Distinguishing Between Chemovars with Mass Spectrometry and Chemometrics

The luxurious resin of the cannabis plant is made up of secondary metabolites like cannabinoids and terpenes that help the plant survive. Each plant contains a unique consortium of these molecules, they, in turn, comprising the resin’s chemical fingerprint, or chemovar. To know the chemovar provides us with the molecular story written in a given plant’s trichomes, and the data to determine why the organoleptic experience is what it is as well as what it may offer from a medicinal perspective. Just as the chemovar helps the plant endure, the plant extends those defensive chemical gifts to us.

Cannabis chemovars have been previously determined using several analytical and chemometric methods. Doctors Arno Hazekamp and Justin Fischedick, for example, evaluated 460 cannabis “accessions” using gas chromatography/mass spectrometry (GC-MS) and principal component analysis (PCA). [1,2] They found that “the presence of the terpenes trans-bergamotene, trans-beta-farnesene, delta-3-carene, and terpinolene was most strongly associated with the Sativa-type samples, while beta- and gamma-eudesmol, guaiol, myrcene, and gamma-elemene were the most prominent markers for the Indica-type samples.” What’s more, terpene alcohols were all associated with “Indica-type” cultivars.

Other studies evaluated the chemovars of cannabis flowers available in Nevada and demonstrated some rather startling findings. [3,4] It turns out, for example, that while there were 396 differently named flower products in the state, they boiled down to three basic chemistries. [4] In other words, three chemistries were named almost 400 different things! While some of these names may give farmers the chance to show off their products and novel genetics, it can be seriously confusing for the consumer.

A recent application note published by the instrumentation manufacturer SCIEX demonstrated how their quantitative time-of-flight (qTOF) MS and chemometrics can be used to evaluate your cultivar’s chemovar. In addition to aiding consumers with product selection, SCIEX identifies several other implications for better understanding a plant’s chemovar, including “intellectual property development, metabolomic profiling, and economically motivated adulteration.”

The experiment considered seven cannabis varieties, each of which was extracted into acetonitrile and subsequently diluted 200-fold with methanol. Separation of the phytomolecules was achieved using high-performance liquid chromatography. Mass spectrometry (MS) enabled the determination of m/z (mass to charge) ratios that aid in identifying a molecule.

SCIEX used chemometrics such that they didn’t need to target specific cannabinoids and terpenes. Principal component analysis (PCA) enabled the identification of key marker compounds that best differentiated the plants. Those plants that cluster closer to each other in a PCA score plot are more chemically similar.

This process helped define peaks of interest that could be searched within MSMS libraries. If nothing came up in the library, the next phase of the protocol considered accurate mass and isotope data that enabled an empirical formula to be proposed using the FormulaFinder algorithm. This formula can then be searched for using the ChemSpider database.

Using this process, the application note pointed to cannabinol (CBN) and the sesquiterpenoid xanthorrizol as being two key differentiators between the cultivars, highlighting how this technique can be utilized to identify what molecules distinguish one cannabis plant from another.



[1] Hazekamp A, Fischedick JT. Cannabis – from cultivar to chemovar. Drug Test Anal. 2012;4(7-8):660-667. [journal impact factor = 3.345; times cited = 99] [2] Hazekamp A, Tejkalová K, Papadimitriou S. Cannabis: From cultivar to chemovar II—A metabolomics approach to Cannabis classification. Cannabis & Cannabinoid Research. 2016;1(1). [journal impact factor = N/A; times cited = 50] [3] Orser C, Johnson S, Speck M, Hilyard A, Afia I. Terpenoid chemoprofiles distinguish drug-type Cannabis sativa L. cultivars in Nevada. Natural Products Chemistry & Research. 2018;6:1-7. [journal impact factor = 2.158; times cited = 9] [4] Reimann-Philipp U, Speck M, Orser C, et al. Cannabis chemovar nomenclature misrepresents chemical and genetic diversity; Survey of variations in chemical profiles and genetic markers in Nevada medical Cannabis samples. Cannabis Cannabinoid Res. 2020;5(3):215-230. [journal impact factor = N/A; times cited = 5]

About the author

Jason S. Lupoi, Ph.D.

Jason S. Lupoi, Ph.D.

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