on a team of microbiology consultants. Performance and cost are both important and need to be weighed. They focused on reducing the cost of feedstock. In cannabis, for every kilo of dried flower produced there is also 8 kilos of associated plant waste material, which the licensees have to destroy. Testing was done on the feedstock material to better understand its properties. It was hard to get access to the biomass and also hard to find a facility. They used an agricultural testing partner in Canada. 

The plant material contains cellulose and fatty acids. The stalk material is mostly cellulose and not much fatty acid. They finally were able to secure a small, shared lab space. Using acid, they converted finely ground particles of cellulose into glucose, and then fermented the glucose to PHB polymer followed by extraction from a bacterial pellet. They worked with Poly[Analytick]. They analyzed the polymer by FTIR and NMR, and then tested its physical properties using DSC and TGA. The PHB polymer had a melting point of 165°C, and decomposed over 270°C. In the road ahead, there will be additional tests, the acquiring of other material components, and expanding to a commercial scale. Their work has been featured in Forbes, Marijuana Business Magazine, Cannabis Tech, and Waste 360.

“Investigation of chocolate matrix interference on cannabinoid analytes” by David Dawsonof CW Analytical, Oakland, California, United States 

David Dawson discusses his insights and research into chocolate matrix interferences. The Bureau of Cannabis Control regulates 6 cannabinoids, pesticides, solvents, heavy metals, and more. Focusing in on potency testing, there are some pretty high stakes. Recreational edibles cannot exceed 10 mg per serving or 100 mg per package. Concentrates and topicals cannot exceed 1g of delta-9-THC per package. If the potency tests come back too low, the batch must be relabeled, which makes the product slightly less appealing. But if the potency is too high, the batch must be destroyed with no chance for remediation. This puts a lot of pressure on producers to hit their numbers. It’s also a big responsibility for testing labs considering the many matrices of edibles and concentrates, combined with the need to be efficient and scientifically accurate in the analysis. 

He started looking at chocolate matrices and found an unexpected potency variation that depending on the mass of sample used for prep, there would be a different potency result. Using the same extraction procedure, a 1g-extraction procedure gave a higher amount of THC than a 2g chocolate extraction. Chocolate is one of the more difficult matrices because of its high fat, polyphenols, and tannin. There are also at least 51 organic flavor molecules in milk chocolate, and at least 72 in dark chocolate. He made stock solutions of cannabinoids, and then added un-dosed chocolate (milk chocolate, dark chocolate, and cocoa powder) at 1, 2 and 3 gram sample sizes, with 10 replicates for each combination. The recovery of delta-9-THC was found to be more affected by adding more chocolate than the recovery of CBD was. Furthermore, data with CBN were found to be even more affected than delta-9-THC. CBG showed almost no loss of recovery, more similar to what was seen with CBD. Overall, the recovery of di-ol cannabinoids CBD and CBG were shown to be hardly affected by chocolate interference, but the single phenol THC and CBN cannabinoids were affected a bit more. The same trend was seen in both milk and dark chocolate. 

They wanted to consider a cannabinoid with zero hydroxyl groups, so they did a chemical reaction on CBD, methylation of both OH groups with Methyl Iodide, potassium carbonate, in DMF at room temperature for 24 hours, giving 78% yield of CBDD, cannabidiol dimethyl ether. This reaction was achieved in collaboration with Liz Jarvo at UC Irvine, and can be seen by a color change from purple to orange. The purified CBDD subjected to chocolate was very suppressed, only 85% recovery with 3 g of milk chocolate, and 80% recovery in dark chocolate, compared to the 95% and 100% recovery seen with the THC and CBD cannabinoids at 3 g chocolate. However, in cocoa powder, CBN’s recovery is more akin to CBDD levels, so they are looking at possible cleanup procedures that might help with that. The extraction procedure used was dissolution in Methanol solvent, so it would be very interesting to look at how a cleanup step could change things. Also, the matrices of topicals and baked goods are of concern in future studies. 

“Cannabidiol isolation including cannabis-relevant pesticides removal by centrifugal partition chromatography”

Róbert Örkényi¹, Kristóf Gazda¹, Erika Tamás¹, László Óvári¹, Márió Nacsa¹, Eszter Blanár¹, Dóra Rutterschmid¹, László Lorántfy¹, Balázs Krámos³, Andrea Böszörményi², Szabolcs Béni², Árpád Könczöl¹
1. Research Laboratory, RotaChrom Technologies, Dabas, Pest, Hungary. 2. Department of Pharmacognosy, Semmelweis University, Budapest, Hungary. 3. Gedeon Richter Plc, Budapest, Hungary 

Árpád Könczöl, PhD, discusses their unique and patented CPC, or centrifugal partition chromatography, technology. RotaChrom was established in 2014 in Hungary. 

With the testing requirements for solvents, pesticides, microbes, mycotoxins, and heavy metals, the question is: what can be done with all this contaminated, high value cannabis? CPC is a unique preparative liquid-liquid separation technique that can be operated in two different directions: ascending and descending mode. The driving force is the density difference of phases. CBD contaminated with pesticides was isolated by CPC using an n-hexane and water system. Out of 115 pesticides, the Canadian list, 90 were fully removed, 18 were co-eluting and problematic, and 7 were irregular or outliers. When extreme loading is applied, it can be more prone to co-elution of pesticides. Even when the methods are fine tuned, some co-elution still remains. However, the most commonly seen pesticides such as myclobutanil and bifenazate are completely removed, and the generic method still removed 92% of pesticides. This work was submitted for publication in the Journal of Chromatography A. Similar studies are in progress for the isolation of THC. 

“Applying physicochemical chromatography techniques to optimize adsorbent use in the processing and analysis of cannabis” by Jerry King of CFS, Fayetteville, Arkansas, United States 

Jerry King is a consultant with Critical Fluid Symposia, or CFS. Adsorbents have many uses, for example the trapping of terpenes or cannabinoids solutes by sorbates, odor abatement in greenhouses, and the removal of contaminants. Extraction vessels may have adsorbent columns. Also, adsorbents and a mini pump are used to monitor personal or occupational exposure of a substance. Carbon dioxide is a chemically active gas, compared to helium, meaning that it displaces molecules on surfaces. As temperature increases, the adsorbent trapping ability decreases. Sorbents can get degraded or fused over time, especially when exposed to carbon dioxide, they lose their surface area. Adsorbents have limited service lifetimes. 

“Making the case for biosynthesis to create & isolate cannabinoids at scale” by Jeremy Friedberg, Istok Nahtigal, and Alexia Blake of LAVVAN, Toronto, Ontario, Canada

Dr. Jeremy Friedberg is the CSO of LAVVAN. In terms of quality, some of the old industry metrics were basic THC content, taste, smell, and personal effects. Just looking at these basic metrics is not enough of an indication for the quality of cannabis. Now there is a new definition of quality: 11 canabinoids, heavy metals, pesticides, microbial testing, GMP production, stability testing, mycotoxins, total yeast and mold, not to mention the actual yield of the crop. This is a new series of challenges for farmers and the industry. The plant produces the acidic form CBDA, which is chemically distinct from CBD and CBDV, the shortened chain analogue. The CBD market is expected to grow to $25 billion by 2025, so can plant production meet this demand?

Isolates can be made from plants, from chemical synthesis, or cellular agriculture, also known as biosynthesis: It’s a large-scale fermentation and isolation from an organism. Cannabis plants can be grown in the field, in a greenhouse, or completely indoor. In a finished cannabis plant, the usable flower makes up 5-10% of the biomass. In biosynthesis, however, each cell can be its own production factory. Almost all organisms have the biosynthetic pathway for GPP, but the olivetolic acid pathway needs to be added, along with the cannabinoid synthase enzymes. Large fermentation vessels can be used, and overall only 10% of land is used compared to farming. Standard purification techniques are used following the biosynthesis. There is a high demand for minor cannabinoids at larger quantities, and price has been a limiting factor for meeting this demand. Biosynthesis could address the cost issue because of it’s ability to scale and to be fine tuned with specific enzymes. 

Conclusions

The ACS National Meeting Cannabis Chemistry Subdivision symposium was a huge success and a valuable source of the current cannabis science landscape. Scientists from around the world all proudly presented their cannabis research, from the United States, Canada, Hungary, and Italy, they gave talks from the perspective of industry professionals, producers, testing labs, consultants, academic researchers, regulators, standards institutes, and even legal considerations. Overall, I felt like a common theme was that we have made great strides in this industry, not just in the last 10 years, but specifically in the last year, perhaps in response to the EVALI crisis, and also as testing labs are becoming more streamlined and in-demand. The importance of safety and control cannot be stressed enough. There was also the common theme that there is still much work to be done, and this will require cooperation and collaboration of many intuitions, particularly government, industry, and academia. The cannabis landscape draws its rules and regulations from other industries, so there is really no particular reason as to why the cannabis industry cannot become as legitimate and controlled as other industries in the long run. It is mostly the issues with inconsistent regulations, namely lack of federal regulations so far, that create difficulties with the progress of industry maturation. Hopefully we will be able to look back on this time as a pivotal point. I think many people are getting ready for federal legalization of cannabis, which would undoubtedly change the cannabis landscape once again. No matter what happens with federal legalization, though, it seems as though advances in technology and quality control will be crucial.

[1] Ramesh Jagannathan, ACS Omega 2020 5 (1), 281-295. DOI: 10.1021/acsomega.9b02663

2) Michele Protti, Virginia Brighenti, Maria Rita Battaglia, Lisa Anceschi, Federica Pellati, and Laura Mercolini ACS Medicinal Chemistry Letters 2019 10 (4), 539-544

DOI: 10.1021/acsmedchemlett.8b00571

[3] Nandakumara D. Sarma, Andrew Waye, Mahmoud A. ElSohly, Paula N. Brown, Sytze Elzinga, Holly E. Johnson, Robin J. Marles, Jeremy E. Melanson, Ethan Russo, Lawrence Deyton, Christopher Hudalla, Gordon A. Vrdoljak, Joshua H. Wurzer, Ikhlas A. Khan, Nam-Cheol Kim, and Gabriel I. Giancaspro

Journal of Natural Products 2020 83 (4), 1334-1351

DOI: 10.1021/acs.jnatprod.9b01200