Figure 1. Biosynthesis of phytocannabinoid acids in Cannabis begins with a common precursor called olivetolic acid that then undergoes a series of enzymatic transformations. When exposed to heat, the phytocannabinoid acids readily decarboxylate into their neutral forms.

Processing

The process of extracting phytocannabinoids from Cannabis inflorescences is both time- and labor-intensive work. While each processor may follow a different method to obtain Cannabis extracts, the overall model of harvest, extraction, winterization, filtration, and distillation has been shown to provide isolated material with >90% phytocannabinoid content. After harvesting, Cannabis inflorescences are dried and removed from the harvested plant material. At this point some extractors choose to decarboxylate the acidic phytocannabinoids by oven-drying them at temperatures >125°C prior to solvent extraction. The plant matter is then extracted with solvents such as butane, ethanol, or supercritical fluid carbon dioxide. The biomass extract is cooled in a process known as winterization to induce lipid solidification, which is then removed by filtration. If necessary, the extract may be subjected to additional filtrations through clays or other solid support filter aids. Finally, the extract is vacuum distilled at very low vapor pressures and high temperatures, where decarboxylation occurs if it has not already. The lack of uniformity in standard operating procedures for the extraction and distillation processes provides multiple pathways for forming phytocannabinoid byproducts. For example, it is suspected that the acidic or basic nature of various filtration media may be responsible for byproduct formation.³ Additionally, the heating required for distillation increases the potential formation of byproducts through oxidative degradation. Identification of these byproducts provides a necessary quality control check for the extraction and distillation processes to avoid byproduct contamination.

Isomerization Degradants

Under basic conditions, the double bond in Δ⁹-THC isomerizes from the Δ9 to the Δ10 position, becoming conjugated to the resorcinol core.^1,4 This can form two different diastereomeric structures of Δ¹⁰-THC: (6aR,9R)-Δ¹⁰-THC and (6aR,9S)-Δ¹⁰-THC (Figure 2). Neither seems to exhibit any abnormal behavioral effects according to animal studies.⁵ Under acidic conditions, CBD cyclizes to Δ⁹-THC and further to Δ⁸-THC, due to it being the more thermodynamically stable isomer (Figure 2). Δ⁸-THC exhibits similar pharmacological effects to Δ⁹-THC on the CB₁ and CB₂ receptors, except with less potency.¹ Acidic conditions also drive the double bond of either Δ¹⁰-THC diastereomer to isomerize further to the Δ^6a,10a position.⁴ This results in the formation of two potential enantiomers: 9(R)-Δ^6a,10a-THC and 9(S)-Δ^6a,10a-THC (Figure 2). Animal studies involving these two compounds revealed that the (S)-enantiomer produces a Δ⁹-THC-like behavioral effect and that the (R)-enantiomer produces no noticeable effects.⁵ Binding assays have shown that while both enantiomers exhibit partial agonist activity at the CB₁ and CB₂ receptors, the potency of the (S)-enantiomer is six times that of the (R)-enantiomer.⁶

Figure 2. Isomerization can occur under acidic (H+) or basic (OH-) conditions.

Oxidative Byproducts

Some phytocannabinoids can be altered upon exposure to oxygen, generating various oxidative byproducts. Cannabinol (CBN) forms as the oxidative byproduct of Δ⁹-THC (Figure 3).^1,7 Studies have shown that Δ⁹-THC oxidation occurs at a rate of up to 5% loss per month at room temperature.¹ However, the rate of CBN formation is not equal to the oxidative degradation rate of Δ⁹-THC.⁷ This seemingly missing Δ⁹-THC could be explained due to the proposed presence of hydroxylated and epoxidized intermediates generated during the Δ⁹-THC oxidation process.¹ Additionally, formation of Δ⁸-THC may account for part of the Δ⁹-THC loss as it can also be generated from Δ⁹-THC oxidatively.¹ CBN exhibits very mild psychoactivity when compared to Δ⁹-THC.² A heat map of the relative potency of various isomers is depicted in Figure 4.

Figure 3. Atmospheric oxygen oxidizes Δ⁹-THC to CBN and Δ⁸-THC, as well as to other intermediates not shown above.

Figure 4. Reported psychoactivity displayed in relative terms of potency.

In the presence of ultraviolet light, (±)-cannabichromene (CBC) undergoes [2+2] cycloaddition to form (±)-cannabicyclol (CBL) (Figure 5). Formation of CBL is based on the concentration of CBC and may serve as a marker for storage in the presence of light. Currently, no pharmacological data is available concerning the bioactivity of CBL.

Figure 5. Photo-oxidation induces a [2+2] cyclization in (±)-CBC, resulting in (±)-CBL. 

Under pyrolytic conditions such as smoking, CBD is oxidized to cannabielsoin (CBE) (Figure 6).^1,8 However, in rare instances, the carboxylic acid CBEA has been reportedly isolated from hashish, suggesting that some other form of degradation to CBE or CBEA may be possible.⁹ Currently, little pharmacological data exists concerning the bioactivity of CBE.

Figure 6. Pyrolysis of CBD leads to the oxidative formation of CBE. 

Another oxidative series of phytocannabinoid byproducts is the richly colored quinone series. Phytocannabinoid quinones are designated with a “Q” and include those formed from CBD, CBN, Δ⁹-THC, Δ⁸-THC, (±)-CBC, and cannabigerol (CBG) (Figure 7).

Figure 7. Common phytocannabinoid quinones.

A forensic technique known as the Beam test utilizes the oxidative conversion of CBD to CBDQ, better known as HU-331, to detect the presence of Cannabis through treatment with a base to reveal a deep purple color (Figures 8 & 9).¹⁰ The Beam test conditions are typically selective for phytocannabinoids with two free hydroxyls on the resorcinol ring system. Other phytocannabinoid quinones are also known to be highly colored species and may be formed by alternative oxidative mechanisms. Scientists have been able to form Δ⁹-THCQ through electrochemical oxidation, but there is still the need for development of a reliable assay that can conveniently confirm the presence of Δ⁹-THC in a sample through rapid conversion to Δ⁹-THCQ.¹¹ Some phytocannabinoid quinones have been found to have medicinal properties. Pharmacological research conducted into the quinone series thus far has revealed anticancer potential for HU-331, Δ⁸-THCQ (HU-336), and CBNQ (HU-345), as well as anti-inflammatory potential for CBGQ (VCE-003).^1,12

Conclusion

The field of phytocannabinoid testing and research is experiencing rapid growth mainly resulting from the USDA Farm Bill legalization of regulated hemp products and from other changes to legalization at the state level. Many of these degradant byproducts have unknown or incomplete studies on their pharmacological and toxicological effects. Identification of these byproducts and a better understanding of the chemistry involved in their formation is paramount to providing the highest quality Cannabis products.

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