The analysis of legal cannabis is a new field, and not surprisingly beset with growing pains. As a result, misconceptions exist about how to test, what to test, and when to test. In this column, I will put my PhD and 40+ years of analytical chemistry experience to work to dispel several of these myths. This matters because these misconceptions are preventing the industry from meeting its full potential.

One of the challenges in the legal cannabis industry is the newness of it all. Regulations are constantly changing, and many people with no previous experience in the manufacture of medicines—and yes everyone in this business is somehow involved in the production of cannabis-based medicines—are entering the business. When it comes to cannabis analysis, these newbies may easily succumb to myths and half-truths peddled by some of the less scrupulous members of our business community. My goal in this column is to dispel some of those myths to help the cannabis analysis community make informed decisions about what analyzers to buy and how to properly use them. Here is the first of
seven myths.

As discussed in a previous column, accuracy is how far an analysis is from its true value (1). Given the importance of accuracy, it is not uncommon for people to demand all cannabis analyses have the maximum amount of accuracy that is technically possible. The problem here is that accuracy comes at a cost. For example, liquid chromatography–mass spectrometry (LC–MS) systems can accurately determine pesticide amounts in samples with an accuracy in the parts-per-billion (ppb) range. These instruments cost hundreds of thousands of dollars. Whereas if you wanted to measure the potency of a marijuana bud, I doubt ppb accuracy for the determination of total tetrahydrocannabinol (THC) is needed to optimize growing conditions or determine when to harvest. Probably an error bar of ±1 weight percent (wt.%) may be needed to make rational business decisions. The chromatographs (2) and spectrometers (3) used to measure potency in cannabis samples cost tens of thousands of dollars, not hundreds of thousands of dollars. In this example, the obsession with accuracy at all costs might prompt you to buy a potency analyzer that costs hundreds of thousands of dollars, when an analyzer a fraction of this cost may be sufficient.

The morale of this story is that the accuracy to demand of your cannabis analyzer is determined not by the maximum accuracy available, but by the needs of your business

 You need to think about how you will use the data it generates, what kinds of decisions will be made based on that data, and then use this information to determine the needed accuracy. Lastly, as I have pointed out in previous columns (1,2) in addition to accuracy, speed, and cost, the ability to readily perform representative sampling should be used to evaluate cannabis analyzers.

On the other hand, there are companies peddling cannabis potency analyzers that claim to accurately determine cannabinoids and terpenes with high accuracy at a cost of around $1000 or less. Many of these systems work off a cell phone and need an internet connection to the cloud to work. My first concern here is data security—your company’s proprietary sample data is being sent over the internet, via a connection of who knows what security, to a server somewhere in the cloud where the weight percents are mysteriously generated and downloaded to your phone. Many of these companies claim high accuracy but refuse to disclose what their algorithms are, how they work, or how they calibrate and validate their units. I know because I have called these companies, talked to their chief technical officers, and they have absolutely refused to discuss how their systems work. This is not how science works folks—we have to show our work to be taken seriously.

My other criticism here is of their accuracy claims. Some claim to be able to accurately determine total THC in hemp at the 0.3% total THC legal limit as legislated here in the US. This requires a margin of error of 0.05% or better to confidently determine legality. In any other field of chemical analysis I have never seen an analyzer capable of second decimal place accuracy at a cost of $1000. Why is it then it is only in cannabis analysis that these miraculous $1000 devices capable of second decimal place accuracy are found? And where is the peer-reviewed data backing up your accuracy claims? Nowhere to be found of course.

The morale of the story here is that you get what you pay for. If you only need ±10% accuracy an inexpensive analyzer may work fine. But if you need anything better than this it is going to cost you tens of thousands of dollars or more. This is simply the state of analytical technology in 2021.

If I had a dime for every time I have heard this myth I would be a rich man. It appears the thought here is that since governments only require final products to be tested before sale, that this is the only testing that needs to be performed and that in-house testing is not needed. This is wrong on so many levels it is hard to know where to start. I am constantly using this column to point out that cannabis businesses make medicines and hence should test like they are making medicines. That is, “Cannabis is Medicine…Test it Like Medicine,” which as I have stated previously was almost the title for this column series (4). My goal for the cannabis industry is for us to adopt the pharmaceutical testing paradigm, which means cradle-to-grave testing (4). This means whether you are growing, extracting, or formulating final products you need to be testing at every step in your process and testing your final products as well. Here is why:

Quality control—You cannot make any chemical product reproducibly without testing. You can’t do the same thing twice if you don’t what you did or how you did it. A definition of quality is being able to reproduce your process and product on a consistent basis. This will never happen without in-house testing.

Business knowledge—You cannot make cannabis products profitably unless you optimize your grow, extraction, formulation, and so forth. And you cannot do this without in-house testing. For example, I have seen extractors increase oil yield by upwards of 20% by optimizing their extractions and distillations by using in-house analyzers. In many cases, the payback period here is only a few weeks. I have seen hemp farmers use in-house analyzers to pick the right harvest time where the total cannabidiol (CBD) is maximized but the total THC level is still legal to get the best price for their crop. In-house testing is the profitable thing to do

Customer safety­—Almost all cannabis products end up going into or onto a human being. Thus, we have a legal and moral obligation to make sure our products are safe. Not surprisingly, killing or injuring customers is bad for business. We cannot make cannabis products that are safe and effective unless we know what we are making and how we make it. The only route to this goal is in-house testing.

We Only Need to Test During Process Development

I run across this myth particularly with extractors and processors. The thought here is that once all the temperatures, times, amounts, and so forth for an extraction are optimized no further testing is needed. Hence, you only need to rent an analyzer for a few weeks, optimize the process, and then give it back to save money. The problem here is that something may always go wrong with a chemical process such as an extraction or distillation, so you need to test every batch you make not just during product development.

A further problem is that physical variables such as temperature and pressure are stand-ins for what we truly care about, the chemical composition of the product we are actually making. It is thus best to test the chemical composition of the product, rather than stuff that might be related to it.

A final problem here is that the definition of quality control is monitoring your process every time and in real time. By not monitoring chemical composition this way you leave yourself open to making bad batches that may need to be scrapped, or worse yet letting bad product go out the door for sale that may harm people.

I have found end users using cannabis analyzers to test paper, food, or most anything else they can get their hands on, and then claim their cannabis analyzer “sucks” because it “found cannabinoids in samples I know don’t contain them.” The problem here is that analyzers can only accurately analyze that for which they have been calibrated. Cannabis analyzers are not typically designed to analyze anything else. When used to analyze non-cannabis samples a cannabis analyzer may produce results, but they will be meaningless. Some people expect their new fangled cannabis analyzer to be smart enough to know when it is presented with a cannabis sample and when it isn’t. As I like to say in these situations, the tricorder will not be invented for another 300 years (5) and there is not one analyzer that can analyze everything.

There are millions of possible samples one can try to study with a cannabis analyzer. I am afraid science does not yet know how to discriminate 100% of the time between cannabis and non-cannabis samples. In the meantime, please only use your analyzer for the samples it is designed to work on.

I hear this said in the cannabis and nutraceutical industries. The thought here is since cannabis products are derived from all natural plant material that anything derived from it is innocuous and harmless and that minimal or no testing is needed in our industry. In fact, nothing could be further from the truth. For example, moldy cannabis upon extraction can yield aflatoxins which are known poisons, carcinogens, and mutagens (6). The reason we test biomass for mold is to prevent this. So much for natural things being safe.

Adulteration, lying, and counterfeiting takes place in every industry and the cannabis industry is no exception. For example, I had a hemp extractor customer who took his hemp analyzer to a farmer to purchase biomass. The farmer trotted out a certificate of analysis (COA) saying the total CBD value was 14.6%. An on the spot test with the analyzer showed 9.2% total CBD. The farmer then said, “I am sorry, I gave you the wrong COA,” and he trotted one out that said 9.2% total CBD. By performing an onsite test the extractor saved tens of thousands of dollars by not paying for CBD that wasn’t there.

Regardless of the relationship you may have with your vendors, people make mistakes either intentionally or unintentionally, by not testing what you are buying it can cause great damage to your business. Given this, I believe every cannabis industry transaction should feature an on the spot test with a potency analyzer to insure people are getting what they pay for. As an old Russian proverb says, “Trust, but verify” (7).

There are a number of dangerous cannabis analysis myths floating around our industry. Using science and my own experience, I have tried to dispel these myths.

M.W. Giese, M.A. Lewis, L. Giese, and K.M. Smith, 

, https://en.wikipedia.org/wiki/Tricorder.

, https://en.wikipedia.org/wiki/Aflatoxin.

, https://en.wikipedia.org/wiki/Trust,_but_verify.

, is Founder, CEO, and Chief Technical Officer of Big Sur Scientific. He is the inventor of the BSS series of patented mid-infrared based cannabis analyzers. Dr. Smith has done pioneering research and published numerous peer-reviewed papers on the application of mid-infrared spectroscopy to cannabis analysis, and sits on the editorial board of 

. He has worked as a laboratory director for a cannabis extractor, as an analytical chemist for Waters Associates and PerkinElmer, and as an analytical instrument salesperson. He has more than 30 years of experience in chemical analysis and has written three books on the subject. Dr. Smith earned his PhD on physical chemistry from Dartmouth College. Direct correspondence to: 

Articles

Cannabis Analysis 

A number of misconceptions people have about how to test, what to test, and when to test in the cannabis industry are dispelled.

Dispelling Cannabis Analysis Myths, Part I

Extraction and refinement of cannabis has a wide range of steps and variables. Strong record keeping and integrity at each stage of the process creates a wealth of data. This data can be harnessed to better understand strengths and weaknesses within methods, equipment, employee performance, and source material. With data, better decisions can be made to improve efficiency and open the door to increased profitability.

Processing cannabis has many critical steps along the way to producing target cannabis extracts and final products. Many production facilities operate with inherited procedures. However, as operations continue to scale up and the industry moves closer to federal legalization it is vital to take into consideration standardizing processes and continuous process improvement. Without analyzing metrics and data at each stage in the process, it is a shot in the dark as to where to tackle optimization. In this column, we discuss the steps during cannabis extraction to better understand where and how to implement better record keeping and data analysis practices in your facility.

In Figure 1, general steps of cannabis extraction and refinement are outlined in a process flow diagram. While each extraction and processing facility may use different techniques, generally these are the main steps in producing refined cannabis extract. Accompanied with the process flow diagram in Figure 1 are vital metrics that can and should be tracked at each stage of the process. Each of these metrics can impact the end result of processing by way of quality, yield, and ultimately the cost to produce.

Cannabis extraction methodology has been developed enough such that there are now industry standard parameters for a generic extraction. Parameters such as pressure, temperature, input weight, and extraction time are some metrics that will impact the end result of an extraction. Each of these metrics can be manipulated independently to create a different result, whether that be an increase in efficiency or quality of the extract.

Some data points reveal much more than others with respect to the end product and overall process efficiency. Whether input materials are cultivated within the organization or sourced from outside the organization, it is important to analyze them for concentration of target compounds. For example, upon intake the cannabinoid and terpene potency and profile can be tested with an in-house or third party analytical testing device. This data sets the stage for what can be expected throughout the processing steps.

Understanding and regularly assessing extraction data establishes baseline performance for a technician, equipment, or input source. This baseline performance creates data-supported metrics for a facility to target. One term that is often utilized in the cannabis industry is “yield.” The term yield is consistently used to qualify value or quality. 

 is defined as “to produce or provide,” especially within agricultural and manufacturing industries. How much product is this plant going to yield? How much weight is this extraction going to yield? Cultivators, within or outside an organization, will comment on extraction yields for specific chemovars or negotiate a price on a lot of materials. This term is too vague to make a solid business decision as one can ask:

Why does this particular product yield well in extraction?

Is the yield defined as the bulk weight or the output of
target compounds?

In this industry, “yield” often means ‘how much extract was extracted from a set weight of biomass.’ See Formula 1 as an example.



 Extract weight (g)/Input weight (g) x 100% = Yield (%) 450 g extract/4500 g of biomass = 10%


In Figure 2, Formula 1 was used to calculate the yield as represented in the chart to demonstrate yield by batch of extracted material. Green circles indicate two areas of interest. One circle highlighting a series of batches that appear to represent a “good” yield and a second circle indicating a series of batches that appear to represent a “bad” yield.

A question remains unanswered: how much of the target compounds were extracted? If the target is tetrahydrocannabinol (THC), then it is important to know how much THC was present in the starting materials and how much THC was in the final extract. If the targets are terpenes and terpenoids, it is essential to know the concentration in the starting and final materials. This focused parameter can be referred to as 

. Formula 2 demonstrates how to calculate extraction efficiency based on the known concentration of target compounds.



 (Extract weight (g) X Potency of target compound in final extract)/(Input weight (g) X Potency of target compound in input material) = Extraction Efficiency (%)

600 g extract X 70% THC)/(4500 g of biomass X 10% THC) = 93.3%
extraction efficiency



From this metric, it is known what percentage of the target compounds were extracted. In Figure 3, the same batches are shown as in Figure 2 however the extraction efficiency was measured instead of basic yield. The same two circles are shown hovering over the batches that appear to be a set of “good” and “bad” yields. After looking at the extraction efficiency, it is clear that the “good yield” batches could have produced even more extract and the “bad yield” batches produced at their peak efficiency.

With these metrics, production facilities can make more informed decisions around sourcing material, cost of operations and product, and extraction parameters to optimize extraction and production for targets. There is ample opportunity for more granular data analysis on cost to produce or process optimization.

Some operators are using physical log books to keep records of batch run metrics while others are relying on traceability software for minimum required record keeping. Without easy data tracking systems and data analysis reporting, operators are unable to foster a team culture with data integrity at its core. Data must be consistently and accurately tracked to generate meaningful information. Extraction and refinement technology has advanced and automation has been integrated into multiple phases of the process. Many operators believe the data that is housed on automated equipment is a wealth of information. However, there are often no processes in place to filter, organize, and utilize the data. This is useless. I am confident that solutions will surface as organizations continue to scale up and search for ways to optimize efficiency.

In the next parts of this series, we will review the post-processing and refinement steps of producing cannabis extracts and how data during the extraction and refinement phase can translate into higher profit margins.

 is the founder, CEO, and Chief Extractor of Heylo. With a background in chemistry and clinical research, Lo was inspired to explore cannabis as a medicine and to enter the emerging industry. She joined Eden Labs, a leading CO

 extraction equipment manufacturer to support and expand a Research and Development department. There she managed the development of their latest and greatest CO

 extraction system. In 2017, after working with Eden Labs and another cannabis processor, Lo launched Heylo with a mission to help people get more out of life with cannabis.

Extraction Science

A look at how extraction data can be harnessed to better understand strengths and weaknesses within methods, equipment, employee performance, and source material.

Harnessing the Power of Data in Extraction and Refinement of Cannabis, Part I

In this installment of “Cannabis Crossroads” I interview Sheila Gibson, the founder of Aura IP Law. She started her career in intellectual property (IP) at a nationally recognized IP boutique and practiced at several top-tier law firms during her first 16 years as an attorney. She worked in the biotech industry prior to attending law school and pursuing a career in IP. She brings a unique combination of strong science and IP experience with a solid understanding of her clients’ business needs. Sheila is able to use her unique skill set and approach to provide more efficient and tailored legal services for her clients. Here, she shares her insights, knowledge, and advice on cannabis IP, including emerging topics.

Can you give a brief overview of how IP and patent law is applicable to the cannabis industry?

In every industry, cannabis included, a company’s most valuable assets are its unique strengths that provide a competitive edge over others in the same space. These strengths can include, for example, a new discovery, a new product, a secret formulation, a new product design, a new plant variety, a new method of making a product, a new method of treatment, manufacturing know how, a brand that consumers reach for, a great slogan, distinct packaging, a loyal following, client lists, or supplier lists. Whatever the strengths may be, every cannabis entrepreneur, innovator, and company should specifically identify them and have a plan in place for protecting each of these valuable assets. This is what intellectual property (IP) is all about—protecting the intangible assets that provide a competitive edge. Even though IP is protected at the federal level and most cannabis remains federally illegal, it is absolutely possible to obtain meaningful IP protection for all facets of the cannabis industry.

Could you share with our readers what led you to get involved and apply your skills to the cannabis industry?

Prior to focusing on cannabis IP, some of my biggest clients were in the cancer immunotherapy space. I was working with world-renowned cancer researchers to obtain global patent protection for their cancer immunotherapeutics. Working in patent law can be a grind. It’s very intense and deadline heavy and it’s quite easy to lose sight of your why. To stay connected to the bigger picture of the work I was doing, I would follow cancer patient journeys online to keep the patients and my contribution to improving their lives at the forefront.

Around 2015 I started following a pediatric patient account on Instagram, @prayersforsophie. Her parents, Josh and Tracy Ryan, were treating their infant daughter’s optic pathway glioma with cannabis. I followed Sophie’s journey in real time and was immediately captivated by her spirit, her energy, and her journey with cancer and cannabis. This ignited in me a desire to learn more about cannabis as medicine, which in turn fueled my desire to help facilitate patient access to cannabis.

With my practice being a federal practice and cannabis being federally illegal, it was not immediately clear to me how my skill set could be useful to the cannabis industry. However, as I continued to think through the issues, it became apparent that the industry not only needed IP protection but that meaningful protection could indeed be obtained with the right amount of determination, mental gymnastics, and legal maneuvering. While patent protection for cannabis innovation is fairly straightforward, including plant patent protection for cannabis cultivars (or strains), federal trademark protection requires “use in commerce,” which is commerce that is regulated by Congress. Given the federal illegality of cannabis, we are not always able to demonstrate “lawful” use in commerce for all goods and services offered even where those goods and services are being lawfully offered in a particular state.

IP serves to both foster innovation and protect consumers from confusion among brands. Fostering cannabis science research and having reliable and trusted cannabis brands will ultimately help create better patient access. This is what drives me to really listen and understand the unique issues the cannabis industry faces with respect to IP and to think through them creatively and through the lens of my extensive experience helping other industries protect their IP. Figuring out how to obtain meaningful IP protection before a federal agency for a federally illegal plant has sparked a passion in me unlike any other in my career.

What type of clients and projects do you currently work with in the cannabis space?

I currently work with innovators and startups in the cannabis space on their patent and trademark matters. On the patent side, I’ve worked with both ancillary and plant-touching companies on matters ranging from simple mechanical inventions to complex product and nutrient formulations. On the trademark side, I’ve developed comprehensive trademark strategies to obtain meaningful trademark protection of brands for cannabis operators, researchers, product developers, and innovators.

Can you talk a little about your science degree and how you utilize it in your work?

My science degree is in microbiology and molecular genetics and I had a brief stint in the biotech industry in a good manufacturing practice (GMP) manufacturing facility for a gene therapeutic for hemophilia patients. My technical background allows me to speak the same language as the innovators I represent and to get up to speed quickly on their new technologies so that I can develop an IP strategy specific to their ideas and business goals.

Having a science degree (or equivalent course work) is a requirement to become a patent practitioner. The United States Patent and Trademark Office (USPTO) requires all patent practitioners to demonstrate “the necessary qualifications to render to applicants or other persons valuable service, advice, and assistance in the presentation or prosecution of their applications or other business before the [USPTO]” (1,2). 

The necessary qualifications include both scientific and technical training and knowledge of applicable patent laws, rules, and procedures. Scientific and technical training is demonstrated through possession of a bachelor’s degree in science or engineering from an accredited university (or equivalent) and knowledge of patent rules and procedures is demonstrated by passing a rigorous examination, the “patent bar.” While you have to demonstrate scientific and technical training in order to sit for the patent bar, you do not need to be an attorney. Patent agents are non-attorney patent practitioners who possess the requisite scientific and technical training and have passed the patent bar.

What emerging topics or new areas of interest are you seeing in the cannabis IP and patent law space? Is there anything our readers should be aware of?

The question I’m probably asked most frequently is whether it’s actually possible to patent a cannabis plant and the answer is yes, asexually reproduced cannabis plants. A patented plant must differ from known plants by at least one distinguishing characteristic that is more than that caused by different growing conditions or fertility. There is nothing in US patent law that excludes cannabis plants from being patented. While a plant patent protects against asexual reproduction of genetically identical clones, it does not prevent a patented plant from being used for breeding. Thus, plant patents are most useful in combination with strong IP licensing agreements to control the activities of those you trust your plants to. A search of USPTO patent records revealed at least 25 issued plant patents and almost 50 pending plant patent applications that include the term “cannabis.”

Utility patent protection is also available for cannabis innovation. In fact, the United States government holds a now-expired patent for a method of treatment that dates back to 1998. So far this year, I am seeing an uptick in cannabis patent filings generally.

The most common cannabis IP myth I encounter is that trademark protection is not available for cannabis due to the “use in commerce” requirement. While it is still not possible to obtain federal trademark protection for certain plant-touching goods and services, a lot has opened up in terms of being able to obtain meaningful trademark protection at the federal level even for plant-touching businesses. Where initial cannabis trademark strategies involved obtaining protection for apparel, we are able to obtain protection for services such as providing information, including information about consuming, growing, processing, and infusing with cannabis, or about cannabis products. It is also possible to obtain state trademark protection for plant touching goods and services in many states where cannabis is legal. If your cannabis trademark strategy started and ended with apparel, it’s time to revisit your trademark portfolio.

As for trends in cannabis IP litigation, we are seeing a lot of litigation brewing around trademarks. Initially, the litigation primarily involved established and famous brands suing cannabis parody brands (such as Gorilla Glue, UPS, Tapatio, and most recently, Skittles). We are now seeing cannabis brands sue each other over similar marks and partners within the same entity sue each other over rights to their existing trademarks.

While there is still an outstanding question about the enforceability of cannabis patents in federal courts, thus far, courts have moved forward with lawsuits involving cannabis patents without throwing them out based on ineligible subject matter. Even if cannabis patents were to be deemed unenforceable due to the current federal status of cannabis, patents are enforceable for 20 years from their earliest filing date. Provided the federal status changes within the 20-year term, there is still a lot of value to be had from early cannabis patents.

Do you have any advice for anyone interested in a career in cannabis IP or patent law?

One of the most exciting things about a career in patent law is the ability to work on a wide variety of technologies even within a single sector, such as cannabis. You must be able to get up to speed very quickly on a new invention and be able to describe the invention in words and drawings in a way that meets all of the patent requirements. On any given day, you may be working on several completely different technologies and you must be able to switch between them easily. In my experience, most scientists who become patent practitioners are those who love science, but did not enjoy being in a laboratory setting or being devoted to a single subfield of science.

Patent law can be very demanding, requiring you to juggle multiple inventions of differing technologies, each with its own unique set of issues and non-extendable deadlines. Missing patent deadlines can have dire consequences, so organization and attention to detail are key. In addition, as a patent practitioner, you bridge the gap between science and law. Scientists and researchers can become frustrated with the tedious legal requirements of patent procedure and a successful patent practitioner must be able to communicate effectively with scientists and researchers in a way that makes the process understandable for inventors. On the other end, patent examiners may be unfamiliar with an inventor’s technology making examination of the application unnecessarily difficult. Patent practitioners must be able to communicate with patent examiners in a way that helps them understand the invention quickly.

Anyone thinking about a career in cannabis patent law should start with a degree in a science (such as biology, botany, chemistry, molecular biology, or microbiology) or engineering (such as agricultural engineering, biomedical engineering, chemical engineering, electrical engineering, or mechanical engineering). From there, I recommend finding opportunities to gain familiarity with patents and the patent process whether through work experience with a technology transfer office, an in-house position with a biotech company, a patent scientist position with a law firm, or through law school. Law firms will often train qualified candidates without having yet passed the patent bar.

Do you have any closing thoughts to share with our readers about IP, patent, and plant law?

First, all IP protections—patents, trademarks, copyrights, and trade secrets—are available to the cannabis industry. Even where IP rights, such as trademark rights, are not available for plant-touching goods and services, meaningful protection is still available for other aspects of cannabis businesses.

Second, publicly disclosing your invention or bringing it to market before having IP protections in place can waive your rights to certain types of IP protection. Thus, it is extremely important to at least have the conversation about IP before any public disclosure or offer for sale. It is always best to know you are forgoing certain rights than to inquire about them later only to find out you are no longer eligible to obtain protection. Ideally, you should be having IP conversations either internally or with an IP professional as early in the process as possible and certainly before going to market or otherwise publicly disclosing your IP.

Third, even if you never plan to enforce your patent rights, keep in mind that your competitors may be obtaining patents that may someday be enforced against you. While the US patent system was previously a first-to-invent system—meaning if you could prove you invented first, you could obtain a patent to an invention—the US moved to a first-to-file system in 2013. It is now a literal race to the patent office. Unlike trademark rights, obtaining patent protection does not require you to enforce your rights in order to keep them. Be sure to consider patents as a defensive strategy as well. With the right patents in place, a potential threat of litigation from a competitor could turn into a cross-license situation. There is so much more to intellectual property than meets the eye!

United States Patent and Trademark Office (USPTO), 35 U.S.C. § 2(b)(2)(D). 

https://www.uspto.gov/web/offices/pac/mpep/consolidated_laws.pdf

https://www.uspto.gov/web/offices/pac/mpep/mpep-9015-appx-l.html

 is the columnist and editor of “Cannabis Crossroads”
and a contributing editor to 

 magazine.
Crossney is also the president and CEO of Cannabis Science Conference.

 is the founder of Aura IP Law, an intellectual property (IP) law firm focused on the IP needs of entities specializing in cannabis, life sciences, medical devices, cosmetics, food and beverage, retail, and sports innovation. Prior to law school, Sheila studied microbiology and molecular genetics and worked in the biotech industry manufacturing gene therapeutics in a GMP facility. After law school, she began her career in IP at a nationally recognized IP boutique and practiced at several top-tier law firms prior to starting her own firm. She has counseled clients around the world and at every stage from startup to large, publicly traded companies. She advises her cannabis clients in all aspects of IP, including patents, trademarks, copyrights, branding, licensing, IP transactions, IP agreements, opinion work, due diligence, product launch, and pre-litigation strategy for trademark ownership disputes.

https://www.auraiplaw.com/sheila-gibson-esq

Cannabis Crossroads

Sheila Gibson, the founder of Aura IP Law, shares her insights, knowledge, and advice on cannabis IP, including emerging topics.

Intellectual Property and Cannabis: A Look at the Current Landscape and Emerging Topics

The current state of hemp regulations in the United States begs the question: why is the current hemp compliance threshold set to an arbitrary value of 0.3% w/w total tetrahydrocannabinol (THC)? This represents a lack of understanding of Group III cannabis/hemp genetics, which is causing farms, extractors, and ancillary businesses two- to three-times as much capital to get the same results in an oil or isolate product. This also impacts land use, nutrients application, and pesticide use; ultimately having deleterious effects on the environment. Our extensive research into hemp genetics has revealed that across the board a simple increase in cannabidiol (CBD) also causes a proportional increase in THC, irrespective of provenance. This increase does not affect the final potency of raw oils as extraction equalizes to the same ratio; however, the current compliance threshold of 0.3% total THC hinders the amount of oil that can be produced from a given acreage. As such, everyone in the industry is being punished by archaic laws that have little to no real-world application in the modern hemp markets. Farmers, breeders, and extractors should not be punished for creating and cultivating superior genetics. Isolated CBD, full-spectrum oil, and even smokable hemp are all being diluted down by a draconian regulatory framework that needs to be changed to facilitate growth, innovation, and prosperity throughout the industry. 

For the better part of the last 10 years 

 varietals have been categorized as either 

 being more closely associated with the latter two sub-species. Nonetheless, the widespread hybridization that has resulted from systemic interbreeding of these varietals has led to a comparatively new classification system, whereby the relative expression of Delta-9-tetrahydrocannbinol (D

-THC) and cannabidiol (CBD) dictates a particular strain’s sub-type. For example, Group I cannabis refers to THC-dominant strains (>20:1 THC/CBD); Group II cannabis corresponds with ratioed genetics (~1:1 THC/CBD); and Group III cannabis (hemp) is comprised of CBD-dominant strains (>20:1 CBD/THC). Interestingly, Groups I–III cannabis can all express similar morphological structures (Figure 1), flavonoid and terpene profiles; with the primary differentiating factor being the final concentration of THC. Additionally, all three Groups can be observed coexisting together in putative wild landrace strains. This is to say even with the inbreeding of, for example, two Group II landrace plants for the production of “selfed” feminized seed that is homogeneous, the progeny will follow the principles of Mendelian inheritance, whereby 25% will be expressed as Group I cannabis, 50% will resemble Group II, and 25% will be representative of Group III cannabis (hemp). This particular genetic feature of cannabis and hemp presents a whole other issue plaguing the hemp industry; however, the topic of genetic stability of commercially-available hemp seed is a discussion for another day.

While the regulation of Groups I and II falls under the jurisdiction of cannabis; the regulation of Group III genetics is somewhat variable depending on the final THC content of the cured flower material. For example, prior to the recent changes to the United States Department of Agriculture’s (USDA) hemp guidelines in October 2019, the threshold for determining whether or not a plant was considered hemp was a D

-THC concentration of 0.3% weight/weight (w/w). However, the aforementioned changes maintained the value of 0.3% while now requiring a cumulative measure of total THC, which is comprised of the amount D

-THC plus the amount of its acidic precursor, tetrahydrocannabinolic acid (THCA), accounting for the loss of mass during decarboxylation (total THC = D

-THC + 0.877*THCA). This has left the American hemp industry in a highly restricted state, whereby most commercially-available genetics are no longer compliant and thus are considered THC-rich cannabis, which inherently requires different permitting and licensure.

In fact, our own research indicates that because CBD and THC levels are linearly correlated in Group III cannabis, it is nearly impossible to produce compliant hemp material with a CBD concentration greater than 10% w/w (Figure 2). By comparison, the hemp regulations in select European markets such as Switzerland allow for up to 1.0% w/w total THC. This in turn presents Swiss cultivators with the opportunity to produce much more phytochemically dense flower material, with compliant genetics exceeding 25% w/w CBD in many cases. These more favorable regulations also allow for the enhanced expression of secondary cannabinoids such as cannabichromene (CBC) and cannabichromenic acid (CBCA), which demonstrates multicollinearity with CBD and THC (Figure 1). Additionally, the more accommodating regulations, whether they be 1.0% total THC or 0.3% D9-THC, allow for a greater expression of ancillary cannabinoid species such as cannabidivarin (CBDV) and cannabidivarinic acid (CBDVA), tetrahydrocannabivarin (THCV/THCVA), and cannabicyclol (CBL) and cannabicyclolic acid (CBLA) as these molecules are expressed in the same fragile, hair-like trichome structures that produce and house CBD and THC. As such, the more restricted hemp regulations (0.3% total THC) result in the production of less efficacious plant material, which is a tremendous disservice to both the cultivator and the end user.

Financial, Operational, and Environmental Implications

While the regulation-mediated reduction of phytochemical content of American hemp is certainly an injustice to the medicinal value of this newly-founded commercial crop, the pains associated with the new 0.3% total THC regulation also resonate with commercial growers. As illustrated in Table I, the amount of CBD isolate that can be derived from a given acreage is directly correlated with the CBD content in the plant material being cultivated. And while our own research has revealed that a final CBD concentration of 10% w/w represents the limit of compliance under the 0.3% total THC guideline, many farmers aren’t willing to push this threshold to potentially face regulatory enforcement (controlled crop destruction, fines, or incarceration), and as such produce safe, yet inferior, plants (Table I, left scenario). Unfortunately, this has a tremendous impact on operations, logistics, expenditures, and the environment. Growing “weaker” plants requires that a larger acreage be utilized to produce enough CBD for a set amount of isolate. This equates to large capital expenditures (real estate, equipment, labor), and the use of more water, nutrients, and power. For example, those operating under the jurisdiction of the 1.0% total THC guideline are effectively able to produce as much as five times the amount of hemp-based medicine in a given space compared to those following the 0.3% total THC requirement (Table I). Outdoor cultivation is often regarded as a more environmental-friendly form of hemp cultivation compared to indoor or greenhouse cultivation based on the simple fact that supplemental lighting and HVAC are not required to grow hemp in the open atmosphere. Nonetheless, the need to occupy more land, use more water, and spread more exogenous nutrients to facilitate the cultivation of 5–10% CBD hemp, almost negates the environmental stewardship of outdoor cultivation.

Additionally, larger commercial cultivation facilities present a larger degree of risk. Inherently, larger farms are more susceptible to rogue pollination events than small farms; the occurrence of which can severely compromise yield and phytochemical production as pollinated plants will divert cellular resources to the production of seeds as opposed to the expression of cannabinoids and terpenes. The management of larger allotments of vegetative matter also has a greater potential for spoilage and loss in the harvesting, drying, and curing phases. This very phenomenon has been an Achilles’ heel in the American hemp industry whereby several entities have experienced significant crop losses due to inexperience with large scale drying.

Lastly, the production of less phytochemically-dense hemp material also makes distribution and logistics a challenge for farmers. If the plant material is produced with the intention to generate oils, concentrates, or isolates, then many extractors will demand a much more substantial split of any profit sharing since the margins to produce a viable end product are so thin. Conversely, if the hemp flower is cultivated for sale as “smokeable” flower, every percentage point of CBD or total terpenes that is lost renders the commodity less valuable.

Collectively, the implementation of the 0.3% total THC regulation in the American hemp industry has made the production of CBD-rich hemp more expensive, more precarious, and less lucrative for farmers. Generally, the goal of having regulatory framework in place is to help facilitate the production of consistent products that are reliable and safe. However, when the regulatory entities promulgate new regulations that are unsubstantiated and are not based on the latest scientific data, we find ourselves in a perilous situation that hinders quality, dissuades innovation, and makes hemp cultivation seem like a fool’s errand. Our hope with this commentary is to convey the importance of taking genetics and available horticultural data into account when trying to regulate this new frontier of medicine. Implementing more accommodating cultivation requirements, such as the 1.0% total THC requirement utilized in parts of Europe, will not adversely affect product safety or increase the risk of producing hemp products that trigger significant psychoactivity. On the contrary, these more favorable guidelines present farmers with a greater opportunity for success, and would make hemp cultivation a more sustainable pursuit from both a financial and environmental perspective.

Feature

Isolated CBD, full-spectrum oil, and even smokable hemp are all being diluted down by a draconian regulatory framework that needs to be changed to facilitate growth, innovation, and prosperity throughout the industry.

Hemp Regulations and the Restrictions on Innovation

As the emphasis on sustainability grows to battle climate change, grow house owners want to improve their energy-efficiency and reduce energy waste. Energy-efficient cooling equipment vastly improves grow house sustainability and even saves operating costs. To develop a cooling strategy that reduces energy waste, cooling experts consider everything from high-tech, automated controls to the heating, ventilation, and air conditioning (HVAC) equipment itself. For indoor growers to maximize sustainability in their farms, they can invest in systems that save more water and more energy than others. This article outlines types of cooling equipment that contribute to sustainability and energy-efficiency; how to develop a customized cooling strategy that meets growers’ sustainability goals; and steps growers can take to simultaneously improve energy efficiency and operating costs.

As the world seeks ways to tackle climate change, sustainability has become increasingly popular, not just for large corporations. Although sustainability may not be the primary consideration for all growers shopping for heating, ventilation, and air conditioning (HVAC) equipment, there are also business reasons to strive for greater energy efficiency.

HVAC equipment and lighting tailored to indoor legally licensed cannabis production can help decrease a grower’s carbon footprint while minimizing disruptions and expense from future equipment upgrades to replace under-performing off-the-shelf equipment.

The concern is growing over the substantial electrical needs of the cannabis industry. Many believe it’s just a matter of time until municipalities and states implement regulations aimed at increasing energy efficiency. Forward-thinking growers and their investors can anticipate regulations and be energy-efficient, which saves operational costs. Efficiency can also translate into a competitive advantage in some saturated sales markets experiencing downward pricing pressures.

At present, a grow operation can feasibly become carbon-neutral by adding renewable (solar or wind) energy, either on-site or remote. But as with any lofty goal, taking steps—even small ones—will move a grower closer to carbon neutrality. Here we present seven key steps to staying ahead.

Anticipate regulations: Get familiar with your municipality’s or state’s sustainability goals

An October 2018 report from New Frontier Data identified cannabis as the most energy-intensive agricultural crop produced in the United States (1). The report predicted a 162% increase in electricity consumption from cannabis cultivation between 2017 and 2022, with an estimated 472,000 tons of electricity-related carbon produced in 2017 alone.

The rapid growth of energy-intensive indoor cultivation facilities has strained the infrastructure of electric utilities. In the summer of 2015, shortly after Oregon legalized recreational cannabis, Portland’s Pacific Power experienced seven blackouts traced to cannabis production facilities, according to an article by the National Conference of State Legislatures (2). The piece also noted that 45% of Denver’s increase in energy demand is for electricity to power marijuana facilities.

Because the expanding cannabis cultivation industry is drawing attention to its energy use, it’s a matter of when, not if, regulators across the country implement efficiency standards to manage energy use.

Implement LED lighting and other efficient technologies

California’s Building Energy Efficiency Standards (Title 24, Parts 6 and 11) is updated every three years, with the next code change slated for January 1, 2023. One expected change would prohibit energy-intensive lighting technologies, such as high-pressure sodium (HPS) and high-intensity-discharge (HID), in favor of light-emitting diode (LED) lights for all new installations or significant upgrades.

LED grow lights reduce a grow operation’s electricity use in two ways. One, they use less electricity by having greater luminous efficacy, or how well a light source produces visible light, measured in lumens per watt. Second, they also produce less heat than legacy designs, resulting in less heat that must be removed by air conditioning, which saves on cooling
operational costs.

Invest in cooling and dehumidification strategies to improve dividends

It can tempt some growers to take the easy way out and purchase HVAC equipment off the shelf. It’s readily available, less expensive, and there are fewer options to consider.

But HVAC equipment that isn’t designed for indoor agriculture is often not up to the task of maintaining a low enough humidity in the facility, which is necessary to prevent mold, micropests, and poor yields. So, a grower often resorts to buying multiple portable dehumidifiers at a big box store. As a result, the HVAC system “fights” to remove the heat output of the dehumidifiers.

Lower costs of equipment and installation, known as “first costs,” quickly turn into long-term headaches and additional operating expenses. Instead, consult with an HVAC system expert who’s aware of an indoor grower’s unique needs. Collaboratively, they can design an integrated system that performs better and uses less electricity, which adds to its bottom line while aiding sustainability.

Purchase equipment specified for a grower’s unique needs

As an example of purpose-built equipment, one type of unit designed for a smaller indoor agriculture operation is a packaged rooftop unit. Such packaged rooftop units include all equipment needed in one unit and are available in varying capacity ranges, capable of removing up to 600 lbs of water per hour. They can also be paired with modulating hot gas reheat, which means the heat produced as compression during the dehumidification process is recaptured to use as building heat instead of being lost to the ambient environment.

That said, there’s not a one-size-fits-all solution. Facility size, number of plants, and watering strategy are all parameters that factor into the appropriate equipment choice. For some large facilities needing high cooling capacity, it will be more cost-effective to install and operate an efficient chilled-water system rather than a “sea” of rooftop units.

Collaborate with a cooling and dehumidification expert

Assess your needs as a grower. Have realistic goals for the facility’s needs. During a needs assessment consultation with an HVAC expert, be upfront about operational plans for the facility. The building configuration, including how to designate rooms for various growth cycles, will influence how to equip it.

Growers are often reluctant to share too many details of their environmental recipe for fear they’ll share their “secret sauce,” but this hampers the supplier’s ability to install the equipment that’s just right for them. More space and more plants mean more heat and humidity that must be removed, requiring equipment of the proper capacity.

Use automation controls to boost energy efficiency

Electric utilities often implement peak demand charges, charging a higher rate when there is a high demand on the electrical grid. Because most of the grow processes can be scheduled when electricity costs are lower, automated controls can automate this instead of relying on staff to turn switches on and off manually.

Day cycles, with lights on, use much more energy than night cycles. The mechanical and electrical systems must have adequate capacity to handle the maximum number of rooms in a day cycle at any time. By staggering the day cycles between growing rooms, the cultivator can dramatically reduce peak demand and system capacity. Many utilities offer cheaper rates at night and peak demand charges during the day. Electricity available at night is not only cheaper it is typically “cleaner” because the overall demand on the grid is lighter and peak plants are not being used to produce electricity at night.

Using controls helps reduce electrical demand charges and allows a grower to capitalize on the most affordable time of day electricity rates.

Use a building automation system (BAS), a web-based user interface, to streamline facility management, scheduling, and turning on and off equipment. A BAS is accessible from most PCs, tablets, and smartphones and it’s a flexible, cost-effective solution for programming and managing the facility conditions. It can also control lighting, monitor energy consumption, and integrate with equipment that controls other processes, such as carbon dioxide (CO2) injection and fertigation of the plants.

Make a plan now to strive to be carbon neutral

Growers weighing their HVAC options should look for a supplier that values sustainability. Does it publicize those goals? A supplier focused on sustainability likely designs products with a priority on high efficiency, and they likely ensure that their supply chain partners also employ sustainable practices. Aligning with such a supplier can help growers meet their green goals and help them market to their investors and customers.

Even if sustainability is not part of the company’s social responsibility goal or mission statement, growers should plan now to get as close to carbon-neutral as possible. Investing in systems that have a lower cost of ownership over a long period of time can ultimately reduce electricity costs, adding to the bottom line. The effort also makes you more resilient to potential future regulatory changes.

https://newfrontierdata.com/product/2018-cannabis-energy-report/

J. Durkay and D. Freeman, “Electricy Use in Marijuana Production,” 

National Conference of State Legislatures

 optimizes indoor agriculture environments by providing expertise on cooling strategies, automation controls, energy-efficiency, and services. In collaboration with Trane engineers, he designs systems for indoor growing facilities that help achieve high-yield growing environments and energy efficiency.

This article outlines types of cooling equipment that contribute to sustainability and energy-efficiency; how to develop a customized cooling strategy that meets growers’ sustainability goals; and steps growers can take to simultaneously improve energy efficiency and operating costs.

Is Carbon-Neutral Cannabis Possible?

Cannabinoids can be analyzed using different techniques. The aim of this review was to identify and compare analytical methods used for the determination of cannabinoids in different matrices using liquid chromatography (LC)-based systems. A systematic literature review was carried out using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) method. In the results, 41 relevant articles were identified. The most commonly used methods for the analysis of cannabinoids were high performance liquid chromatography-photodiode array (HPLC-DAD) (n= 8), ultrahigh-pressure liquid chromatography–mass spectrometry (UHPLC–MS) (n= 8), and HPLC–tandem mass spectrometry (HPLC–MS/MS) (n= 8). Matrices from which cannabinoids were extracted included plants, oil, hair, human biological fluids, resin, honey, wastewater, and commercial products (n= 41). The most commonly used stationary phases were C18 Poroshell (n= 9) and C18 Kinetex (n= 8). The identification and comparison of methods used for the determination of cannabinoids can help in the development of more efficient and effective methods of analysis.

 is an annual dioecious flowering plant (2) known for its medicinal and textile uses since ancient times (1,3). 

 contains chemically active compounds called cannabinoids, which have a wide range of therapeutic effects in humans (3). Medicinal uses of cannabinoids include management of spasticity related to multiple sclerosis (MS), chronic neuropathic and cancer pain, nausea and vomiting, sleep disorders, anxiety, epilepsy, and Tourette syndrome (4).

The principal cannabinoids known to have medicinal properties are 

-tetrahydrocannabinol (THC) and cannabidiol (CBD) (5). THC and CBD are synthesized and accumulate in their acidic form in 

 (6). The alkylation of olivetolic acid (OLA) with geranyl-pyrophosphate (GPP) by olivatolate geranyltransferase produces cannabigerolic acid (CBGA) (3,4,7). The catalysis of CBGA by three oxidocyclases—

-tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), and cannabichromenic acid synthase (CBCAS)—produces 

-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromenic acid (CBCA), respectively (3,4,7). The decarboxylation of THCA, CBDA, and CBCA due to high temperatures (8) produces THC, CBD, and cannabichromene (CBC), respectively (4,8). Cannabinol (CBN) is produced as a result of oxidation of THC (4,8) and is a sign of deterioration of the plant (9).

THC is the main psychoactive component in cannabis and has been used in the management of chemotherapy-induced nausea and vomiting, for appetite stimulation in patients with acquired immunodeficiency syndrome (AIDS) (10), for suppressing spasticity related to MS (6), and in the treatment of migraines (1).

CBD is known to have the largest number of therapeutic properties (7) and is the main nonpsychoactive component in cannabis (11). CBD presents potent antioxidant and anti-inflammatory properties (6). CBD has anticonvulsive, neuroprotective, anxiolytic, antipsychotic, and antidepressant properties (12). CBD is used principally in children in the treatment of drug-resistant epilepsy, Dravet and Lennox-Gastaut syndromes (13,14).

The endocannabinoid system has two principal receptors CB1 (type-1) and CB2 (type-2) (15) connected to G-proteins, endogenous cannabinoids called arachidonoylethanolamine (anandamide) and 2-arachidonoyl glycerol (2-AG) and enzymes that are involved in synthesis and degradation of endocannabinoids (9,16). CB1 receptors are present in different regions of the human brain (17). Distribution of these receptors are in areas involved in cognitive function and mood (4,17). CB1 receptors can be also found in the liver, testes, and small intestine (16).

There are different analytical techniques for the determination and quantification of cannabinoids (10,18). Gas chromatography (GC) has been the method of choice for analysis of cannabinoids (10), but chemical derivarization is required to avoid decarboxylation of acid cannabinoids (18). Liquid chromatography (LC) allows determination of cannabinoids in neutral and acidic forms without the need for derivarization. LC has become more popular with the introduction of high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) (7,18,19). LC, HPLC, and UHPLC can be coupled to different detectors: fluorescence, diode-array detection (DAD), mass spectrometry (MS), or an ultraviolet (UV) detector (20). The use of MS coupled to HPLC and UHPLC increases the selectivity and the sensitivity of analysis (7), but the cost is higher and requires more skilled expertise to operate (20). CBN does not have a fluorophore and therefore use of a fluorescence detector is unfavorable (2). HPLC and UHPLC coupled to a UV-visible detector is a method commonly used because it can be economic and more convenient than other methods of analysis. DAD offers a range of detection wavelengths but can be more expensive than UV (21). The aim of this study was to conduct a systematic literature search to compare and identify analytical methods and parameters used in the determination of naturally occurring cannabinoids in different matrices.

A systematic literature review was carried out using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) method. (Note: PRISMA. Transparent reporting of systematic review and meta-analysis. [Internet] PRISMA, 2021 [cited 2021, 23 April] Available from: 

http://prisma-statement.org/PRISMAStatement/FlowDiagram

.) The systematic literature review included methods used for separation and determination of cannabinoids using LC. Sources included open access peer-reviewed journal articles published in English between the years 2015 and 2020. Databases used for the literature search were Pubmed and Scopus. Keywords used in the search were: analysis, cannabinoids, cannabis, tetrahydrocannabinol, cannabidiol, cannabinol, and LC. Data collected was presented in tables, according to the matrix in which the cannabinoids were presented. Data in each table compared the type of matrix, cannabinoids analyzed, sample preparation method, stationary phase, mobile phase, and detector.

In the study, 41 articles were identified. The articles were classified depending on the matrices used for the analysis: 18 articles analyzed cannabinoids from plant material and four articles in plant material and other matrices. Ten articles analyzed cannabinoids from biological fluids and hair, and one article from biological fluids and hair and other matrices. Four articles analyzed cannabinoids in oil, and four articles, in oil and other matrices. Ten articles analyzed cannabinoids from miscellaneous matrices.

Methods of Analysis of Cannabinoids from Plant Material

LC, HPLC, and UHPLC have been performed for the separation, determination and quantification of different cannabinoids in 

. The samples included aerial parts of the plant (n=1), male and female inflorescences (n=5), leaves (n=2), roots (n=1), colas (n=1), resins (n=1), buds (n=2), and flowers (n=8) (7,22–42). HPLC was the most popular analytical technique used for the analysis of cannabinoids in plants (n=13) (22–27,32,34,36,38–42). HPLC can be coupled to UV, DAD, MS, or fluoresence detectors (20).

The detectors most commonly used for the analysis of cannabinoids in plant material were UV or DAD (n=13) (23–27,32,34,36,40,41). For example, Križman completed a study using HPLC-UV (34). Križman carried out a simple isocratic HPLC method for the analysis of THC, CBD, CBN, cannabigerol (CBG), THCA, tetrahydrocannabivarin (THCV), CBGA, CBDA, and d8-THC. The mobile phase consisted of water and actonitrile (ACN) in the ratio of 9:31 (v/v), with 0.1% formic acid (v/v) and 10 mM ammonium formate, using a Luna C18 (150 mm × 3 mm i.d., 3 µm) column and UV at 275 nm (34).

In recent years, UHPLC has become more popular (n=7) because of the small quantity of solvent needed in the mobile phase and a shorter analysis time (43). Bala and colleagues carried out a study using UHPLC coupled to MS to detect THC, CBD, and THCA which are present in large amounts in the cannabis plant and have therapeutic properties (22). The most commonly used detector to analyze cannabinoids from plants with UHPLC was DAD (n=3) (7,28,31). Elkins and colleagues analyzed THC, CBD, CBN, CBDA, CBC, and THCA using a simple method consisting of a mobile phase based in water containing 0.1% formic acid (HCOOH) and ACN containing 0.1% HCOOH (ranging between 40–100%) with gradient elution mode using a Phenomenex Luna Omega C18 (150 × 2.1 mm × 1.6 µm) column and DAD detection monitored at 280 nm (7).

While the use of DAD as a detector for HPLC and UHPLC is quite common, combined detectors such as UV-DAD, ESI-MS, and MS/MS have also increased in their popularity. One of the advantages of using ESI-MS or MS/MS is that the analysis can be performed in negative and positive ion mode. Neutral cannabinoids give a better signal in the positive ion-mode while acidic cannabinoids give better signal in the negative ion-mode (24). Brighenti and colleagues developed a method for the analysis of nonpsychoactive cannabinoids using the three combined detectors (24).

Other methods used for the analysis of cannabinoids were a fast-HPLC-DAD (25), UHPLC-travelling wave ion mobility (TWIM)-MS (30), and HPLC-Q-Exactive-Orbitrap-MS (39). Burnier and colleagues analyzed THC, CBD, CBN, and THC-A in a total run time of less than 5 min using a fast-HPLC-DAD method that could be an alternative to UHPLC but with a lower cost (25).

LC presents less sensitivity than HPLC and UHPLC. The use of LC requires less expensive and simpler equipment (33). Dong and colleagues developed a thermal desorption direct analysis in real time mass spectrometry method and compared the results with those obtained using a simple LC–MS (29).

One of the limiting factors in the analysis of compounds using an LC system is the solubility of cannabinoids prior to analysis. The analysis of cannabinoids from plant material using LC, HPLC, or UHPLC requires an extraction method to determine the presence of cannabinoids qualitatively and quantitatively (2).

Solvent extraction is the most commonly used analytical extraction method to extract cannabinoids from plant material. In our findings, 20 studies used solvent extraction as a part of the sample preparation method for the analysis of cannabinoids. The most commonly used solvents for the extraction of cannabinoids from plants are ethanol (EtOH) and methanol (MeOH), used in 7 out of 22 and 7 out of 22 studies, respectively. Ethanol is an organic solvent commonly used because of its higher eco-friendly behavior, even if it is more viscous than MeOH (31) and due to its high extraction efficacy because of its high affinity for the molecular structure of cannabinoids (44). MeOH is also commonly used because it presents a high extraction efficiency (24). Other extraction methods with solvents make use of ACN or a mix of solvents. Deville and colleagues performed the extraction technique with a mix of methanol/chloroform (90:10 v/v). The long-term use of chloroform by the analyst can cause liver and kidney injury to the operator of the method of analysis. Reducing the use of chloroform will increase safety in the laboratory and decrease costs of reagent disposal while improving the impact in the environment (36).

Sample preparation is usually accompanied by dynamic maceration (DM), which consists of extraction of analytes of interest from plant material using a solvent and vortex or stirring at ambient temperature (24). Brighenti and colleagues compared four different extraction techniques DM, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE). UAE and MAE are extraction techniques that use ultrasound waves and microwave energy for a faster and higher extraction of the secondary metabolites of cannabinoids (24). SFE is a more environmentally friendly technique than the extraction techniques of cannabinoids from plant material that use organic solvents (24). Elkins and colleagues extracted the resin from cannabis using a biobotanical SFE liquid CO2 extractor (7). DM is the best method to extract acidic cannabinoids such as CBDA and MAE for CBD (24). Ribeiro Grijó and colleagues carried out the extraction process using solid phase extraction (SPE) with supercritical carbon dioxide (scCO

) avoiding trace of organic solvents in the sample prepared (41).

The majority of the analysis of cannabinoids in plant material were carried out using an Agilent system (n=11) with different modular model systems (7,23,24,26,27,32,35–37,40,42). Among those studies using Agilent systems, the modular models 1100 and 1290 were the most popular and were used in three studies: (23–25,40,7,32,37) two studies used modular model 1200 system (35,36), one study used modular model 1260 system (42), and another study used modular model 1220 system (27). Ciolino and colleagues conducted the analysis using Agilent 1100, 1200, or 1260 HPLC-DAD systems (26). Another HPLC unit used was the Waters system, this unit was used in four studies (22,25,28,30). Other HPLC systems used were Thermo LTQ XL by Dong and colleagues, Finnigan Surveyor by Križman, and Nexera LC20AD XR system by Palmieri and colleagues (29,34,38).

The majority of the studies used C18 Poroshell (n=4), Kinetex (n=3), and Ascentis (n=3) columns. Gradient mode elution of the mobile phase was the most common method chosen for the analysis of cannabinoids from plants and only four studies out of 22 used an isocratic mode of elution. The majority of the mobile phases are composed of water and an organic solvent (n=16), usually MeOH and ACN. ACN was preferred because it decreases the total run time with respect to MeOH. The flow rate of the mobile phase ranged from 0.3 mL/min to 3 mL/min, with 0.4 mL/min (n=5) and 0.3 mL/min (n=5) being the most commonly used.

Methods of Analysis of Cannabinoids from Biological Fluids and Hair

Cannabis can be determined in biological fluids and hair (45). The analysis of cannabinoids in human fluids is important to understand their pharmacology in humans and to be able to establish the correct dosage (46). The availability of analytical techniques to detect and quantify THC in blood, saliva, hair, and urine is necessary to demonstrate consumption of illicit preparations (47).

The concentration of THC and its metabolites from blood and urine depends on the amount and route of administration and the time of analysis following consumption (48).

LC, HPLC, and UHPLC methods are used for the analysis of cannabinoids from different biological fluids (46,48–56). Six studies were carried out using UHPLC, three used HPLC, and one study by Toennes and colleagues used an Agilent 1290 Infinity LC system. Analysis was performed using a Kinetex XB-C18, 100 Å, (100 × 2.1 mm) column with a gradient mode mobile phase composed of 0.01% formic acid with 5 mM ammonium formate and ACN with 0.1 % formic acid ranging between 50–100%, the flow rate was 0.5 mL/min (53). The samples included urine (n=4), hair (n=1), human plasma (n=3), human serum (n=2), blood (n=1), and sweat (n=1) (46,48–55).

The detector most commonly used in the analysis of cannabinoids from biological fluids was MS/MS (n=9) because it presents higher selectivity and sensitivity allowing for the detection of major and minor cannabinoids in small quantities (2,7). THC and its metabolites (THC-OH and THC-COOH) are, in general, the cannabinoids analyzed in blood and urine because of the psychoactive effects of THC (2). Analysis of other cannabinoids such as CBD, CBN, CBG, CBDV, and CBDA were also identified in the literature (46,50–54,56).

Sample preparation is an important step in the analysis of compounds from biological fluids and has an effect on reproducibility, efficiency, and selectivity and eliminates interferences (2). Different techniques were performed to extract cannabinoids from biological fluids and hair. Protein precipitation (PP) is a popular technique used for the sample preparation in blood and can eliminate up to 98% of the protein (57). Dybowski and colleagues, Dziadosz and colleagues, and Klawitter and colleagues, carried out protein precipitation studies (46,48,51). Dybowski and colleagues analyzed CBD using an UHPLC–MS/MS system with a Gemini C18 column (4.6 x 100 mm, 3 µm) and an isocratic mode mobile phase consisting of 60% 25 mM formic acid with water and 40% 25 mM formic acid with ACN with a flow rate of 0.5 mL/min (46). Dziadosz and colleagues used an HPLC–MS/MS system with a gradient mode mobile phase and MeOH as organic solvent for the analysis of THC, 11-OH-THC, and THC-COOH (48). Klawitter and colleagues performed protein precipitation for plasma and urine and carried out analysis from both matrices using an HPLC–MS/MS (51). Moorthy and colleagues used volumetric absorptive microsampling (VAMS) devices in the sample preparation technique. VAMS is a relatively new microsampling tool used for obtaining dried biological matrices, which improves the accuracy of the sample volume (52,58). Pires de Silva and colleagues used salting-out assisted liquid-liquid extraction (SALLE), another recent extraction technique where the extraction solvent is a water miscible organic solvent (54,59). SALLE is cheaper and easier to use than SPE (59). Toennes and colleagues, and Weit and colleagues, performed sample preparation using SPE (55,56).

Chang and colleagues performed hydrolysis of the urine specimen before the extraction method to improve sample accuracy (50). Pichini and colleagues carried out the study in oral fluid, serum, urine, and sweat patch samples. Sample preparation from oral fluid, serum, and urine were the same with further alkaline hydrolysis for urine samples for the quantification of CBD as it appears as glucuronide in urine. The extraction of cannabinoids from sweat patch samples was performed with MeOH as the extraction solvent (53).

Hair is also used as a matrix because traces of some compounds can be present in hair (49). Hair is a complex matrix that requires longer sample preparation times because washing and digestion steps are required (2,49). Cho and colleagues carried out the sample preparation washing the hair twice with MeOH to eliminate any external contaminants and performed the digestion with 1 M NaOH to free the cannabinoids from the matrix. Analysis was carried out using a system consisting of a binary pump, Agilent 1290 UHPLC pump (pump 1), and an additional Agilent 1260 pump (pump 2) (49).

Klawitter and colleagues, Chang and colleagues, and Toennes and colleaguesl also performed analysis with an Agilent HPLC unit. Ten studies used C18 columns. Three out of ten studies used Acquity and two out of ten used Kinetex. The majority of the studies used gradient mobile phase (n=9). Dybowski and colleagues performed an isocratic method of elution. The mobile phases were composed of ammonium formate or water and an organic solvent. ACN and MeOH are the organic solvents more commonly used for the mobile phase, with ACN being preferred (n=7) because of shorter elution times for cannabinoids (60). The flow rate ranged from 0.15 mL/min to 1 mL/min. A flow rate of 0.4 mL/min was the most commonly used (n=3).

Methods of Analysis of Cannabinoids from Oil

In recent years, CBD oil has become popular for use in different conditions (61). There is a lack of standardized extraction regulation (2,61). Different carrier oils on the market are olive oil, medium chain triglyceride (MCT), hemp seed oil, and black cumin seed oil.

HPLC is the method of analysis most commonly used (n=5) for the determination and quantification of cannabinoids in olive oil (n=2) and hemp seed oil (n=2) (26,27,36,60,66). The detectors most commonly used were UV (27,36,60) and DAD (26,64). Two studies carried out the analysis using UHPLC (37,53). Nemeškalová and colleagues carried out analysis in a wide variety of oils—paraffin oil, sunflower oil, castor oil, jojoba oil, shea oil, argan oil, almond oil, coconut oil, and aviril baby massage oil—using an UHPLC-UV-MS/MS method. UV-visible detection was used for the analysis of cannabis with a high amount of cannabinoids, while MS/MS was used for low quantities of major cannabinoids such as THC and CBD and for minor concentrations of cannabinoids (37).

Efficient extraction procedures are required for the analysis of cannabinoids in oil because oil cannot be injected directly in the HPLC due to its high viscosity (62). Bettiol and colleauges and Deidda and colleagues performed the same method to extract different cannabinoids from olive oil, consisting of 40 µL of sample in olive oil added to 960 µL of tetrahydrofuran (TFH) and vortex-mixed. Next, 50 µL of this solution was added to 950 µL of ACN in the study of Bettiol and colleagues, and in MeOH in the study by Deidda and colleagues (60,63). Mudge and colleagues carried out a solvent extraction with MeOH while Nemeškalová and colleagues used isopropanol/ethyl acetate (1:1, v/v). Ciolino and colleagues used EtOH or isopropyl alcohol (26,36,37). Araneda and colleagues performed analysis of cannabinoids using benchtop nuclear magnetic resonance (NMR) instruments to compare the results with the ones obtained using HPLC-UV. Analysis was carried out for five different concentrates of cannabinoids. The relative standard deviation for the samples analyzed with benchtop NMR was higher than that with the HPLC-UV. In the analyses performed with benchtop NMR, the amount of CBD in sample 1 and THC in sample 2 could not be quantified while in the analysis with HPLC both samples were quantified (64).

Three different brands of HPLC units were used among the articles published in the literature for the extraction of cannabinoids from oil. The HPLC unit most commonly used was Agilent (26,27,36,37). The second brand used was Thermo Fisher Surveyor and Pichini and colleagues used a Waters Xevo TQ-S. Bettiol and colleagues and Deidda and colleagues used a Thermo Fisher Surveyor Plus HPLC system using an Agilent PoroshellR 120 SB-C18 column, (2.1 mm × 150 mm; 2.7 µm) as a stationary phase and an isocratic mode mobile phase composed of ACN/5 mM phosphate buffer rate 75/25 v/v with a flow rate of 0.38 mL (60,63). Ciolino and colleagues also performed analysis using the isocratic mode for the mobile phase, but used two types of mobile phases 66:34 ACN: 0.5% acetic acid and 83:17 MeOH:50 mM citrate both using a flow rate of 1 mL/min. Analysis was carried out using an ACE column (26).

Seven studies used a C18 column as a stationary phase. The majority of the studies were performed using the brand Agilent Poroshell (n=4). The methods used for the mobile phase were gradient in four studies and isocratic in the other three.

The majority of the methods used ACN (n=5) as organic solvent and the amount ranged from 60–100% in mobile phase composition. The flow rate ranged from 0.38 mL/min to 1 mL/min.

HPLC Methods of Analysis of Cannabinoids from Miscellaneous Matrices

There is a need for quantitative analyses to determine cannabinoids such as CBD and THC in commercial products such as honey, capsules, and serum to calculate the amount of each cannabinoid and evaluate the dosage and the exposure of the patient when the product is consumed (26). Studies were carried out in different matrices such as cannabis concentrates, honey (n=1), hemp nut (n=1), vaporized fluid (n=1), milk (n=1), liver (n=1), capsules (n=2), wastewater (n=1), cotton cloths (n=1), and gummies (n=1).

Ciolino and colleagues carried out analysis of cannabinoids in different commercial products (26). Methods of sample preparation were the same for all the matrices: the sample was weighed and MeOH (95% or 100%) was added as an extraction solvent. The sample was then vortex-mixed and filtered with nylon membrane filter of 0.45 µm. Depending on the quantity of cannabinoids, the sample was further diluted or directly injected in an Agilent 1100, 1200, or 1260 HPLC-DAD system with an ACE 5 C18-AR analytical column (5 µm, 4.6 mm i.d. x 250 mm) (26).

Jornet-Martínez and colleagues detected traces of cannabinoids in different matrices such as plastic bags, cotton tip, aluminium foil, office paper, piece of cotton cloth, and skin. Due to the complex nature of the matrices and the small quantity of cannabinoids, Jornet-Martínez and colleagues performed analysis using an in-tube solid-phase microextraction (IT-SPME) coupled on-line to nanoliquid chromatography (nanoLC), which improved the selectivity of the analysis. The study was carried out using a Zorbax 300SB C18 (50 × 0.075 mm i.d., 3.5 µm) column with a simple gradient mode mobile phase consisting of water and ACN ranging between 55–75%. Jornet-Martínez and colleagues performed an ultrasound assisted extraction for the preparation of the sample using just 1 mL of MeOH per sample making the sample preparation an eco-friendly technique (63).

Nemeškalová and colleagues performed analysis of cannabinoids in oils and plant materials as well as in cosmetics and gelatinous gummies. The large amount of therapeutic benefits of CBD has led to a varied market of CBD based-products such as candies and cosmetics, which contain smalls amounts of THC that need to be quantified due to its psychoactive effects. The method proposed by Nemeškalová and colleagues demonstrated its feasibility on 13 CBD-based products using an UHPLC-UV-MS/MS with a Poroshell 120 EC-C18 (100 mm × 2.1 mm, 2.7 µm) column. The sample preparation was different for hydrophilic liquids, gummies, and hydrophobic cosmetics, but it consisted of dissolution and dilution (37).

A systematic literature review to identify and compare analytical methods used for the determination of cannabinoids in different matrices using LC-based systems.

June 2021

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