Tag Archives: terpene

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

By Ingo Mueller
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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

 

Who’s Afraid of Biotech Institute LLC?

By Brett Schuman, Daniel Mello, Nicholas Costanza, Olivia Uitto
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While cannabis patenting activity is still in its infancy, relatively speaking, a lot has been written already about the cannabis patenting activity of an entity called Biotech Institute LLC (BI) of Westlake Village, California.1 BI is building a sizable portfolio of utility and plant patents covering various aspects of the cannabis plant. According to some commentators, BI’s patents have “many in the cannabis industry concerned.”2

But how concerned should members of the cannabis industry really be about BI’s patents? Generally, patents are susceptible to numerous challenges in multiple fora. From 2012-2016, approximately 80% of challenged patents were invalidated by the Patent Trial and Appeal Board (PTAB) each year.3 The PTAB was created in 2011 by the Leahy-Smith America Invents Act, 35 U.S.C. § 6, to create a process for eliminating improvidently issued patents. And the statistics suggest that the process may be working as intended by Congress.

BI may be building its portfolio by taking advantage of some unique challenges in the cannabis patenting area. First, even though cannabis has been cultivated and consumed by humans for thousands of years, there is a relative lack of published prior art available to patentees and patent examiners examining patent applications.4 Second, patent examiners are not as familiar with cannabis patent applications as they may be with other types of patent applications.

So, we examined carefully BI’s earliest and arguably broadest utility patent, U.S. Patent No. 9,095,554, and concluded that maybe the cannabis industry need not be so concerned about this and some of BI’s other utility patents. Although the ’554 patent is lengthy – 247 columns of text and over an inch thick when printed in hardcopy – there appears to be little if any novelty to the claimed invention. Alternatively, the patent appears to be obvious in light of the available prior art.

In a patent, the claims define the metes and bounds of the patentee’s intellectual property. Claim 1 of the ’554 patent recites:

  1. A hybrid cannabis plant, or an asexual clone of said hybrid cannabis plant, or a plant part, tissue, or cell thereof, which produces a female inflorescence, said inflorescence comprising:
  1. a BT/BD genotype;
  2. a terpene profile in which myrcene is not the dominant terpene;
  3. a terpene oil content greater than about 1.0% by weight; and
  4. a CBD content greater than 3%;
  5. wherein the terpene profile is defined as terpinolene, alpha phelladrene, beta ocimene, careen, limonene, gamma terpinene, alpha pinene, alpha terpinene, beta pinene, fenchol, camphene, alpha terpineol, alpha humulene, beta caryophyllene, linalool, cary oxide, and myrcene, and wherein the terpene oil content is determined by the additive content of the terpenes in the terpene profile; and wherein the terpene contents and CBD content are measured by gas chromatography-flame ionization detection (GC-FID) and calculated based on dry weight of the inflorescence; wherein a representative sample of seed producing said plants has been deposited under NCIMB Nos. 42246, 42247, 42248, 42249, 42250, and 42254.

While claim elements define the metes and bounds of the invention, typically only certain claim elements are intended to distinguish the claimed invention from the prior art. Other claim elements merely help to describe the invention. For example, the preamble in the ‘554 patent, or the part of the claim before subpart (a), describes the flowering part of the cannabis plant. This is not intended to describe anything novel about the claimed invention, but rather it simply describes the part of the cannabis plant that is relevant to the invention.

The structure of cannabidiol (CBD), one of 400 active compounds found in cannabis.

Before the priority date of the ’554 patent, it was known in the prior art that BT/Bgenotypes produce nearly equal amounts of THC and CBD (both are dominant; one is not recessive).5 Thus, it is not unexpected to have a CBD content greater than 3% in a genotype that can produce large amounts of CBD (known references state as high as 21% in CBD-dominant strains and 3%-15% in BT/Bgenotypes).6 Further, it was known in the prior art that terpenes generally constitute more than 1.0% percent by weight (usually between 2-4%) of the flower.7

As these databases continue to grow and studies of cannabis are publicly disclosed, cannabis patents like BI’s ’554 patent will become more and more susceptible to patent challenges and invalidation.Claim element (b), reciting a terpene profile in which myrcene is not the dominant terpene, appears to be one of – if not the only – claimed element of novelty of the BI invention. Terpenes are aromatic compounds produced in plants, and the cannabis plant has more than 100 different terpenes. Claim element (e) simply lists the most abundant terpenes in the cannabis plant. A majority of cannabis strains express high levels of myrcene; however, there are known prior art strains that express high levels of other terpenes, such as caryophyllene, limonene, pinene, etc. Additionally, it is well known in the art that terpenes have different therapeutic effects. For example, pinene and linalool are known to have antidepressant activity.8 Thus, a prior disclosure of a BT/Bgenotype that has a terpene profile where myrcene is not the dominate terpene very likely invalidates this claim. And even assuming there is any novelty to a high-CBD strain where myrcene is not the dominant terpene, there is a motivation to breed for a dominant terpene besides myrcene.

Because cannabis has been and remains a Schedule I drug under the Controlled Substances Act, previously known and used strains generally have not been chemically characterized, studied, researched, and the subject of publications that can be used as prior art for purposes of challenging cannabis patents. But that is changing. For example, the Open Cannabis Project (OCP) attempted to characterize and publish chemical details of cannabis plants. Even though OCP closed as of May 31, 2019, is database is still publicly available. Another example is CANNA, a non-profit initiative of the CANNA Espana Fertilizantes SL company, which carries out studies and conducts research on cannabis and its active compounds.9 In one study,10 CANNA found that some strains have terpene profiles where myrcene is not the dominant terpene, which could be relevant to a novelty-based or obviousness challenge to claim 1 of the ‘554 patent. As these databases continue to grow and studies of cannabis are publicly disclosed, cannabis patents like BI’s ’554 patent will become more and more susceptible to patent challenges and invalidation.


References

  1. See, e.g.,Amanda Chicago Lewis, The Great Pot Monopoly Mystery, GQ (August 23, 2017), https://www.gq.com/story/the-great-pot-monopoly-mystery;  Brian Wroblewski, Utility Patents on Marijuana? Who is BioTech Institute LLC?, The National Marijuana News, https://thenationalmarijuananews.com/utility-patents-marijuana-biotech-institute-llc/; Eric Sandy, Biotech Institute Has Applied for Patents on 8 Individual Cannabis Cultivars, Cannabis Business Times(June 24, 2019), https://www.cannabisbusinesstimes.com/article/biotech-institute-cannabis-patent-applications/.
  2. Nicole Grimm, George Lyons III, and Brett Scott, Biotech Institute’s Growing Patent Portfolio — U.S. Patent No. 9,095,554 and the Path Forward, JD Supra (November 17, 2017), https://www.jdsupra.com/legalnews/biotech-institute-s-growing-patent-17433/.
  3. World Intellectual Property Organization, An overview of patent litigation systems across jurisdictions,World Intellectual Property Indicators 2018, https://www.wipo.int/edocs/pubdocs/en/wipo_pub_941_2018-chapter1.pdf.
  4. Brett Schuman et al., Emerging Patent Issues In The Cannabis Industry, Law360(February 20, 2018), https://www.goodwinlaw.com/-/media/files/publications/emerging-patent-issues-in-the-cannabis-industry.pdf.
  5. Chandra, et al. Cannabis sativa L. – Botany and Biotechnology, pages 142-144, Springer, 2017 (citing de Meijer, Genetics163: 225-346 (2003)). See alsoMolecular Breeding (2006) 17:257-268, doi/10.1007/s11032-005-5681-x. 
  6. American Journal of Botany 91(6): 966:975 (2004). doi.org/10.3732/ajb.91.6.966; See e.g., Jikomes, Peak THC: The Limits on THC and CBD Levels for Cannabis Strainshttps://www.leafly.com/news/science-tech/peak-thc-cbd-levels-for-cannabis-strains.
  7. PLoS One. 2017; 12(3): e0173911. doi: 10.1371/journal.pone.0173911.  See also, Fischedick J. T., Hazekamp A., Erkelens T., Choi Y. H., Verpoorte R. (2010). Phytochemistry712058–2073 (2010). 10.1016/j.phytochem.2010.10.001
  8. J Ethnopharmacol. 2012 Sep 28;143(2):673-9. doi: 10.1016/j.jep.2012.07.026. Epub 2012 Jul 31.
  9. Retrieved from https://www.fundacion-canna.es/en/about-us, on August 6, 2019.
  10. Retrieved from https://www.fundacion-canna.es/en/variations-terpene-profiles-different-strains-cannabis-sativa-l, on August 6, 2019.

The Need for Standardization in Medical Cannabis Testing

By Andrew James
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There has been a move towards the legalization of cannabis for medical and/or recreational use across many countries and US states in recent years, leading to greater demand for accurate potency and safety testing. Despite this, there are currently no standardized regulations between states or countries for quality control including content, composition, adulterants, potency or levels of toxic residues. As such, in many cases where regulations are in place, testing is generally carried out at a small number of approved independent testing laboratories.

The need for self-regulation has led to the growth of portable gas chromatography (GC) being used in the field of cannabis testing.This lack of clarity makes it difficult for consumers to make informed decisions about what they are purchasing, an issue which could be damaging to the industry’s changing reputation. As it stands, producers of cannabis and cannabis-derived products can supply goods with potentially harmful contaminants such as fungi or pesticide residues, which are potentially threatening to human health. Most cannabis products sold legally in the US are required to be tested and labelled for THC, the chemical responsible for most of cannabis’s psychoactive effects. A US study found that as few as 20% of recreational cannabis products are accurately labelled with only 17% of products reviewed accurately labelled for THC content (i.e. within 10% of the labeled value). It also found 23% were under labelled, and 60% were over labelled.

If cannabis were to be categorized as a regulated pharmaceutical drug, it would be rigorously tested to comply with stringent rules and regulations regarding quality and safety of the product, as are all other drugs. However, as there is currently no centralized regulatory body that oversees this, the responsibility of quality assurance falls to the grower, manufacturer and sometimes the consumer.

The Need for Cannabis Analysis

The most common requirement when testing cannabis is positive identification and quantification of the total THC:CBD ratio. In a highly competitive marketplace, this information is important, as cannabis consumers tend to equate THC levels with price. In many instances, lower THC products are cheaper and higher THC concentrations make products more expensive. Without robust systems in place for sufficient testing, this information cannot be accurately determined, meaning the customer often cannot make an informed decision.

Pesticide use is surprisingly common in the cannabis cultivation industry

In addition to potency testing, one of the core issues facing the industry and by extension, the end consumer, is the prevalence of pesticides in cannabis products. In the Netherlands, the Ministry of Environment and Health reported that over 90% of cannabis plants tested had pesticides on them. While steps have been taken to tackle this, the lack of cohesion in testing standards combined with the onus on individual labs to carry out testing, has led to some issues within the industry.

Many individual retailers in the U.S. and internationally are self-testing for impurities such as pesticides, heavy metals and microbials. While there is a clear need for standard testing across all locations, the need for self-regulation at present has led to the growth of portable gas chromatography (GC) being used in the field of cannabis testing.

Using GC as an analytical tool 

With the increased need for quality control and quality assurance in the largely unregulated cannabis industry, technology is now more accessible to smaller companies and to people with minimal laboratory experience. There are a range of cannabis testing packages available for smaller individual labs which offer more mobile testing with affordable packages. The lower entry price makes GC analysis affordable for more laboratories while still delivering reliable, high quality results.

Portable GC instruments can offer high quality potency testing, pesticide screening, terpene and flavor profiling, and residual solvents analysis. These instruments can give growers and processors an accurate result of cannabinoid percentages, a fundamental piece of information for growers and dispensaries. Systems can be configured for manual injection or a range of autosampler options can be added.

The structure of cannabidiol (CBD), one of 400 active compounds found in cannabis.

GC enables the rapid and accurate identification and quantification of the THC:CBD ratio. This is important for companies which are carrying out self-testing as it allows their customers to have assurances in the short term over the quality of their product, as well as reducing any potential risks to public health.

An example of this in practice is the use of GC by Dutch company Shamanics which carries out testing service for coffee shops in the Netherlands. The company conducts terpene analysis and potency testing to assure the quality of the products it supplies, with a portable GC, which offers the flexibility required without any established guidelines on testing in place.

When testing for potency using the GC, they look for total THC and CBD by converting the acidified versions of the cannabinoids into neutral forms within the heat of the GC injector. The process has flexibility which means that if they need to see both the acidified and neutral versions, they can do this by derivatizing the sample. The accuracy of this process is crucial to Shamanics and similar companies within the industry so that they can accurately judge the quality of a product, and relay this information to retailers and consumers.

The future of GC in standardized testing

While the growing availability of portable GC instruments and the increasing sophistication of individual labs means more companies are able to self-test products, there is still a significant hurdle to overcome in terms of standardising and regulating both the medical and recreational cannabis markets. Where regulation is brought in it should be consistent across states and countries and most importantly, it should be monitored and enforced. In the meantime, responsible producers are using the technology available to them to provide consumers with guarantees that their cannabis products are safe and of a high quality.

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

Orange Photonics Introduces Terpenes+ Module in Portable Analyzer

By Aaron G. Biros
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Last week at the National Cannabis Industry Association’s (NCIA) Cannabis Business Summit, Orange Photonics unveiled their newest product added to their suite of testing instruments for quality assurance in the field. The Terpenes+ Module for the LightLab Cannabis Analyzer, which semi-quantitatively measures terpenes, Cannabichromene (CBC) and degraded THC, adds three new chemical analyses to the six cannabinoids it already reports.

CBC, a cannabinoid typically seen in hemp and CBD-rich plants, has been linked to some potentially impactful medical applications, much like the findings regarding the benefits of CBD. The module that tests for it, along with terpenes and degraded THC, can be added to the LightLab without any changes to hardware or sample preparation.

Dylan Wilks, chief technology officer of Orange Photonics
Dylan Wilks, chief technology officer of Orange Photonics

According to Dylan Wilks, chief technology officer of Orange Photonics, this could be a particularly useful tool for distillate producers looking for extra quality controls. Cannabis distillates are some of the most prized cannabis products around, but the heat used to create them can also create undesirable compounds,” says Wilks. “Distillate producers can see potency drop more than 25% if their process isn’t optimized”. With this new Terpenes+ Module, a distillate producer could quantify degraded THC content and get an accurate reading for their QC/QA department.

We spoke with Stephanie McArdle, president of Orange Photonics, to learn more about their instruments designed for quality assurance for growers and extractors alike.

Stephanie McArdle, president of Orange Photonics
Stephanie McArdle, president of Orange Photonics

According to McArdle, this could help cultivators and processors understand and value their product when terpene-rich products are the end goal. “Rather than try to duplicate the laboratory analysis, which would require expensive equipment and difficult sample preparation, we took a different approach. We report all terpenes as a single total terpene number,” says McArdle. “The analyzer only looks for monoterpenes (some common monoterpenes are myrcene, limonene and alpha-pinene), and not sesquiterpenes (the other major group of cannabis terpenes, such as Beta- Caryophyllene and Humulene) so the analysis is semi-quantitative. What we do is measure the monoterpenes and make an assumption that the sesquiterpenes are similar to an average cannabis plant to calculate a total terpene content.” She says because roughly 80% of terpenes found in cannabis are monoterpenes, this should produce accurate results, though some exotic strains may not result in accurate terpene content using this method.

The LIghtLab analyzer on the workbench
The LIghtLab analyzer on the workbench

As growers look to make their product unique in a highly competitive market, many are looking at terpenes as a source of differentiation. There are a variety of areas where growers can target higher terpene production, McArdle says. “During production, a grower may want to select plants for growing based on terpene content, or adjust nutrient levels, lighting, etc. to maximize terpenes,” says McArdle. “During the curing process, adjusting the environmental conditions to maximize terpene content is highly desirable.” Terpenes are also beginning to get recognized for their potential medical and therapeutic values as well, notably as an essential piece in the Entourage Effect. “Ultimately, it comes down to economics – terpene rich products have a higher market value,” says McArdle. “If you’re the grower, you want to prove that your product is superior. If you’re the buyer, you want to ensure the product you buy is high quality before processing it into other products. In both cases, knowing the terpene content is critical to ensuring you’re maximizing profits.”

Orange Photonics’ LightLab operates very similarly to instruments you might find in a cannabis laboratory. Many cannabis testing labs use High Performance Liquid Chromatography (HPLC) to analyze hemp or cannabis samples. “The primary difference between LightLab and an HPLC is that we operate at lower pressures and rely on spectroscopy more heavily than a typical HPLC analysis does,” says McArdle. “Like an HPLC, LightLab pushes an extracted cannabis sample through a column. The column separates the cannabinoids in the sample by slowing down cannabinoids by different amounts based on their affinity to the column.” McArdle says this is what allows each cannabinoid to exit the column at a different time. “For example, CBD may exit the column first, then D9THC and so on,” says McArdle. “Once the column separates the cannabinoids, they are quantified using optical spectroscopy- basically we are using light to do the final quantification.”

extractiongraphic

The Four Pillars of Cannabis Processing

By Christian Sweeney
2 Comments
extractiongraphic

Cannabis extraction has been used as a broad term for what can best be described as cannabis processing. A well-thought-out cannabis process goes far beyond just extraction, largely overlapping with cultivation on the front-end and product development on the back-end1. With this in mind, four pillars emerge as crucial capabilities for developing a cannabis process: Cultivation, Extraction, Analytics and Biochemistry.

The purpose and value of each pillar on their own is clear, but it is only when combined that each pillar can be optimized to provide their full capacities in a well-designed process. As such, it is best to define the goals of each pillar alone, and then explain how they synergize with each other.

At the intersection of each pillar, specific technology platforms exist that can effectively drive an innovation and discovery cycle towards the development of ideal products.Cultivation is the foundation of any horticultural process, including cannabis production. Whether the goal be to convert pigments, flavors or bioactive compounds into a usable form, a natural process should only utilize what is provided by the raw material, in this case cannabis flower. That means cultivation offers a molecular feedstock for our process, and depending on our end goals there are many requirements we may consider. These requirements start as simply as mass yield. Various metrics that can be used here include mass yield per square foot or per light. Taken further, this yield may be expressed based not only on mass, but the cannabinoid content of the plants grown. This could give rise to a metric like CBD or THC yield per square foot and may be more representative of a successful grow. Furthermore, as scientists work to learn more about how individual cannabinoids and their combinations interact with the human body, cultivators will prioritize identifying cultivars that provide unique ratios of cannabinoids and other bioactive compounds consistently. Research into the synergistic effect of terpenes with cannabinoids suggests that terpene content should be another goal of cultivation2. Finally, and most importantly, it is crucial that cultivation provide clean and safe materials downstream. This means cannabis flower free of pesticides, microbial growth, heavy metals and other contaminants.

Extraction is best described as the conversion of target molecules in cannabis raw material to a usable form. Which molecules those are depends on the goals of your product. This ranges from an extract containing only a pure, isolated cannabinoid like CBD, to an extract containing more than 100 cannabinoids and terpenes in a predictable ratio. There are countless approaches to take in terms of equipment and process optimization in this space so it is paramount to identify which is the best fit for the end-product1. While each extraction process has unique pros and cons, the tunability of supercritical carbon dioxide provides a flexibility in extraction capabilities unlike any other method. This allows the operator to use a single extractor to create extracts that meet the needs of various product applications.

Analytics provide a feedback loop at every stage of cannabis production. Analytics may include gas chromatography methods for terpene content3 or liquid chromatography methods for cannabinoids 3, 4, 5. Analytical methods should be specific, precise and accurate. In an ideal world, they can identify the compounds and their concentrations in a cannabis product. Analytics are a pillar of their own due simply to the efforts required to ensure the quality and reliability of results provided as well as ongoing optimization of methods to provide more sensitive and useful results. That said, analytics are only truly harnessed when paired with the other three pillars.

extractiongraphic
Figure 1: When harnessed together the pillars of cannabis processing provide platforms of research and investigation that drive the development of world class products.

Biochemistry can be split into two primary focuses. Plant biochemistry focuses back towards cultivation and enables a cannabis scientist to understand the complicated pathways that give rise to unique ratios of bioactive molecules in the plant. Human biochemistry centers on how those bioactive molecules interact with the human endocannabinoid system, as well as how different routes of administration may affect the pharmacokinetic delivery of those active molecules.

Each of the pillars require technical expertise and resources to build, but once established they can be a source of constant innovation. Fig. 1 above shows how each of these pillars are connected. At the intersection of each pillar, specific technology platforms exist that can effectively drive an innovation and discovery cycle towards the development of ideal products.

For example, at the intersection of analytics and cultivation I can develop raw material specifications. This sorely needed quality measure could ensure consistencies in things like cannabinoid content and terpene profiles, more critically they can ensure that the raw material to be processed is free of contamination. Additionally, analytics can provide feedback as I adjust variables in my extraction process resulting in optimized methods. Without analytics I am forced to use very rudimentary methods, such as mass yield, to monitor my process. Mass alone tells me how much crude oil is extracted, but says nothing about the purity or efficiency of my extraction process. By applying plant biochemistry to my cultivation through the use of analytics I could start hunting for specific phenotypes within cultivars that provide elevated levels of specific cannabinoids like CBC or THCV. Taken further, technologies like tissue culturing could rapidly iterate this hunting process6. Certainly, one of the most compelling aspects of cannabinoid therapeutics is the ability to harness the unique polypharmacology of various cannabis cultivars where multiple bioactive compounds are acting on multiple targets7. To eschew the more traditional “silver bullet” pharmaceutical approach a firm understanding of both human and plant biochemistry tied directly to well characterized and consistently processed extracts is required. When all of these pillars are joined effectively we can fully characterize our unique cannabis raw material with targeted cannabinoid and terpene ratios, optimize an extraction process to ensure no loss of desirable bioactive compounds, compare our extracted product back to its source and ensure we are delivering a safe, consistent, “nature identical” extract to use in products with predictable efficacies.

Using these tools, we can confidently set about the task of processing safe, reliable and well characterized cannabis extracts for the development of world class products.


[1] Sweeney, C. “Goal-Oriented Extraction Processes.” Cannabis Science and Technology, vol 1, 2018, pp 54-57.

[2] Russo, E. B. “Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects.” British Journal of Pharmacology, vol. 163, no. 7, 2011, pp. 1344–1364.

[3] Giese, Matthew W., et al. “Method for the Analysis of Cannabinoids and Terpenes in Cannabis.” Journal of AOAC International, vol. 98, no. 6, 2015, pp. 1503–1522.

[4] Gul W., et al. “Determination of 11 Cannabinoids in Biomass and Extracts of Different Varieties of Cannabis Using high-Performance Liquid Chromatography.” Journal of AOAC International, vol. 98, 2015, pp. 1523-1528.

[5] Mudge, E. M., et al. “Leaner and Greener Analysis of Cannabinoids.” Analytical and Bioanalytical Chemistry, vol. 409, 2017, pp. 3153-3163.

[6] Biros, A. G., Jones, H. “Applications for Tissue Culture in Cannabis Growing: Part 1.” Cannabis Industry Journal, 13 Apr. 2017, www.cannabisindustryjournal.com/feature_article/applications-for-tissue-culture-in-cannabis-growing-part-1/.

[7] Brodie, James S., et al. “Polypharmacology Shakes Hands with Complex Aetiopathology.” Trends in Pharmacological Sciences, vol. 36, no. 12, 2015, pp. 802–821.

Refining Techniques for Growing Cannabis

By Cannabis Industry Journal Staff
No Comments

As the cannabis industry in the United States and throughout the world develops, the market is getting more competitive. Markets in a number of states are experiencing disruptions that will have lasting effects for cultivators, including oversupply and supply chain bottlenecks. Now more than ever, growers need to look for ways to differentiate their product or gain a bigger market share. Looking at yield efficiency, quality improvements and analyzing the cost of inputs versus value of the crop can help growers make the right choices in technology for lighting, irrigation and pest control among other technologies.

adamplants
Adam Jacques, co-founder of Growers’ Guild Gardens and Sproutly

A series of free webinars in two weeks can help growers learn about some of the more advanced techniques in improving yield and quality. The Cannabis Cultivation Virtual Conference on May 23rd will explore a variety of tips and tricks for taking their cultivation operation to the next level. This event is free to attendees, made possible by sponsors VividGro and CannaGrow Expo.

Dr. Allison Justice
Dr. Allison Justice, vice president of cultivation at Outco

Attendees will hear from experts in cannabis cultivation on a range of topics, including breeding, drying, curing, environmental monitoring and micropropagation. Adam Jacques, co-founder of Growers’ Guild Gardens and Sproutly, will discuss some of his experience with breeding high-CBD strains in Oregon. His talk will delve into some of the proper breeding procedures, along with how to hunt for particular phenotypes and developing specific cannabinoids and terpenes.

Dr. Allison Justice, vice president of cultivation at Outco, is going to present some of her findings in drying and curing at the company. She plans on sharing her research on how the post-harvest stages can affect and control the chemical makeup of flower. She’ll also discuss some new protocols to monitor the dry and cure of cannabis flowers so we are able to modulate the terpene and cannabinoid profiles.

More information on the other speakers at this event and how to register for free can be found here.

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From The Lab

The Other Side of Cannabis: Terpenes

By Dr. Zacariah Hildenbrand, Allegra Leghissa, Dr. Kevin A. Schug
2 Comments
Terpene_KAS2

Have you ever wondered why all beers have that strong, characteristic smell? Or why you could tell the smell of cannabis apart from any other plant? The answer is simple – terpenes.

These 55,000 different molecules are responsible for a majority of the odors and fragrances around us, from a pine forest, to the air diffuser in your house 1–3. They all share the same precursor, isoprene, and because of that, they are all related and have similar molecular structures. Unfortunately, it is this uncanny similarity that makes their analysis so challenging; we still lack a complete list of which terpenes expected to be found in each given plant species 1,2.

Many different methods have been developed in an effort to provide a time-optimized and straightforward analysis. Gas chromatography (GC) is usually center stage due to the volatility of the terpenes. Therefore, there is significant concern with the type of GC detector used 2.

The flame ionization detector (FID) is a good quantitative detector for GC, but qualitatively it does not provide any information, except for retention time; the differentiation between terpene species is achieved solely by use of retention indices (RI), which are based on elution times from a particular GC stationary phase. The best part of the FID is its low cost, reliability, and relatively easy interface, which make it an effective tool for quality control (QC) but less so with respect to research and discovery 2.

The primary choice for a research setting is the mass spectrometer (MS) detector. It is more expensive and complicated than FID, but importantly, it provides both good quantitative capabilities, and it provides mass spectra for each species that elutes from the chromatograph. However, for terpene analysis, it may still not be the best detector choice. Since terpene class molecules share many structural and functional similarities, even their fragmentation and sub-sequential identification by MS may lead to inconsistent results, which need to be confirmed by use of RI. Still, MS is a better qualitative analysis tool than the FID, especially for distinguishing non-isobaric terpenes 2.

Recently, new technology based on vacuum ultraviolet spectroscopy (VUV) has been developed as a new GC detector. The VUV detector enables analysis of virtually all molecules; virtually all chemical compounds absorb light in the range in the 125-240 nm wavelength range probed by the detector, making it an essentially universal detector 4–11. Previously, spectroscopic absorption detectors for GC have lacked sufficient energy to measure absorption of most GC-amenable species. The VUV detector fills a niche, which is complementary to MS detection in terms of the qualitative information it provides.

Terpene_KAS2
Figure 1: A, Section of the chromatographic separation of a terpenes standard mix; B, highlight of the co-eluting terpenes, camphor and (-)-isopulegol; C, differences in the absorbance spectra of camphor and (-)-isopulegol.

With the VUV detector, each compound exhibits its own unique absorbance spectrum. Even isomers and isobars, which are prevalent in terpene mixtures and can be difficult to distinguish different species by their electron ionization mass spectra, can be well differentiated based on their VUV spectra 6,9,10.  Nevertheless, because analytes exhibit different spectra, it is not required to achieve a perfect chromatographic separation of the mixture components. Co-eluting peaks can be separated post-run through the use of library spectra and software inherent to the instrument 4,10. This ability is called “deconvolution”, and it is based on the fact that two co-eluting terpenes will give a peak with an absorbance spectrum equal to the sum of the two single absorbance spectra 4. Figure 1 shows the deconvolution process for two co-eluting terpenes, camphor and (-)-isopulegol. Due to their different absorbance spectra (Figure 1C), it is possible to fully separate the two peaks in post-run, obtaining sharp peaks for both analytes 6.

The deconvolution process has been shown to yield precise and accurate results. Thus, chromatographic resolution can be sacrificed in favor of spectroscopic resolution; this enables the development of methods with faster run times. With the ability to deconvolve unresolved peaks, a long temperature ramp to chromatographically separate all isomeric terpenes is not required 6. Additionally, the presence of coeluting components, which might normally go undetected with some GC detectors, can be easily judged based on comparison of the measured spectra with pure reference spectra contained in the VUV spectral library.

The other issue in terpenes analysis is the extraction process. Terpenes can be extracted with the use of solvents (e.g., methanol, ethanol, hexane, and cyclohexane, among others), but the process is usually time-consuming, costly and not so environmentally-friendly 2. The plant needs to be manually crushed and then aliquots of solvent are used to extract components from the plant, ideally at least 3 times and combined to achieve acceptable results. The problem is that some terpenes may respond better to a certain solvent, making their extraction easier and more optimized than for others 2. The choice of solvent can cause discrimination against the extraction some terpenes, which limits the comprehensiveness of analysis.

Headspace is another technique that can be used for the sample preparation of terpenes. Headspace sampling is based on heating the solid or liquid sample inside a sealed vial, and then analyzing the air above it after sufficient equilibration. In this way, only volatile analytes are extracted from the solid/liquid sample into the gas phase; this allows relatively interference-free sampling 12–14.

How do we know whether our extraction analysis methods are correct and comprehensive for a certain plant sample? Unfortunately, there is not a complete list of available molecules for each plant species, and even if two specimens may smell really similar to our nose, their terpenes profiles may be notably different. When working with a new plant material, it is difficult to predict the extraction efficiency for the vast array of terpenes that may be present. We can only perform it with different extraction and detection methods, and compare the results.

The route for a comprehensive and fast analysis of terpenes is therefore still long; however, their intoxicating aromas and inherent medicinal value has provided a growing impetus for researchers around the world. Considering the evolving importance of Cannabis and the growing body of evidence on the synergistic effects between terpenes and cannabinoids, it is likely that newly improved extraction and analysis methods will be developed, paving the way for a more complete list of terpene species that can be found in different cultivars. The use of new analytical technologies, such as the VUV detector for GC, should aid considerably in this endeavor.


References:

[1]          Breitmaier E., Terpenes: Flavors, Fragrances, Pharmaca, Pheromones. John Wiley & Sons 2006.

[2]          Leghissa A., Hildenbrand Z. L., Schug K. A., A Review of Methods for the Chemical Characterization of Cannabis Natural Products. J. Sep. Sci.2018, 41, 398–415 .

[3]          Benvenuto E., Misra B. B., Stehle F., Andre C. M., Hausman J.-F., Guerriero G., Cannabis sativa: The Plant of the Thousand and One Molecules. Front. Plant Sci2016, 719, DOI: 10.3389/fpls.2016.00019.

[4]          Schug K. A., Sawicki I., Carlton D. D., Fan H.,Mcnair H. M.,Nimmo J. P., Kroll P.,Smuts J.,Walsh P., Harrison D., Vacuum Ultraviolet Detector for Gas Chromatography. Anal. Chem.2014, 86, 8329–8335 .

[5]          Fan H.,Smuts J., Walsh P.,Harrison D., Schug K. A., Gas chromatography-vacuum ultraviolet spectroscopy for multiclass pesticide identification. J. Chromatogr. A2015, DOI: 10.1016/j.chroma.2015.02.035.

[6]          Qiu C.,Smuts J., Schug K. A., Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J. Sep. Sci.2017, 40, 869–877 .

[7]          Leghissa A., Smuts J., Qiu C., Hildenbrand Z. L., Schug K. A., Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Sep. Sci. Plus2018, 1.

[8]          Bai L.,Smuts J., Walsh P., Fan H., Hildenbrand Z., Wong D., Wetz D., Schug K. A., Permanent gas analysis using gas chromatography with vacuum ultraviolet detection. J. Chromatogr. A2015,1388, 244–250 .

[9]          Skultety L., Frycak P., Qiu C.,Smuts J., Shear-Laude L., Lemr K., Mao J. X., Kroll P., Schug K. A., Szewczak A., Vaught C., Lurie I., Havlicek V., Resolution of isomeric new designer stimulants using gas chromatography – Vacuum ultraviolet spectroscopy and theoretical computations. Anal. Chim. Acta2017, 971, 55–67 .

[10]       Bai L., Smuts J., Walsh P., Qiu C., McNair H. M., Schug K. ., Pseudo-absolute quantitative analysis using gas chromatography–vacuum ultraviolet spectroscopy–a tutorial. Anal. Chim. Acta2017, 953, 10–22 .

[11]       Schenk J., Nagy G., Pohl N. L. B., Leghissa A., Smuts J., Schug K. A., Identification and deconvolution of carbohydrates with gas chromatography-vacuum ultraviolet spectroscopy. J. Chromatogr. A2017, 1513, 210–221 .

[12]       Van Opstaele F., De Causmaecker B., Aerts G., De Cooman L., Characterization of novel varietal floral hop aromas by headspace solid phase microextraction and gas chromatography-mass spectrometry/olfactometry. J. Agric. Food Chem.2012, 60, 12270−12281 .

[13]       Hamm S., Bleton J., Connan J., Tchapla A., A chemical investigation by headspace SPME and GC-MS of volatile and semi-volatile terpenes in various olibanum samples. Phytochemistry2005,66, 1499–1514 .

[14]       Aberl A., Coelhan M., Determination of volatile compounds in different hop Varieties by headspace-trap GC/MS-in comparison with conventional hop essential oil analysis. J. Agric. Food Chem.2012, 60, 2785−2792 .

oregon

Turning the Oregon Outdoor Market into a Research Opportunity

By Dr. Zacariah Hildenbrand, Dr. Kevin A. Schug
No Comments
oregon

Much has been made about the plummeting market value of cannabis grown outdoors in Oregon. This certainly isn’t a reflection of the product quality within the marketplace, but more closely attributable to the oversaturation of producers in this space. This phenomenon has similarities to that of ‘Tulip Mania’ within the Dutch Golden Age, whereby tulip bulbs were highly coveted assets one day, and almost worthless the next. During times like these, it is very easy for industry professionals to become disheartened; however, from a scientific perspective, this current era in Oregon represents a tremendous opportunity for discovery and fundamental research.

Dr. Zacariah Hildenbrand
Dr. Zacariah Hildenbrand, chief technical officer at Inform Environmental.

As we have mentioned in previous presentations and commentaries, our research group is interested in exploring the breadth of chemical constituents expressed in cannabis to discover novel molecules, to ultimately develop targeted therapies for a wide range of illnesses. Intrinsically, this research has significant societal implications, in addition to the potential financial benefits that can result from scientific discovery and the development of intellectual property. While conducting our experiments out of Arlington, Texas, where the study of cannabis is highly restricted, we have resorted to the closet genetic relative of cannabis, hops (Humulus lupulus), as a surrogate model of many of our experiments (Leghissa et al., 2018a). In doing so, we have developed a number of unique methods for the characterization of various cannabinoids and their metabolites (Leghissa et al., 2018b; Leghissa et al., 2018c). These experiments have been interesting and insightful; however, they pale in comparison to the research that could be done if we had unimpeded access to diverse strains of cannabis, as are present in Oregon. For example, gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) is a relatively new tool that has recently been proven to be an analytical powerhouse for the differentiation of various classes of terpene molecules (Qiu et al., 2017). In Arlington, TX, we have three such GC-VUV instruments at our disposal, more than any other research institution in the world, but we do not have access to appropriate samples for application of this technology. Similarly, on-line supercritical fluid extraction – supercritical fluid chromatography – mass spectrometry (SFE-SFC-MS) is another capability currently almost unique to our research group. Such an instrument exhibits extreme sensitivity, supports in situ extraction and analysis, and has a wide application range for potential determination of terpenes, cannabinoids, pesticides and other chemical compounds of interest on a single analytical platform. Efforts are needed to explore the power and use of this technology, but they are impeded based on current regulations.

Dr Kevin Schug
Dr. Kevin A. Schug, Professor and the Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry and Biochemistry at The University of Texas at Arlington (UTA)

Circling back, let’s consider the opportunities that lie within the abundance of available outdoor-grown cannabis in Oregon. Cannabis is extremely responsive to environmental conditions (i.e., lighting, water quality, nutrients, exposure to pest, etc.) with respect to cannabinoid and terpene expression. As such, outdoor-grown cannabis, despite the reduced market value, is incredibly unique from indoor-grown cannabis in terms of the spectrum of light to which it is exposed. Indoor lighting technologies have come a long way; full-spectrum LED systems can closely emulate the spectral distribution of photon usage in plants, also known as the McCree curve. Nonetheless, this is emulation and nothing is ever quite like the real thing (i.e., the Sun). This is to say that indoor lighting can certainly produce highly potent cannabis, which exhibits an incredibly robust cannabinoid/terpene profile; however, one also has to imagine that such lighting technologies are still missing numerous spectral wavelengths that, in a nascent field of study, could be triggering the expression of unknown molecules with unknown physiological functions in the human body. Herein lies the opportunity. If we can tap into the inherently collaborative nature of the cannabis industry, we can start analyzing unique plants, having been grown in unique environments, using unique instruments in a facilitative setting, to ultimately discover the medicine of the future. Who is with us?


References

Leghissa A, Hildenbrand ZL, Foss FW, Schug KA. Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry. J Sep Sci 2018a; 41: 459-468.

Leghissa A, Hildenbrand ZL, Schug KA. Determination of the metabolites of Δ9-Tetrahydrocannabinol using multiple reaction monitoring gas chromatography – triple quadrapole – mass spectrometry. Separation Science Plus 2018b; 1: 43-47.

Leghissa A, Smuts J, Changling Q, Hildenbrand ZL, Schug KA. Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Separation Science Plus 2018c; 1: 37-42.

Qiu C, Smuts J, Schug KA. Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J Sep Sci 2017; 40: 869-877.

Multi-analyte Configuration for Cannabis Testing Services

Managing Cannabis Testing Lab Workflows using LIMS

By Dr. Susan Audino
No Comments
Multi-analyte Configuration for Cannabis Testing Services

With the state led legalization of both adult recreational and medical cannabis, there is a need for comprehensive and reliable analytical testing to ensure consumer safety and drug potency. Cannabis-testing laboratories receive high volumes of test requests from cannabis cultivators for testing quantitative and qualitative aspects of the plant. The testing market is growing as more states bring in stricter enforcement policies on testing. As the number of testing labs grow, it is anticipated that the laboratories that are now servicing other markets, including high throughput contract labs, will cross into cannabis testing as regulations free up. As the volume of tests each lab performs increases, the need for laboratories to make effective use of time and resource management, such as ensuring accurate and quick results, reports, regulatory compliance, quality assurance and many other aspects of data management becomes vital in staying competitive.

Cannabis Testing Workflows

To be commercially competitive, testing labs offer a comprehensive range of testing services. These services are available for both the medical and recreational cannabis markets, including:

  • Detection and quantification of both acid and neutral forms of cannabinoids
  • Screening for pesticide levels
  • Monitoring water activity to indicate the possibility of microbiological contamination
  • Moisture content measurements
  • Terpene profiling
  • Residual solvents and heavy metal testing
  • Fungi, molds, mycotoxin testing and many more

Although the testing workflows differ for each test, here is a basic overview of the operations carried out in a cannabis-testing lab:

  1. Cannabis samples are received.
  2. The samples are processed using techniques such as grinding and homogenization. This may be followed by extraction, filtration and evaporation.
  3. A few samples will be isolated and concentrated by dissolving in solvents, while others may be derivatized using HPLC or GC reagents
  4. The processed samples are then subjected to chromatographic separation using techniques such as HPLC, UHPLC, GC and GC-MS.
  5. The separated components are then analyzed and identified for qualitative and quantitative analysis based on specialized standards and certified reference materials.
  6. The quantified analytical data will be exported from the instruments and compiled with the corresponding sample data.
  7. The test results are organized and reviewed by the lab personnel.
  8. The finalized test results are reported in a compliant format and released to the client.

In order to ensure that cannabis testing laboratories function reliably, they are obliged to follow and execute certain organizational and regulatory protocols throughout the testing process. These involve critical factors that determine the accuracy of testing services of a laboratory.

Factors Critical to a Cannabis Testing Laboratory 

  • Accreditations & Regulatory Compliance: Cannabis testing laboratories are subject to regulatory compliance requirements, accreditation standards, laboratory practices and policies at the state level. A standard that most cannabis testing labs comply to is ISO 17025, which sets the requirements of quality standards in testing laboratories. Accreditation to this standard represents the determination of competence by an independent third party referred to as the “Accreditation Body”. Accreditation ensures that laboratories are adhering to their methods. These testing facilities have mandatory participation in proficiency tests regularly in order to maintain accreditation.
  • Quality Assurance, Standards & Proficiency Testing: Quality assurance is in part achieved by implementing standard test methods that have been thoroughly validated. When standard methods are not available, the laboratory must validate their own methods. In addition to using valid and appropriate methods, accredited laboratories are also required to participate in appropriate and commercially available Proficiency Test Program or Inter-Laboratory Comparison Study. Both PT and ILC Programs provide laboratories with some measure of their analytic performance and compare that performance with other participating laboratories.

    Multi-analyte Configuration for Cannabis Testing Services
    CloudLIMS Cannabis Testing LIMS: Multi-analyte Configuration for Cannabis Testing Services
  • Real-time Collaboration: Testing facilities generate metadata such as data derived from cannabis samples and infused products. The testing status and test results are best served for compliance and accessibility when integrated and stored on a centralized platform. This helps in timely data sharing and facilitates informed decision making, effective cooperation and relationships between cannabis testing facilities and growers. This platform is imperative for laboratories that have grown to high volume throughput where opportunities for errors exist. By matching test results to samples, this platform ensures consistent sample tracking and traceability. Finally, the platform is designed to provide immediate, real-time reporting to individual state or other regulatory bodies.
  • Personnel Management: Skilled scientific staff in cannabis-testing laboratories are required to oversee testing activities. Staff should have experience in analytical chromatography instruments such as HPLC and GC-MS. Since samples are often used for multi-analytes such as terpenes, cannabinoids, pesticides etc., the process often involves transferring samples and tests from one person to another within the testing facility. A chain of custody (CoC) is required to ensure traceability and ‘ownership’ for each person involved in the workflow.

LIMS for Laboratory Automation

Gathering, organizing and controlling laboratory-testing data can be time-consuming, labor-intensive and challenging for cannabis testing laboratories. Using spreadsheets and paper methods for this purpose is error-prone, makes data retrieval difficult and does not allow laboratories to easily adhere to regulatory guidelines. Manual systems are cumbersome, costly and lack efficiency. One way to meet this challenge is to switch to automated solutions that eliminate many of the mundane tasks that utilize valuable human resources.. Laboratory automation transforms the data management processes and as a result, improves the quality of services and provides faster turnaround time with significant cost savings. Automating the data management protocol will improve the quality of accountability, improve technical efficiency, and improve fiscal resources.

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Real Time Test Status in CloudLIMS

A Laboratory Information Management System (LIMS) is a software tool for testing labs that aids efficient data management. A LIMS organizes, manages and communicates all laboratory test data and related information, such as sample and associated metadata, tests, Standard Operating Procedures (SOPs), test reports, and invoices. It also enables fully automated data exchange between instruments such as HPLCs, GC-FIDs, etc. to one consolidated location, thereby reducing transcription errors.

How LIMS Helps Cannabis Testing Labs

LIMS are much more capable than spreadsheets and paper-based tools for streamlining the analytical and operational lab activities and enhances the productivity and quality by eliminating manual data entry. Cloud-enabled LIMS systems such as CloudLIMS are often low in the total cost of acquisition, do not require IT staff and are scalable to help meet the ever changing business and regulatory compliance needs. Some of the key benefits of LIMS for automating a cannabis-testing laboratory are illustrated below [Table 1]:

Key Functionality Benefit
Barcode label designing and printing Enables proper labelling of samples and inventory

Follows GLP guidelines

Instant data capture by scanning barcodes Facilitates quick client registration and sample access
3600 data traceability Saves time and resources for locating samples and other records
Inventory and order management Supports proactive planning/budgeting and real time accuracy
Custodian management Promotes overall laboratory organization by assigning custodians for samples and tests

Maintains the Chain-of-custody (CoC)

Test management Accommodates pre-loaded test protocols to quickly assign tests for incoming samples
Accounting for sample and inventory quantity Automatically deducts sample and inventory quantities when consumed in tests
Package & shipment management Manages incoming samples and samples that have been subcontracted to other laboratories
Electronic data import Electronically imports test results and metadata from integrated instruments

Eliminates manual typographical errors

Report management Generates accurate, customizable, meaningful and test reports for clients

Allows user to include signatures and additional sections for professional use

21 CFR Part 11 compliant Authenticates laboratory activities with electronic signatures
ISO 17025 accreditation Provides traceable documentary evidence required to achieve ISO 17025 accreditation
Audit trail capabilities Adheres to regulatory standards by recording comprehensive audit logs for laboratory activities along with the date and time stamp
Centralized data management Stores all the data in a single, secure database facilitating quick data retrieval
Workflow management Promotes better data management and resource allocation
High-configurability Enables modification of screens using graphical configuration tools to mirror testing workflows
State compliance systems Integrates with state-required compliance reporting systems and communicates using API
Adheres to regulatory compliance Creates Certificates of Analysis (CoA) to prove regulatory compliance for each batch as well as batch-by-batch variance analysis and other reports as needed.
Data security & confidentiality Masks sensitive data from unauthorized user access

 

Cloud-based LIMS encrypts data at rest and in-transit while transmission between the client and the server

Global accessibility Cloud-based LIMS provides real-time access to laboratory data from anytime anywhere
Real-time collaboration Cloud-based LIMS enhances real-time communication within a laboratory, between a laboratory and its clients, and across a global organization with multiple sites

Table 1. Key functionality and benefits of LIMS for cannabis testing laboratories

Upon mapping the present day challenges faced by cannabis testing laboratories, adopting laboratory automation solutions becomes imperative. Cloud-based LIMS becomes a valuable tool for laboratory data management in cannabis testing laboratories. In addition to reducing manual workloads, and efficient resource management, it helps labs focus on productive lab operations while achieving compliance and regulatory goals with ease.

For more information on this, check out a webinar here: Webinar: How to Meet Cannabis Testing Standards and Regulatory Requirements with LIMS by Stephen Goldman, laboratory director at the State of Colorado certified Cannabis testing facility, PhytaTech.