Performing Accurate and Reproducible Analyses of Cannabis Flower Samples

How to Catalog 16 Cannabinoids in 6 Minutes

To those unfamiliar with the cannabis industry, it may seem like industry professionals only know five letters: D, B, C, H and T. Some would be surprised to learn that cannabis contains more than the two famous compounds: Cannabidiol (CBD) and tetrahydrocannabinol (THC).

The cannabis plant is packed with more than 480 unique compounds,¹ of which is more than enough chemicals to keep analysts busy for days.

Of the 480 unique compounds, around 60 are cannabinoids. Cannabinoids are chemicals that interact with the CB2 and CB1 receptors of the central nervous system. Some cannabinoids are so delicate that standard techniques can alter their chemical structure, making them a big challenge for analysts.

An example of this is how gas chromatography techniques will often decarboxylate cannabidiolic acid (CBDA) into CBD due to the high temperatures involved.

Luckily, high-pressure liquid chromatography, or HPLC, does not require the sky-high temperatures of gas chromatography. HPLC is a chromatographic method that uses pressurized liquid instead of gas to separate compound mixtures.

Since this method is cooler, it ensures that chemicals like CBDA can be both measured and retained. Within the industry, it is widely considered that HPLC is the gold standard for the measurement of cannabis potency.

Evidence of the reverence for this technique is that the HPLC photodiode array method described in this article was recently awarded the Potency in Solution award from The Emerald Test, which is a well-regarded inter-laboratory proficiency and comparison test program for hemp and cannabis testing facilities.

Shining a Light on Total THC and CBD With Near-Infrared Spectroscopy

The cannabis plant is both chemically and biologically complex. The plant has unique toxicological and pharmacological properties, with more than 400 chemical components, of which more than 60 are cannabinoids.^2-3

Two of the most researched and well-characterized cannabinoids are cannabidiol (CBD) and Delta-9-tetrahydrocannabinol (Δ⁹-THC), which have both garnered much attention over the years.

CBD is a nonpsychotropic agent and is regarded as the primary therapeutic component,⁴ while THC is considered the main psychoactive ingredient of cannabis.

These different effects mean that it is important for patients, healthcare professionals and researchers to understand the THC:CBD ratio, while cannabis-derived products and cannabis itself are increasingly used for medicinal purposes.

It is essential for cannabis products to have precise labeling, which is enabled through the quantification of CBD, THC and other ‘major’ cannaboids using potency testing.

Cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) decarboxylate to form CBD and THC, respectively. These precursors are naturally occurring and are often preferred for extracted mixtures and edible materials.

HPLC, or high performance liquid chromatography, is an analytical technique that is used to test the potency of cannabis.⁴ However, it does have some limitations, such as long sample preparation time, complex instrumentation and sample destruction.

This means that despite providing a full cannabinoid profile, researchers have begun to seek faster and more user-friendly solutions.⁵

One alternative technique is Fourier transform near-infrared (FT-NIR) spectroscopy. Materials are analyzed by NIR instruments using infrared light to measure the proportion of light that is reflected by the sample.

More infrared light is absorbed by higher concentrations, and therefore, less is reflected to the NIR instrument. Sample analysis time is reduced in FT-NIR spectroscopy through the use of an infrared beam containing many frequencies of light (polychromatic) at once. This enables the simultaneous measurement of all wavelengths.

FT-NIR is an approach is robust, accurate and remarkably versatile. It also allows the sample to be reused in other analyses, involves zero hazardous chemicals, and requires little to no sample preparation.

This means it is a more cost-effective and rapid method for the quantitative determination of cannabis potency for cannabis cultivators, which reduces the overall development and research costs.⁷

The Importance of Pesticide Residue Analysis

There will always be negative factors that affect the optimal growth of a plant in any agricultural setting. There are various forms in which threats can come: from pathogenic fungi that reduce the yield by infecting and killing the crop to insect pests that consume the plants.

Growers typically apply pesticides to combat this. “Pesticides” can mean a wide range of compounds, including plant growth regulators, nemticides, rodenticides, herbicides, fungicides and insecticides.⁸ These chemical compounds provide a vector disease control method and kill the pests, thus improving productivity and protecting the crop.

There was an acceleration in the use of synthetic pesticides in the 1940s. Organochlorine insecticides were found to effectively control human diseases like typhus and malaria by killing insect vectors.

However, they were found to accumulate in the food chain and so were banned in the 1960s. More synthetic pesticides have been produced since the ban. However, some pest species are starting to build resistance.

As demonstrated above, pesticides can enable growers to optimize the yield of their cannabis crops, but some of the chemicals may be harmful to humans, other organisms and the wider environment.

Cannabis can be delivered by smoke inhalation of the dried cannabis flowers in both medicinal and recreational use. Any pesticide residues remaining on the flower can cause toxicity because they can be transferred into the smoke produced by cannabis.

Myclobutanil is a persistent fungicide used by growers and is one example of where the toxic effects of pesticides can come from. The fungicide has a boiling point of 205 ºC and is stable at room temperature.

This can cause an issue because when cannabis is heated with a butane lighter, it can produce temperatures above 450 ºC, which can cause the release of hydrogen cyanide.^9,10 Large doses of hydrogen cyanide can prevent cells from using oxygen, thereby causing those cells to die and is very toxic to humans.

In the long term, it can cause loss of consciousness and potentially death because inhalation primarily affects cells in the central nervous system (CNS). Other effects that hydrogen cyanide has on humans include an enlarged thyroid gland, irritation to the skin and eyes and respiratory and cardiovascular effects.¹¹

The U.S. Environmental Protection Agency (EPA) regulates pesticides in agricultural use to protect public health. Cannabis is considered an illegal drug by the federal government, and so there are no federal regulations or agencies (such as EPA) for pesticide residues related to cannabis products.

This leaves the matter to individual states to devise regulations, resulting in wide variations in these regulations.

California has the strictest pesticide regulations for cannabis in the country. The state’s Department of Pesticide Regulation (DPR) has significant expertise in human health risk assessment and toxicology.

They only allow a pesticide to be used on a plant if a specific criterion is met by the active ingredient. The DPR has a list of 66 pesticides. Of those, twenty-one are classed as category I pesticides, meaning they are not registered for food crops or pose a risk to groundwater and so are banned.

The other 45 chemicals on the list are Category II pesticides, which must fall within defined safety limits to be permitted. Pesticides on inhalable cannabis in California have action limits ranging from 0- 10 µg/g. Oregon has “more relaxed” regulations and has issued a regulatory list for 59 pesticides in flower with action limits ranging from 0.1-2 µg/g.¹²

Previously, to perform pesticide analysis in pesticide samples, gas chromatography-mass spectrometry (GC-MS/MS) has been used. GC-MS/ MS can fragment and isolate a desired molecular weight, which allows fragments to be analyzed by mass spectrometry.

However, GC-MS/MS is not suitable for ionic and polar compounds in some cases, as can be the case with daminozide and abamectin.

Liquid chromatography-mass spectrometry (LC-MS/MS) may be considered to overcome this limitation of GC-MS/MS. Liquid chromatography is better-suited for analyzing more polar and larger molecules that the GC may not be able to handle because it separates the compounds in a liquid phase.

PerkinElmer’s QSight^® 400 Series Triple Quad Mass Spectrometer combines atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) with LC-MS/MS for low level analysis of pesticides and mycotoxins and is a highly sensitive analytical instrument.

This dual source (ESI/ APCI) technology means that cannabis testing labs can quantitate and screen residues using only a single instrument while still utilizing significantly greater detection capabilities than other traditional methods.

References

1. Brenneisen, R. (2007). Chemistry and analysis of phytocannabinoids and other Cannabis constituents. Marijuana and the Cannabinoids (pp. 17-49). Humana Press.
2. Atakan, Z. (2020). Cannabis, a complex plant: different compounds and different effects on individuals. [online] Pubmed Central (PMC). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736954/ [Accessed 17 Feb. 2020].
3. Priyamvada Sharma, M. (2020). Chemistry, Metabolism, and Toxicology of Cannabis: Clinical Implications. [online] PubMed Central (PMC). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570572/ [Accessed 17 Feb. 2020].
4. Ruppel, T. and Kuffel, N. (n.d.). Cannabis Analysis: Potency Testing Identification and Quantification of THC and CBD by GC/FID and GC/MS. [online] PerkinElmer. Available at: https://www.perkinelmer.com/lab-solutions/resources/docs/APP_Cannabis-Analysis-Potency-Testing-Identifification-and-Quantification-011841B_01.pdf [Accessed 17 Feb. 2020].
5. Townsend, D., Eustis, I., Lewis, M., Rodgers, S., Smith, K. and Bohman, A. (n.d.). The Determination of Total THC and CBD Content in Cannabis Flower by Fourier Transform Near Infrared Spectroscopy. [online] PerkinElmer. Available at: https://www.perkinelmer.com/lab-solutions/resources/docs/APP_Determination_of_THC_and_CBD_CannabisFlower.pdf [Accessed 17 Feb. 2020].
6. Materials Evaluation and Engineering Inc. (n.d.). Fourier Transform Infrared Spectroscopy. [online] Available at: https://www.mee-inc.com/hamm/fourier-transform-infrared-spectroscopy-ftir/ [Accessed 17 Feb. 2020].
7. PerkinElmer. (2018). IMPLEMENTATION-READY CANNABIS TESTING. [online] Available at: https://www.perkinelmer.com/lab-solutions/resources/docs/BRO_Cannabis_Testing_014042_02.pdf [Accessed 17 Feb. 2020].
8. Aktar, W. et al (2009) Impact of pesticides use in agriculture: their benefits and hazards. [online] Interdisc Toxicol. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984095/pdf/ITX-2-001.pdf [Accessed 18/02/2020]
9. Sullivan N. et al (2013) Determination of pesticide residues in cannabis smoke. [online] Journal of Toxicology. Available at https://pubmed.ncbi.nlm.nih.gov/23737769/[Accessed 18/02/2020]
10. Dow AgroSciences LLC (2011) “Specimen Label: Eagle 20EW - Specialty Fungicide.” Crop Data Management Systems. [online] http://www.cdms.net/LDat/ld6DG004.pdf [Accessed 19/03/2020]
11. U.S. Environmental Protection Agency (2000) “Cyanide Compiunds”. Air toxics. [online] https://www.epa.gov/sites/production/files/2016-09/documents/cyanide-compounds.pdf [Accessed 16/03/2020]
12. Feldman, J. (2014-15) Pesticide use in Marijuana production: Safety issues and sustainable options. [online] Pesticides and You, A quarterly publication beyond pesticides. Available at: https://ehp.niehs.nih.gov/doi/full/10.1289/EHP5265 [Accessed 18/02/2020]

About PerkinElmer Cannabis & Hemp Testing Solutions

With the cannabis and hemp markets continuing to grow rapidly and regulations strengthening, labs increasingly need streamlined access to best-in-class testing solutions geared toward the unique requirements of the industry. Whether your lab is well established or just starting up, PerkinElmer is a single-source vendor for instruments, methods, reagents, and consumables on hand to help enhance your testing capacity and get ahead of the competition.

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