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Leveraging Chemotyping Techniques for Data-driven Classification of Cannabis

By Kimberly Ross

Published: May 31, 2019   
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Cannabis chemotyping might seem, at first glance, to be a trendy new buzz phrase in the industry. Efforts, though, have been underway for decades to establish an objective classification system to distinguish angiosperm specimens of the family Cannabaceae based on their suite of chemical constituents. Most notably these studies have focused on the cannabinoids THC and CBD, which led to common acceptance of chemotypic profiling capable of delineating drug-type marijuana and fiber-type hemp from a forensic standpoint.

The term chemotype refers to the chemical phenotypes found in cannabis and other plants. We can think of cannabis chemotyping as the tallying of chemical compounds with pharmacological (and forensic) relevance, the results of which assist in classifying the vast array of cannabis cultivars into functional groups. In other words, clusters of strains give different physiologically-based experiences to the end-user. Chemotypic profiling of medicinal and culinary plants and the study of chemotypic variation is common, for example, in lavender, peppermint, basil, thyme, sage, and many others. In another example of borrowed horticultural science from more established fields, chemotypes describe the phytochemical diversity of cannabis phenotypes by measuring their chemical constituents. This is inherently useful in re-emerging and novel medicinal applications of cannabis, in addition to the increasing necessity of forensic identification to distinguish industrial hemp from psychoactive and medicinal cannabis.

Chemotype is formally defined as “the subspecies of a plant that have the same morphological characteristics (relating to form and structure) but produce different quantities of chemical components in their essential oils.” The specific suite of chemicals in a particular cultivar are accountable for the plant’s therapeutic properties and degree of efficacy in humans. Since Ethan Russo’s popularization of the entourage effect hypothesis, we now think of cannabis chemotyping as including a suite of secondary compounds beyond THC and CBD, with the ability to enhance the beneficial effects of therapeutic cannabinoids.

The terpene molecules in cannabis are thought to be responsible for augmenting the physiological relevant effects that give rise to a wide variety of user experiences. Efforts to characterize thousands of cannabis strains into chemotaxonomic groups according to terpene content (when cannabinoid content is held relatively constant) is encouraging in terms of potential clustering patterns. We can imagine in order to draw meaningful conclusions, it is paramount the analytical data are accurate and defensible to lay proper foundational database(s).

Wherein lies the usefulness of cannabis chemotyping?

Forensically differentiating hemp that can cross state borders from higher-THC varieties that cannot, under the current US legal definition of hemp, necessitates total THC be less than 0.3 percent of the total biomass. As a consequence, we truly need sensitive and accurate testing capabilities on which to rely. There is no buffer zone written into the language of the law at this point in time, so even a hemp sample that tests at 0.31 percent would fail to be compliant hemp. Due to regulations for cannabis (>0.3 percent THC) and hemp (>0.3 percent THC) that are so drastically different, the most precise analytical approaches must be utilized to delineate these crops.

In pharmacology, chemotyping is especially useful in documenting which combinations of phytochemicals confer significant efficacy when used to mediate a variety of conditions. We know from studies of nervous system responses that fragrance can modulate human psychophysiological activity, presumably through our brains’ genetic network of olfactory receptors. Less is known about the neurological effects of cannabis terpene ingestion, a potentially fruitful arena for scientific inquiry

What are the best available methods for chemotyping?

An orthogonal approach to chemotyping is recommended, in which the sample is quantified and mass-verified. Instrument manufacturers are in support of rigorous testing for the industry. For example, Agilent offers training on an “orthogonal analytical strategy for screening and putative quantification of tetrahydrocannabinol in hemp, followed by speciation and quantitative confirmation.”

The authors of the training describe the need for robust and rigorous testing as follows:

“The Agriculture Improvement Act, also known as the Farm Bill, was signed into law in December 2018. A major provision in the law legalizes hemp as an industrial crop. The United States Federal Register defines industrial hemp as any part or derivative (including seeds) of the plant Cannabis sativa L. with a dry weight concentration of “tetrahydrocannabinols” not greater than 0.3 percent by dry weight of the plant material. Tetrahydrocannabinols specifically refers to salts and isomers of ∆9- tetrahydrocannabinol (THC). Any hemp plant material that exceeds this threshold is defined as marijuana and considered an illegal Schedule I narcotic.”

“Analytically, cannabinoid quantitation is very commonly performed via high-performance liquid chromatography (HPLC) using ultraviolet (UV) detectors. These assays generally identify and quantify THC, tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), Cannabidiolic acid (CBDA) and other cannabinoids where commercially available reference standards are available. With respect to hemp, the purpose of cannabinoid quantitation for hemp plants or products is to assure they contain less than 0.3 percent (wt/wt) THC and therefore comply with federal law. However, the certification or rejection of a crop as hemp will most certainly have forensic implications and as such, the resulting data must be legally defensible. Typically, forensic data incorporates confirmation via two orthogonal analytical methodologies and very often, mass spectrometry for speciation and quantification.”

To what extent does chemotype correlate with taxonomy?

Others have suggested the collection of data characterizing chemical phenotype point to a continuum of chemotypes, rather than discrete groupings. The goal is to derive objective classification systems to help sort specimens based upon their chemical profiles. Taxonomic systems usually group organisms based on morphological characteristics, and more recently genetic elements common to all organisms. Presumably, highly similar organisms morphologically are closely related taxonomically. But taxonomy is tricky within a single species, due to the high degree of similarity among the genomes. Especially in cannabis, due to its recent modern history of rampant hybridization, the family tree or ‘pedigree’ is what phylogeneticists might call “hazy,” meaning the distinct branching pattern remains unresolved at the sub-species level.

Genomic underpinnings of cannabis chemotypes

What will the future of cannabis chemotyping bring? The holy grail of the cannabis experience is the ability to reproduce pharmacologically consistent results. The goal is to go beyond the classic indica-sativa dichotomy in order to more specifically correlate effects with phenotype, and taking into account terpenes and other compounds that contribute to the user experience.  This could potentially distill an infinite number of strain names into several categories based on the user experience, in both therapeutic and recreational capacities. As Dr. Ethan Russo reports:

“There are biochemically distinct strains of Cannabis, but the sativa/indica distinction as commonly applied in the lay literature is total nonsense and an exercise in futility. One cannot in any way currently guess the biochemical content of a given Cannabis plant based on its height, branching, or leaf morphology. The degree of interbreeding/hybridization is such that only a biochemical assay tells a potential consumer or scientist what is really in the plant. It is essential that future commerce allows complete and accurate cannabinoid and terpenoid profiles to be available.”

The genetic fingerprints that encode the biological instructions for the suites of chemical compounds across cultivars provide a confirmatory methodology to characterize and categorize the diversity of strains. Researchers are targeting the sequence of the genetic code within the genes that give rise to cannabinoids and terpenes - called synthases - in order to develop a genetic test akin to a human paternity test. However, cannabis genomes are not straightforward, and we are only beginning to learn the ways gene silencing and gene duplication events affect the ultimate outcome of the plant’s chemical phenotype. In addition to the hybridization resulting from modern breeding, and the range of environmental conditions also affecting the ultimate outcome, our best bet is to develop massive databases in parallel, of both the genomic and chemical fingerprint for each specimen, in order to derive the best possible classification system for this botanically unique and complex plant.

For further reading:

● Cannabaceae | Description, Genera, & Species https://www.britannica.com/plant/Cannabaceae

● Meijer, E. de. The Chemical Phenotypes (Chemotypes) of Cannabis; Oxford University Press, 2014.

● Medina-Holguín, A. L.; Holguín, F. O.; Micheletto, S.; Goehle, S.; Simon, J. A.; O’Connell, M. A. Chemotypic Variation of Essential Oils in the Medicinal Plant, Anemopsis Californica. Phytochemistry 2008, 69 (4), 919–927. https://doi.org/10.1016/j.phytochem.2007.11.006.

● Muñoz-Bertomeu, J.; Arrillaga, I.; Segura, J. Essential Oil Variation within and among Natural Populations of Lavandula Latifolia and Its Relation to Their Ecological Areas. Biochem. Syst. Ecol. 2007, 35 (8), 479–488. https://doi.org/10.1016/j.bse.2007.03.006.

● Clarke, S. Chapter 7 - Composition of Essential Oils and Other Materials. In Essential Chemistry for Aromatherapy (Second Edition); Clarke, S., Ed.; Churchill Livingstone: Edinburgh, 2008; pp 123–229. https://doi.org/10.1016/B978-0-443-10403-9.00007-8.

● Russo, E. B. Taming THC: Potential Cannabis Synergy and Phytocannabinoid-Terpenoid Entourage Effects. Br. J. Pharmacol. 2011, 163 (7), 1344–1364. https://doi.org/10.1111/j.1476-5381.2011.01238.x.

● Ben-Shabat, S.; Fride, E.; Sheskin, T.; Tamiri, T.; Rhee, M. H.; Vogel, Z.; Bisogno, T.; De Petrocellis, L.; Di Marzo, V.; Mechoulam, R. An Entourage Effect: Inactive Endogenous Fatty Acid Glycerol Esters Enhance 2-Arachidonoyl-Glycerol Cannabinoid Activity. Eur. J. Pharmacol. 1998, 353 (1), 23–31.

● Mcpartland, J.; Russo, E. B. Cannabis and Cannabis Extracts: Greater Than the Sum of Their Parts?; 2001. https://doi.org/10.1300/J175v01n03_08.

● Richins, R. D.; Rodriguez-Uribe, L.; Lowe, K.; Ferral, R.; O’Connell, M. A. Accumulation of Bioactive Metabolites in Cultivated Medical Cannabis. PLOS ONE 2018, 13 (7), e0201119. https://doi.org/10.1371/journal.pone.0201119.

● Fischedick, J. T.; Hazekamp, A.; Erkelens, T.; Choi, Y. H.; Verpoorte, R. Metabolic Fingerprinting of Cannabis Sativa L., Cannabinoids and Terpenoids for Chemotaxonomic and Drug Standardization Purposes. Phytochemistry 2010, 71 (17), 2058–2073. https://doi.org/10.1016/j.phytochem.2010.10.001.

● Calvi, L.; Pavlovic, R.; Panseri, S.; Giupponi, L.; Leoni, V.; Giorgi, A. Quality Traits of Medical Cannabis Sativa L. Inflorescences and Derived Products Based on Comprehensive Mass-Spectrometry Analytical Investigation. Recent Adv. Cannabinoid Res. 2018. https://doi.org/10.5772/intechopen.79539.

● Jikomes, N.; Zoorob, M. The Cannabinoid Content of Legal Cannabis in Washington State Varies Systematically Across Testing Facilities and Popular Consumer Products. Sci. Rep. 2018, 8 (1), 4519. https://doi.org/10.1038/s41598-018-22755-2.

● United Nations Office on Drugs and Crime. Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products Manual for Use by National Drug Analysis Laboratories; United Nations: New York, 2009.

● Bahr, T. A.; Rodriguez, D.; Beaumont, C.; Allred, K. The Effects of Various Essential Oils on Epilepsy and Acute Seizure: A Systematic Review https://www.hindawi.com/journals/ecam/2019/6216745/abs/. https://doi.org/10.1155/2019/6216745.

● Seca, A. M. L.; Pinto, D. C. G. A. Plant Secondary Metabolites as Anticancer Agents: Successes in Clinical Trials and Therapeutic Application. Int. J. Mol. Sci. 2018, 19 (1), 263. https://doi.org/10.3390/ijms19010263.

● Sowndhararajan, K.; Kim, S. Influence of Fragrances on Human Psychophysiological Activity: With Special Reference to Human Electroencephalographic Response. Sci. Pharm. 2016, 84 (4), 724–752. https://doi.org/10.3390/scipharm84040724.

● Anthony Macherone, PhD, Sr. Scientist, Agilent Technologies. Is It Hemp? An Analytical Strategy To Make Sure It Is.

● Fischedick, J. T. Identification of Terpenoid Chemotypes Among High (−)-Trans-Δ9- Tetrahydrocannabinol-Producing Cannabis Sativa L. Cultivars. Cannabis Cannabinoid Res. 2017, 2 (1), 34–47. https://doi.org/10.1089/can.2016.0040.

● Merlin, M.; Clarke, R. Cannabis: Evolution and Ethnobotany.

● Small, E. Evolution and Classification of Cannabis Sativa (Marijuana, Hemp) in Relation to Human Utilization. Bot. Rev. 2015, 81 (3), 189–294. https://doi.org/10.1007/s12229-015-9157-3.

● McPartland, J. M. Cannabis Systematics at the Levels of Family, Genus, and Species. Cannabis Cannabinoid Res. 2018, 3 (1), 203–212. https://doi.org/10.1089/can.2018.0039.

● Russo, E. B.; Marcu, J. Cannabis Pharmacology: The Usual Suspects and a Few Promising Leads. Adv. Pharmacol. San Diego Calif 2017, 80, 67–134. https://doi.org/10.1016/bs.apha.2017.03.004.

● Piomelli, D.; Russo, E. B. The Cannabis Sativa Versus Cannabis Indica Debate: An Interview with Ethan Russo, MD. Cannabis Cannabinoid Res. 2016, 1 (1), 44–46. https://doi.org/10.1089/can.2015.29003.ebr.

● Welling, M. T.; Shapter, T.; Rose, T. J.; Liu, L.; Stanger, R.; King, G. J. A Belated Green Revolution for Cannabis: Virtual Genetic Resources to Fast-Track Cultivar Development. Front. Plant Sci. 2016, 7, 1113. https://doi.org/10.3389/fpls.2016.01113.

● Rotherham, D.; Harbison, S. A. Differentiation of Drug and Non-Drug Cannabis Using a Single Nucleotide Polymorphism (SNP) Assay. Forensic Sci. Int. 2011, 207 (1), 193–197. https://doi.org/10.1016/j.forsciint.2010.10.006.

● Russo, E. B. The Case for the Entourage Effect and Conventional Breeding of Clinical Cannabis: No “Strain,” No Gain. Front. Plant Sci. 2018, 9, 1969. https://doi.org/10.3389/fpls.2018.01969.

● Staginnus, C.; Zörntlein, S.; de Meijer, E. A PCR Marker Linked to a THCA Synthase Polymorphism Is a Reliable Tool to Discriminate Potentially THC-Rich Plants of Cannabis Sativa L. J. Forensic Sci. 2014, 59 (4), 919–926. https://doi.org/10.1111/1556-4029.12448.

● Dufresnes, C.; Jan, C.; Bienert, F.; Goudet, J.; Fumagalli, L. Broad-Scale Genetic Diversity of Cannabis for Forensic Applications. PloS One 2017, 12 (1), e0170522. https://doi.org/10.1371/journal.pone.0170522.

● Campbell, L. G.; Dufresne, J.; Sabatinos, S. A. Cannabinoid Inheritance Relies on Complex Genetic Architecture. Cannabis Cannabinoid Res. 2019. https://doi.org/10.1089/can.2018.0015.

● van Bakel, H.; Stout, J. M.; Cote, A. G.; Tallon, C. M.; Sharpe, A. G.; Hughes, T. R.; Page, J. E. The Draft Genome and Transcriptome of Cannabis Sativa. Genome Biol. 2011, 12 (10), R102. https://doi.org/10.1186/gb-2011-12-10-r102.

● Hazekamp, A.; Fischedick, J. T. Cannabis - from Cultivar to Chemovar. Drug Test. Anal. 2012, 4 (7–8), 660–667. https://doi.org/10.1002/dta.407.

Kimberly Ross

Chief Science Officer at Peak Compliance, LLC

Kim is the chief scientific officer at Peak Compliance, LCC, and has written for Analytical Cannabis on lab practices since 2019. She earned her PhD from the University of Colorado's Molecular, Cellular, and Developmental Biology program and is currently a contributing member to ASTM's D37 Committee for development of standards for cannabis products and processes and a participant in the Colorado Marijuana Enforcement Division's cannabis regulatory workgroup.


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