Tag Archives: decarboxylation

extraction equipment

THC Remediation of Hemp Extracts

By Darwin Millard
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extraction equipment

Remediation of delta-9 tetrahydrocannabinol (d9-THC) has become a hot button issue in the United States ever since the Drug Enforcement Agency (DEA) released their changes to the definitions of marijuana, marijuana extract, and tetrahydrocannabinols exempting extracts and tetrahydrocannabinols of a cannabis plant containing 0.3% or less d9-THC on a dry weight basis from the Controlled Substances Act. That is because, as a direct consequence, all extracts and tetrahydrocannabinols of a cannabis plant containing more than 0.3% d9-THC became explicitly under the purview of the DEA, including work-in-progress “hemp extracts” that because of the extraction process are above the 0.3% d9-THC limit immediately upon creation.

The legal ramifications of these changes to the definitions on the “hemp extracts” marketplace will not be addressed. Instead, this article focuses on the amount of d9-THC that is available in the plant material prior to extraction and tracks a “hemp extract” from the point it falls out of compliance to the point it becomes compliant again and stresses the importance of accurate track-n-trace protocols at the processing facility. The model developed to support this article was intended to be academic and was designed to follow the d9-THC portion of a “hemp extract” through the lifecycle of a typical CO2-based extract from initial extraction to THC remediation. A loss to the equipment of 2% was used for each step.

Initial Extraction

For this exercise, a common processing scenario of 1000 kg of plant material at 10% cannabidiol (CBD) and 0.3% d9-THC by weight was modeled. This amount, depending on scale of operations, can be a facility’s total capacity for the day or the capacity for a single run. 1000 kg of plant material at 0.3% d9-THC has 3 kg of d9-THC that could be extracted, purified, and diverted into the marketplace. CO2 has a nominal extraction efficiency of 95%, meaning some cannabinoids are left behind in the plant material. The same can be said about the recovery of the extract from the equipment. Traces of extract will remain in the equipment and this little bit of material, if unaccounted for, can potentially open an operator up to legal consequences. Data for the initial extraction is shown in Image 1.

Image 1: Summary Data Table for Typical CO2-based Extraction of Phytocannabinoids

As soon as the initial extract is produced it is out of compliance with the 0.3% d9-THC limit to be classified as a “hemp extract”, and of the 3 kg of d9-THC available, the extract contains approx. 2.8 kg, because some of the d9-THC remains in the plant material and some is lost to the equipment.

Dewaxing via Winterization and Solvent Removal

Dewaxing a typical CO2 extract via winterization is a common process step. For this exercise, a wax content of 30% by weight was used. A process efficiency of 98% was attributed to the wax removal process and it was assumed that 100% of the loss can be accounted for in the residue recovered from the equipment rather than in the removed waxes. Data for the winterization and solvent recovery are shown in Image 2 and 3.

Image 2: Summary Data Table for Typical Winterization of a CO2 Extract
Image 3: Summary Data Table for Solvent Removal from a CO2 Extract

Two things occur during winterization and solvent removal, non-target constituents are removed from the extract and there is compounded loss from multiple pieces of process equipment. These steps increase the concentration of the d9-THC portion of the extract and produce two streams of noncompliant waste.

Decarboxylation & Devolatilization

Most cannabinoids in the plant material are in their acid form. For this exercise, 90% of the cannabinoids were considered to be acid forms. Decarboxylation is known to produce a mass difference of 87.7%, i.e. the neutral forms are 12.3% lighter than the acid forms. Heat was modeled as the primary driver and a process efficiency of 95% was used for the conversion rate during decarboxylation. To simplify the model, the remaining 5% acidic cannabinoids are presumed destroyed rather than degraded into other compounds because the portion of the cannabinoids which get destroyed versus degrade into other compounds varies from process to process.

Devolatilization is the process of removing low-molecular weight constituents from an extract to stabilize it prior to distillation. Since the molecular constituents of cannabis resin extracts vary from variety to variety and process to process, the extracts were assumed to consist of 10% volatile compounds. The model combines the decarboxylation and devolatilization steps to account for complete decarboxylation of the available acidic cannabinoids and ignores their weight contribution to the volatiles collected during devolatilization. Destroyed cannabinoids result in an amount of loss that can only be accounted for through a complete mass balance analysis. Data for decarboxylation and devolatilization are shown in Image 4.

Image 4: Summary Data Table for Decarboxylation and Devolatilization of a CO2 Extract

As the extract moves along the process train, the d9-THC concentration continues to increase. Decarboxylation further complicates traceability because there is both a known mass difference associated with the process and an unknown mass difference that must be calculated and justified.

Distillation

A two-pass distillation was modeled. On each pass a portion of the extract was removed to increase the cannabinoid concentration in the recovered material. Average data for distilled “hemp extracts” was used to ensure the model did not over- or underestimate the concentration of the cannabinoids in the distillate. The variables used to meet these data constraints were derived experimentally to match the model to the scenario described and are not indicative of an actual distillation. Data for distillation is shown in Image 5.

Image 5: Summary Data Table for Distillation of a Decarboxylated and Devolatilized Extract

After distillation, the d9-THC concentration is shown to have increased by 874% from the original concentration in the plant material. Roughly 2.2 kg of the available 3 kg of d9-THC remains in the extract, but 0.8 kg of d9-THC has either ended up in a waste stream or walking out the door.

Chromatography – THC Remediation Step 1

Chromatography was modeled to remove the d9-THC from the extract. Because there are several systems with variable efficiency rates at being able to selectively isolate the d9-THC peak from the eluent stream, the model used a 5% cut-off on the front-end and tail-end of the peak, i.e. 5% of the material before the d9-THC peak and 5% of the material after the d9-THC peak is assumed to be collected along with the d9-THC. Data for chromatography is shown in Image 6.

Image 6: Summary Data Table for d9-THC Removal using Chromatography

After chromatography, a minimum of three products are produced, compliant “hemp extract”, d9-THC extract, and noncompliant residue remaining in the equipment. The d9-THC extract modeled contains 2.1 kg of the available 3 kg in the plant material, and is 35% d9-THC by weight, an increase of 1335% from the distillation step and 11664% from the plant material.

CBN Creation – THC Remediation Step 2

For this exercise, the d9-THC extract was converted into cannabinol (CBN) using heat rather than cyclized into d8-THC, but a similar model could be used to account for this scenario. The conversion rate of the cannabinoids into CBN through heat degradation alone is low. Therefore, the model assumes half of the available cannabinoids in the d9-THC extract are converted to CBN. The entirety of the remaining portion of the cannabinoids are assumed to convert to some form of degradant rather than a portion getting destroyed. Data for THC destruction is shown in Image 7.

Image 7: Summary Data Table for THC Destruction through Degradation into CBN

Only after the CBN cyclization step has completed does the product that was the d9-THC extract become compliant and classifiable as a “hemp extract.”

Image 8: Summary Data Table for Reconciliation of the d9-THC Portion of the Hemp Extract

Throughout the process, from initial extraction to the final d9-THC remediation step, loss occurs. Of the 3 kg of d9-THC available in the plant material only 2.1 kg was recovered and converted to CBN. 0.9 kg was either lost to the equipment, destroyed in the process, attributable to the mass difference associated with decarboxylation, or was never extracted from the plant material in the first place. All of these potential areas of product loss should be identified, and their diversion risk fully assessed. Not every waste stream poses a risk of diversion, but some do; having a plan in place to handle waste the DEA considers a controlled substance is essential. Without a track-n-trace program following the d9-THC and identifying the potential risk of diversion would be impossible. The point of this is not to instill fear, instead the intention is to shed light on a very real issue “hemp extract” producers and state regulators need to understand to protect themselves and their marketplace from the DEA.

EVIO labs photo
Soapbox

(L)Earning from Failure

By Dr. Markus Roggen, Soheil Nasseri
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EVIO labs photo

The spectacular rise and crash of the Canadian cannabis stock market has been painful to watch, let alone to experience as an industry insider. The hype around the market has vanished and many investors are left disappointed. Large sustainable gains simply haven’t materialized as promised. The producers are clearly suffering. They have consistently been shedding value as they’ve been posting losses every quarter. Stock prices have plummeted along with consumer confidence. Attempts to reduce the cash bleeds through mergers, acquisitions, layoffs, restructures, fund raises, among others, have not resulted in any significant recovery. In short, the current model of a cannabis industry has failed.

Dr. Markus Roggen, Founder of Complex Biotech Discovery Ventures (CBDV)

How could it have been different? What should the industry have done differently? What makes the difference between failure and success? A recent article published in Nature (Volume 575) by Yin et al. titled “Quantifying the Dynamics of Failure Across Science, Startups and Security” analyzes the underlying principles of success. The article studies success rates of many groups after numerous attempts across three domains. One of the domains being analyzed are startup companies and their success in raising funds through many attempts at investment acquisition. The authors point out that the most important factor that determines success is not relentless trying but is actually learning after each attempt. Learning allows successful groups to accelerate their failures, making minute adjustments to their strategy with every attempt. Learning behavior is also seen early in the journey. This means that groups will show higher chances of success early on, if they learn from their mistakes.

If you want to succeed, you need to analyze the current state, test the future state, evaluate performance difference and implement the improved state.

This also needs to happen in the cannabis industry. Producers have been utilizing inefficient legacy systems for production. They have shackled themselves to these inefficient methods by becoming GMP-certified too early. Such certifications prevent them from experimenting with different designs that would enhance their process efficiency and product development. This inflexibility prevents them from improving. This means they are setting themselves up for ultimate failure. GMP is not generally wrong, as it ensures product safety and consistency. Although, at this early stage in the cannabis industry, we just don’t yet have the right processes to enshrine.

How can cannabis producers implement the above-mentioned research findings and learn from their current situation? In an ever-changing business environment, it is companies that are nimble, innovative and fast enough to continually refine themselves that end up succeeding. This agility allows them to match their products with the needs of their consumers and market dynamics. booking.com, a travel metasearch engine, is the prime example of this ethos because they carry out thousands of experiments per year. They have embraced failure through rapid experimentation of different offerings to gauge user feedback. Experimentation has allowed booking.com to learn faster than the competition and build a stronger business.

Soheil Nasseri, Business Associate at Complex Biotech Discovery Ventures (CBDV)

At CBDV, we put the need for iterative experimentation, failure and improvements to achieve breakthroughs at the core of our company. We pursue data to guide our decisions, not letting fear of momentary failure detract us from ultimate success. We continuously explore multiple facets of complex problems to come up with creative solutions.

A good example of how failure and rapid innovation guided us to success is our work on decarboxylation. We were confronted by the problem that the decarboxylation step of cannabis oil was inconsistent and unpredictable. Trying different reaction conditions did not yield a clear picture. We realized that the most important obstacle for improvements was the slow analysis by the HPLC. Therefore, we turned our attention to developing a fast analysis platform for decarboxylation. We found this in a desktop mid-IR instrument. With this instrument and our algorithm, we now could instantaneously track decarboxylation. We now hit another roadblock, a significant rate difference in decarboxylation between THCA and CBDA. We needed to understand the theoretical foundation of this effect to effectively optimize this reaction. So, we moved to tackle the problem from a different angle and employed computational chemistry to identify the origin of the rate difference. Understanding the steric effect on rate helped us focus on rapid, iterative experimentation. Now, with everything in place, we can control the decarboxylation at unrivaled speeds and to the highest precision.

If producers want to regain the trust of the market, they must embrace their failures and begin to learn. They should decrease their reliance on inefficient legacy production methods and experiment with new ones to find what is right for them. Experimentation brings new ways of production, innovative products and happier customers, which will result in higher profits. Producers should strive to implement experimentation into their corporate cultures. This can be done in collaboration with research companies like CBDV or through development of inhouse ‘centers of excellence.’

The Best Way to Remediate Moldy Cannabis is No Remediation at All

By Ingo Mueller
3 Comments

Consumers are largely unaware that most commercial cannabis grown today undergoes some form of decontamination to treat the industry’s growing problem of mold, yeast and other microbial pathogens. As more cannabis brands fail regulatory testing for contaminants, businesses are increasingly turning to radiation, ozone gas, hydrogen peroxide or other damaging remediation methods to ensure compliance and avoid product recalls. It has made cannabis cultivation and extraction more challenging and more expensive than ever, not to mention inflaming the industry’s ongoing supply problem.

The problem is only going to get worse as states like Nevada and California are beginning to implement more regulations including even tougher microbial contamination limits. The technological and economic burdens are becoming too much for some cultivators, driving some of them out of business. It’s also putting an even greater strain on them to meet product demand.

It’s critical that the industry establishes new product standards to reassure consumers that the cannabis products they buy are safe. But it is even more critical that the industry look beyond traditional agricultural remediation methods to solve the microbial problems.

Compounding Risks

Mold and other microbial pathogens are found everywhere in the environment, including the air, food and water that people consume. While there is no consensus yet on the health consequences of consuming these contaminants through cannabis, risks are certainly emerging. According to a 2015 study by the Cannabis Safety Institutei, molds are generally harmless in the environment, but some may present a health threat when inhaled, particularly to immunocompromised individuals. Mycotoxins resulting from molds such as Aspergillus can cause illnesses such as allergic bronchopulmonary aspergillosis. Even when killed with treatment, the dead pathogens could trigger allergies or asthma.

Photo credit: Steep Hill- a petri dish of mold growth from tested cannabis

There is an abundance of pathogens that can affect cannabis cultivation, but the most common types are Botrytis (bud rot, sometimes called gray mold) and Powdery Mildew. They are also among the most devastating blights to cannabis crops. Numerous chemical controls are available to help prevent or stem an outbreak, ranging from fungicides and horticultural oils to bicarbonates and biological controls. While these controls may save an otherwise doomed crop, they introduce their own potential health risks through the overexposure and consumption of chemical residues.

The issue is further compounded by the fact that the states in which cannabis is legal can’t agree on which microbial pathogens to test for, nor how to test. Colorado, for instance, requires only three pathogen tests (for salmonella, E. coli, and mycotoxins from mold), while Massachusetts has exceedingly strict testing regulations for clean products. Massachusetts-based testing lab, ProVerde Laboratories, reports that approximately 30% of the cannabis flowers it tests have some kind of mold or yeast contamination.

If a cannabis product fails required microbial testing and can’t be remedied in a compliant way, the grower will inevitably experience a severe – and potentially crippling – financial hit to a lost crop. Willow Industries, a microbial remediation company, says that cannabis microbial contamination is projected to be a $3 billion problem by 2020ii.

Remediation Falls Short
With the financial stakes so high, the cannabis industry has taken cues from the food industry and adopted a variety of ways to remediate cannabis harvests contaminated with pathogens. Ketch DeGabrielle of Qloris Consulting spent two years studying cannabis microbial remediation methods and summarized their pros and consiii.

He found that some common sterilization approaches like autoclaves, steam and dry heat are impractical for cannabis due the decarboxylation and harsh damage they inflict on the product. Some growers spray or immerse cannabis flowers in hydrogen peroxide, but the resulting moisture can actually cause more spores to germinate, while the chemical reduces the terpene content in the flowers.

Powdery mildew starts with white/grey spots seen on the upper leaves surface

The more favored, technologically advanced remediation approaches include ozone or similar gas treatment, which is relatively inexpensive and treats the entire plant. However, it’s difficult to gas products on a large scale, and gas results in terpene loss. Microwaves can kill pathogens effectively through cellular rupture, but can burn the product. Ionizing radiation kills microbial life by destroying their DNA, but the process can create carcinogenic chemical compounds and harmful free radicals. Radio frequency (which DeGabrielle considers the best method) effectively kills yeast and mold by oscillating the water in them, but it can result in moisture and terpene loss.

The bottom line: no remediation method is perfect. Prevention of microbial contamination is a better approach. But all three conventional approaches to cannabis cultivation – outdoors, greenhouses and indoor grow operations – make it extremely difficult to control contamination. Mold spores can easily gain a foothold both indoors and out through air, water, food and human contact, quickly spreading into an epidemic.

The industry needs to establish new quality standards for product purity and employ new growing practices to meet them. Advanced technologies can help create near perfect growing ecosystems and microclimates for growing cannabis free of mold contamination. Internet of Things sensors combined with AI-driven robotics and automation can dramatically reduce human intervention in the growing process, along with human-induced contamination. Natural sunlight supplemented with new lighting technologies that provide near full-light and UV spectrum can stimulate robust growth more resistant to disease. Computational fluid dynamic models can help growers achieve optimal temperature, humidity, velocity, filtration and sanitation of air flow. And tissue culture micropropagation of plant stock can eliminate virus and pathogen threats, to name just a few of the latest innovations.

Growing legal cannabis today is a risky business that can cost growers millions of dollars if pathogens contaminate a crop. Remediation methods to remove microbial contamination may work to varying degrees, but they introduce another set of problems that can impact consumer health and comprise product quality.

References

i. Holmes M, Vyas JM, Steinbach W, McPartland J. 2015. Microbiological Safety Testing of Cannabis. Cannabis Safety Institute. http://cannabissafetyinstitute.org/wp-content/uploads/2015/06/Microbiological-Safety-Testing-of-Cannabis.pdf

ii. Jill Ellsworth, June 2019, Eliminating Microbials in Marijuana, Willow Industries, https://willowindustries.com/eliminating-microbials-in-marijuana/#

iii. Ketch DeGabrielle, April 2018, Largest U.S. Cannabis Farm Shares Two Years of Mold Remediation Research, Analytical Cannabis, https://www.analyticalcannabis.com/articles/largest-us-cannabis-farm-shares-two-years-of-mold-remediation-research-299842

 

From The Lab

I Was Wrong… und das ist auch gut so!

By Dr. Markus Roggen
3 Comments

I was wrong. And that’s a good thing! Based on all available data, I assumed that evaporating ethanol from a cannabis oil/ethanol solution would result in terpene loss. As it turns out, it doesn’t. There are so many beliefs and assumptions about cannabis: Cannabis cures cancer!1 Smoking cannabis causes cancer!2 Sativas help you sleep; Indicas make you creative!3,4 CBD is not psychoactive!5 But are these ‘facts’ backed by science? Have they been experimentally tested and validated?

I postulated a theory, designed experiments to validate it and evaluated the results. Simply putting “cannabis backed by science” on your label does not solve the problem. Science is not a marketing term. It’s not even a fixed term. The practice of science is multifaceted and sometimes confusing. It evolved from the traditional model of Inductivism, where observations are used in an iterative process to refine a law/theory that can generalize such observations.6 Closely related is Empiricism, which posits that knowledge can only come from observation. Rationalism, on the other hand, believes that certain truths can be directly grasped by one’s intellect.7 In the last century, the definition of science was changed from the method by which we study something, such as Inductivism or Rationalism, and refocused on the way we explain phenomena. It states that a theory should be considered scientific if, and only if, it is falsifiable.8 All that means is that not the way we study something is what makes it scientific, but the way we explain it.

I wonder how can we use empirical observations and rational deliberations to solve the questions surrounding cannabis? And more importantly, how can we form scientific theories that are falsifiable? Cannabis, the plant, the drug, has long been withheld from society by its legal status. As a result, much of what we know, in fact, the entire industry has thrived in the shadows away from rigorous research. It’s time for this to change. I am particularly concerned by the lack of fundamental research in the field. I am not even talking about large questions, like the potential medical benefit of the plant and its constituents. Those are for later. I’m talking about fundamental, mundane questions like how many lumens per square centimetre does the plant need for optimal THC production? What are the kinetics of cannabis extraction in different solvents? What are the thermodynamics of decarboxylation? Where do major cannabinoids differ or align in terms of water solubility and viscosity?

The lack of knowledge and data in the cannabis field puts us in the precarious position of potentially chasing the wrong goals, not to mention wasting enormous amounts of time and money. Here’s a recent example drawn from personal experience:Certainly, I cannot be the only one who has made an incorrect assumption based on anecdotes and incomplete data?

Some of the most common steps in cannabis oil production involve ethanol solutions. Ethanol is commonly removed from extraction material under reduced pressure and elevated heat in a rotary evaporator. I expected that this process would endanger the terpenes in the oil – a key component of product quality. My theory was that volatile terpenes9 would be lost in the rotary evaporator during ethanol10 removal. The close values of vapor pressure for terpenes and ethanol make this a reasonably assumed possibility.11 In the summer of 2018, I finally got the chance to test it. I designed experiments at different temperatures and pressures, neat and in solution, to quantify the terpene lost in ethanol evaporation. I also considered real life conditions and limitations of cannabis oil manufacturers. After all the experiments were done, the results unequivocally showed that terpenes do not evaporate in a rotary evaporator when ethanol is removed from cannabis extracts.12 As it turns out, I was wrong.

We, as an industry, need to start putting money and effort into fundamental cannabis research programs. But, at least I ran the experiments! I postulated a theory, designed experiments to validate it and evaluated the results. At this point, and only this point, can I conclude anything about my hypothesis, even if that is that my working theory needs to be revised. Certainly, I cannot be the only one who has made an incorrect assumption based on anecdotes and incomplete data?

There is a particular danger when using incomplete data to form conclusions. There are many striking examples in the medical literature and even the casual observer might know them. The case of hormone replacement therapy for menopause and the associated risks of cardiovascular diseases showed how observational studies and well-designed clinical trials can lead to contradicting results.13 In the thirties of the last century, lobotomy became a cure-all technique for mental health issues.14 Dr. Moniz even won the Nobel Prize in Medicine for it.15 And it must come as no surprise when WIRED states “that one generation’s Nobel Prize-winning cure is another generation’s worst nightmare.”16 And with today’s knowledge is impossible to consider mercury as a treatment for syphilis, but that is exactly what it was used as for many centuries.17 All those examples, but the last one in particular should “be a good example of the weight of tradition or habit in the medical practice, […] of the necessity and the difficulties to evaluate the treatments without error.”18 There is the danger that we as cannabis professionals fall into the same trap and believe the old stories and become dogmatic about cannabis’ potential.

We, as an industry, need to start putting money and effort into fundamental cannabis research programs. That might be by sponsoring academic research,19 building in-house research divisions,20 or even building research networks.21 I fully believe in the need for fundamental cannabis research, even the non-sexy aspects.22 Therefore, I set up just that: an independent research laboratory, focused on fundamental cannabis research where we can test our assumptions and validate our theories. Although, I alone cannot do it all. I likely will be wrong somewhere (again). So, please join me in this effort. Let’s make sure cannabis science progresses.

References

  1. No, it does not. There are preliminary in-situ studies that point at anti-cancer effects, but its more complicated. The therapeutic effects of Cannabis and cannabinoids: An update from the National Academies of Sciences, Engineering and Medicine report, Abrams, Donald I., European Journal of Internal Medicine, Volume 49, 7 – 11
  2. No, it does not. National Academies of Sciences, Engineering, and Medicine. 2017. The Health Effects of Cannabis and Cannabinoids: The Current State of Evidence and Recommendations for Research. Washington, DC: The National Academies Press. https://doi.org/10.17226/24625.
  3. No, it does not. The chemical profile of the plant dictates the biological effects on humans, not the shape of the leaf.  Justin T. Fischedick, Cannabis and Cannabinoid Research, Volume: 2 Issue 1: March 1, 2017
  4. Indica and Sativa are outdated terms. Piomelli D, Russo EB. The Cannabis sativa versus Cannabis indica debate: An Interview with Ethan Russo, MD. Cannabis Cannabinoid Res 2016; 1: 44–46.
  5. No, it is. CBD’s supposed “calming effects” is indeed a psychoactive effect. However, it is not intoxicating like THC. Russo E.B., Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects.Br. J. Pharmacol. 2011; 163: 1344-1364
  6. As attributed to Francis Bacon.
  7. See the work by philosopher Baruch Spinoza.
  8. As theorized by Karl Popper.
  9. Monoterpenes have a vapor pressure in the low to mid hundreds of Pascals at room temperature.
  10. Vapor pressure of 5.95 kPa at 20˚C.
  11. Furthermore, there is always the possibility of azeotropes in complex mixtures. Azeotropes are mixtures of two or more liquids that have different boiling points individually, but in mixture boil together.
  12. Terpene Retention via Rotary Evaporator Application Note, Heidolph North America
  13. https://www.pharmaceutical-journal.com/research/review-article/establishing-the-risk-related-to-hormone-replacement-therapy-and-cardiovascular-disease-in-women/20202066.article?firstPass=false
  14. https://psychcentral.com/blog/the-surprising-history-of-the-lobotomy/
  15. https://en.wikipedia.org/wiki/António_Egas_Moniz
  16. https://www.wired.com/2011/03/lobotomy-history/
  17. https://www.infezmed.it/media/journal/Vol_21_4_2013_10.pdf
  18. https://www.ncbi.nlm.nih.gov/pubmed/11625051
  19. Canopy Growth funds a professorship of cannabis science at UBC. Tilray collaborates with UCSD on a phase I/II clinical trial.
  20. For examples see: NIBR, PMISCIENCE.
  21. For examples see: CEMI, theAIRnet, Future Sky.
  22. Research that does not lead to short-term stock value spikes but long-term progress
The Practical Chemist

Potency Analysis of Cannabis and Derivative Products: Part 2

By Rebecca Stevens
3 Comments

As mentioned in Part 1, the physiological effects of cannabis are mediated by a group of structurally related organic compounds known as cannabinoids. The cannabinoids are biosynthetically produced by a growing cannabis plant and Figure 1 details the biosynthetic pathways leading to some of the most important cannabinoids in plant material.

Potency figure 1
Figure 1: The biosynthetic pathway of phytocannabinoid production in cannabis has been deeply studied through isotopic labeling experiments

The analytical measurement of cannabinoids is important to ensure the safety and quality of cannabis as well as its extracts and edible formulations. Total cannabinoid levels can vary significantly between different cultivars and batches, from about 5% up to 20% or more by dry weight. Information on cannabinoid profiles can be used to tailor cultivars for specific effects and allows end users to select an appropriate dose.

Routine Analysis vs. Cannabinomics 

Several structurally analogous groups of cannabinoids exist. In total, structures have been assigned for more than 70 unique phytocannabinoids as of 2005 and the burgeoning field of cannabinomics seeks to comprehensively measure these compounds.¹

Considering practical potency analysis, the vast majority of cannabinoid content is accounted for by 10-12 compounds. These include Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), Δ9-tetrahydrocannabivarian (THCV), cannabidivarin (CBDV) and their respective carboxylic acid forms. The cannabinoids occur primarily as carboxylic acids in plant material. Decarboxylation occurs when heat is applied through smoking, vaporization or cooking thereby producing neutral cannabinoids which are more physiologically active.

Potency Analysis by HPLC and GC

Currently, HPLC and GC are the two most commonly used techniques for potency analysis. In the case of GC, the heat used to vaporize the injected sample causes decarboxylation of the native cannabinoid acids. Derivatization of the acids may help reduce decarboxylation but overall this adds another layer of complexity to the analysis² ³. HPLC is the method of choice for direct analysis of cannabinoid profiles and this technique will be discussed further.

A sample preparation method consisting of grinding/homogenization and alcohol extraction is commonly used for cannabis flower and extracts. It has been shown to provide good recovery and precision² ³. An aliquot of the resulting extract can then be diluted with an HPLC compatible solvent such as 25% water / 75% acetonitrile with 0.1% formic acid. The cannabinoids are not particularly water soluble and can precipitate if the aqueous percentage is too high.

To avoid peak distortion and shifting retention times the diluent and initial mobile phase composition should be reasonably well matched. Another approach is to make a smaller injection (1-2 µL) of a more dissimilar solvent. The addition of formic acid or ammonium formate buffer acidifies the mobile phase and keeps the cannabinoid acids protonated.

The protonated acids are neutral and thus well retained on a C18 type column, even at higher (~50% or greater) concentrations of organic solvent² ³.

Detection is most often done using UV absorbance. Two main types of UV detectors are available for HPLC, single wavelength and diode array. A diode array detector (DAD) measures absorbance across a range of wavelengths producing a spectrum at each point in a chromatogram while single wavelength detectors only monitor absorbance at a single user selected wavelength. The DAD is more expensive, but very useful for detecting coelutions and interferences.

References

  1. Chemical Constituents of Marijuana: The Complex Mixture of Natural Cannabinoids. Life Sciences, 78, (2005), pp. 539
  2. Development and Validation of a Reliable and Robust Method for the Analysis of Cannabinoids and Terpenes in Cannabis. Journal of AOAC International, 98, (2015), pp. 1503
  3. Innovative Development and Validation of an HPLC/DAD Method for the Qualitative and Quantitative Determination of Major Cannabinoids in Cannabis Plant Material. Journal of Chromatography B, 877, (2009), pp. 4115

Rebecca is an Applications Scientist at Restek Corporation and is eager to field any questions or comments on cannabis analysis, she can be reached by e-mail, rebecca.stevens@restek.com or by phone at 814-353-1300 (ext. 2154)