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How Are Developments in Terpene Analysis Shaping the Cannabis Industry?

Feb 20, 2019

How Are Developments in Terpene Analysis Shaping the Cannabis Industry?

The cannabis plant is an extremely complex biological material containing over 500 distinct chemical compounds. Of these various compounds, potentially the most well-known are the cannabinoids. The two major cannabinoids, tetrahydrocannabinol (THC) and cannabidiol (CBD), have been extensively researched and investigated for their intoxicating effect and potential medicinal effects, respectively. 

But as anyone who has ever smelled whole cannabis flower could tell you, the cannabis plant has many more properties than simply its intoxicating or medicinal power. Secreted by the same glands that produce THC, CBD, and many other minor cannabinoids, are the terpenes. Terpene compounds are essential oils and are predominantly responsible for the distinctive aroma of cannabis strains. 

Like many other plants and wildflowers, the wild cannabis plant’s strong smell was advantageous as it could repel predators and lure pollinators. Depending on the climate, weather, region, soil type, and many other similar factors determined by the plant’s environment, the development of certain terpenes in the plant has been affected, leading to an incredible diversity of different terpene profiles between cannabis strains. 

Nowadays, cannabis farming operations mean that certain terpene profiles are no longer needed to ensure the survival of domestically cultivated plants, but the distinctive flavor and aroma that these terpene profiles result in are often key to consumers. Certain strains, such as Blueberry and Lemon Haze notably lean into their distinctive smells of berries and lemon respectively when being marketed, and consumers have even been shown to interpret certain aromas as an indication that the cannabis material is more valuable, or that they would be willing to pay more for those products.

In addition to just altering the smell of whole cannabis flower, it is thought that many terpenes may also have beneficial medicinal properties. For example, Linalool, one of the primary terpenes identified in cannabis, has been linked with anti-anxiety and sedative properties. It is also a major component of the lavender plant, which is commonly used in aromatherapy specifically for these relaxing properties. Limonene, which exudes a strong citrus scent, is another of the major cannabis terpenes that shows medicinal promise, having been shown to act as a strong antidepressant through inhibiting the secretion of stress hormones in the body.

As a result of the extreme diversity of terpene expression between different strains, and the relationship between terpene expression and various medicinal properties or potential economic benefit, having accurate and reliable methods of terpene analysis available is beneficial to patients, sellers, and breeders alike. 


Gas Chromatography as a Key Terpene Analysis Method

For the analysis of cannabinoids, high-performance liquid chromatography (HPLC) is the most common analysis technique, reportedly being relied on by almost half of the total cannabis testing market. HPLC cannabinoid analysis is typically done to determine the potency of a given cannabis sample, or to produce a full cannabinoid profile of the plant. However, when it comes to terpene analysis, many believe there is a better way.

Terpenes are particularly volatile compounds, especially compared to the cannabinoids — THC, the main target for this kind of potency analysis, has a boiling point of around 315°F, whereas the most volatile terpenes in the cannabis plant will start to evaporate at around 70°F. For these types of volatile organic compounds, gas chromatography is usually the preferred analysis method.

And this is what happens in terpene analysis — the most common analytical methods in both academic study and commercial use are all variants on gas chromatography.

“Because [terpenes] contribute to the potency and sensory perception of cannabis, having a reliable and efficient analytical method for terpene analysis is extremely important,” explains Cindy Orser, PhD, the Chief Scientific Officer of Digipath Labs in Nevada. “Because terpenes are volatile, they are well-suited for analysis by gas chromatography.”

The traditional method for analyzing terpenes is GC-FID, or gas chromatography with a flame ionization detector. In this method, the first step is to prepare a solvent-based extraction, usually by placing cannabis plant material in some form of liquid solvent (commonly butane, propane, ethanol, or CO2) in order to extract any terpenes or cannabinoids into the solvent. This extraction liquid is then injected into the gas chromatography apparatus, where the cannabinoids and terpenes in the solvent extraction are separated as they move through the gas chromatograph. Finally, the compounds move onto the flame ionization detector, where they are burned over a hot hydrogen-air flame to produce organic ions. Electrodes present in the FID then measure the current from the ions, which when taken with the retention time of the compound in the gas chromatograph, can be used to identify and quantify the terpenes present in the sample. 


Headspace Solid-Phase Microextraction For Non-Destructive Analysis

While GC-FID may be the traditional method for terpene testing, other new methods are starting to gain favor with those who carry out terpene testing — in particular, headspace solid-phase microextraction (HS-SPME) done in conjunction with GC-mass spectrometry (GC-MS) is becoming more common in academic circles. 

Given their high volatility and vapor pressure, terpene compounds are an ideal candidate for headspace analytical methods, where the volatile components in the gas phase above the sample are analyzed. 

In HS-SPME, a special SPME fiber containing a suitable stationary phase is inserted through the rubber septum of a headspace vial holding the sample being studied. The fiber is held close to the sample for a moderate length of time, allowing the volatile compounds in the gas phase above the sample to adsorb to the fiber. The fiber is then inserted into the injection port of the GC-MS machine, where the volatile compounds desorb and can be analyzed with the GC-MS apparatus. 

“The best option [for terpene analysis] currently is GC-Headspace-MS which provides a means of peak identification and purity for terpene speciation with a run time of about 18 minutes,” adds Orser, emphasizing the fast run time of this sort of analysis, with minimal sample prep. 

This sort of analysis method can also be advantageous for many other reasons. Firstly, unlike GC-FID analysis, HS-SPME with GC-MS is non-destructive. By not having to expose the sample to any chemicals in order to make a solvent-extraction, and by not then having to vaporize that extraction, the original sample can be fully preserved. This also means that the GC analysis will not return any solvent peaks that could confuse or obscure the final analysis results. 

By eliminating solvent use in this way, there is also an environmental motivation to using HS-SPME with GC-MS. The most common cannabis solvents, such as butane, propane, or CO2, all have some sort of detrimental environmental effect, whether that is as a result of the compounds themselves being hazardous, or the experimental procedure requiring the use of artificially high-pressure systems. 


The Importance of Terpene Identification

By themselves, terpene compounds are interesting as they can have a large effect on the odor and flavor of the cannabis plant — which can influence consumer behavior and so is an important metric to players in the cannabis market — and many have also been shown to have direct therapeutic properties when isolated. But, as proponents of whole plant cannabis medicine might say, it is also important to look at the interactions between terpenes and cannabinoids, or other trace compounds, when it comes to using cannabis medicinally. 

The “entourage effect” is the term given to the synergistic interactions between the different compounds in cannabis, including terpenes, which lead to an amplification of the plant’s medicinal effects. In a 2011 report authored by Dr Ethan Russo of GW Pharmaceuticals, published in the British Journal of Pharmacology, Russo reports numerous cases where some sort of cannabinoid-terpene interactions have resulted in complementary pharmacological activity that led to stronger therapeutic outcomes than the cannabinoids or terpenes could have achieved alone as pure extracts. 

This may be important for medical professionals who prescribe medical cannabis, and for patients, to keep in mind, much of the current approach to recommending medical cannabis strains for treatment relies on trial and error or assumptions made based on the CBD:THC ratio of the strain. Patients who move between strains of differing CBD:THC content in order to get better relief, may in fact be better served by looking for strains with a different terpene profile that benefits from these synergistic interactions in a more desirable manner. This is why different terpene analysis procedures are needed, to ensure that terpene oil profiles are as accurate and as comprehensive as possible, in order to allow the accurate study of these synergistic mechanisms and their effects.

Many states now require some form of terpene profile to be printed on the labeling or packaging of cannabis products being sold in their jurisdiction. For example, in Connecticut cannabis producers are required to print a terpene profile and a list of all active ingredients that constitute at least 1% of the cannabis batch on the label of the product. In Maryland, even cannabinoids and terpenes present in levels of less than 1 percent are required to be fully detailed on the packaging.

By printing terpene profiles that have been verified by a third-party laboratory on product packaging, cannabis users can be more informed when it comes to choosing a product that is right for them. 


Terpene Genomics and the Cannabis Industry

Naturally, in an environment where terpenes are responsible for odor, flavor, and, to an extent, the resultant effects of the drug on the body, producers and cultivators will look to produce cannabis strains that are the most favorable, whether this means creating strains with a more pleasant smell or flavor for recreational users, or creating more suitable strains to assist in the treatment of specific medical conditions. With this new craft and medicinal breeding business looming on the horizon for the cannabis industry, novel approaches will be needed to support this sort of selective strain breeding. 

An essential starting point for any sort of process like this will be ensuring full understanding of the genetic basis of cannabinoid and terpene oil profile variation. By using a wealth of terpene profile data in conjunction with modern genomic approaches, researchers can identify nearly all of the sets of genes that are responsible for promoting the expression of individual terpenes. With these genes identified, it should be possible to map genetic variants to oil profile variation, and so we can build up a sort of breeding road-map that can guide breeders towards specific terpene endpoints.

There are at least 30 different terpene molecules commonly found in cannabis flowers, and which combination you get is a key differentiator between strains,” explains Dr Keith Allen, the Director of Bioinformatics at the prominent cannabis science and technology company, Steep Hill. Dr Allen’s team has a special focus on the genetic study of terpenes in cannabis, currently geared towards enabling this sort of targeted breeding and exploring the potential limits on what might be possible with these methods.

“As is the case with the cannabinoids, there is a large family of genes responsible for making terpenes. By describing and mapping all of these genes, we will create tools for breeders who want to create strains with very specific oil profiles, something that will become more and more important as we learn more about the effects of these molecules.”

Selective breeding of cannabis has already been a common practice for thousands of years; the infamously potent strains of cannabis found in and around Afghanistan, such as “Afghani”, are a result of targeted human cultivation efforts in the years before Afghanistan moved to make cannabis illegal in light of a local drug war. 

Selective breeding for terpene content, however, is much more complex than selectively breeding for potency. The potency of a cannabis strain is almost solely down to its THC content, but trying to promote an exact terpene profile means manipulating dozens of different compounds within the strains. By utilizing terpene analysis methods alongside modern genomic techniques, what previously seemed like a Herculean task now looks like the near-future for the cannabis industry. 

 

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