Role of Chromatography in Cannabis Analysis

By Vasco Medeiros, BScReviewed by Emily Henderson, B.Sc.

Cannabis is garnering greater popularity as the mores of society change, and it is now rising in the echelons of herbal medicine as one of the most consumed substances on the planet. The united nations now estimate that 158.8 million people partake in marijuana usage globally, giving rise to a whole new market within the pharmaceutical industry. However, like any other drug, with an increase in popularity comes an inherent need for testing and analysis.

Cannabis Analysis. Image Credit: Mitch M/Shutterstock.com

Target Analytes within Cannabis Testing

When testing for Cannabis Sativa (the preferred strain in terms of treating mental ailments such as PTSD, depression, and anxiety), there are six key analytes of interest to be screened for. Cannabinoids, terpenes, microbes and toxins, heavy metals, solvent residues, and pesticides.

The screening for harmful contaminants is inherently important, though the main focus of this article will be on cannabinoids, the active ingredient in cannabis sativa, and terpenes, the fragrant/aromatic component.

Applications of Gas Chromatography in Cannabis Analysis

Gas chromatography would be the superlative form of analysis, assaying molecules in the gas phase. However, the initial sample is not a gas and will have to be analyzed via headspace injection. This is where the primary sample is measured at such high temperatures (50^oC- 70^oC) that the “true point” is achieved, a state that borders between gas and liquid.

Here, all key volatile analytes will be separated and measured within the headspace matrix, a key motif in GC-MS.

The Analysis of Terpenes and Cannabinoids

Terpenes are an archetypical component of all plants in nature, and although more than 5000 variants have been identified, 120 different terpenes can be found in cannabis sativa. Though they are typically known for their odors and fragrances, some terpenes (such as limonene) can even result in therapeutic effects. Terpenes are volatile in nature, so they are well suited for GC-MS analysis.

In contrast to the 5000 variants that terpenes encompass, there are a little over 100 different species of cannabinoids, cataloged into ten different classes. These confer a myriad of psychoactive and medical benefits and require assays to explore potency, trace components, and therapeutic effects.

Δ-9-tetrahydrocannabinol (Δ-9-THC) acts as the key psychoactive cannabinoid. This key component is not easily distinguishable from its isomer, Δ-8-THC, given that the only difference is the migration of the double bond between carbons 8-9 to carbons 9-10. Δ-8-THC acts as the precursor to Δ-9-THC and would act as the acidic component of the plant.

In addition, this acidic component is difficult to quantify given that the carboxyl group on the phenol functional group is highly labile. When exposed to high temperatures within the GC apparatus, this molecule will degrade, transferring the carboxylate so that the original Δ-9-THC is reformed. This will jeopardize the veracity of the resulting chromatograph, yielding higher levels of Δ-9-THC than what is actually found within the plant. For this reason, derivatization is often employed.

Derivatization of Cannabinoids

The methodology of derivatization is commonly associated with gas chromatography, altering key analytes to a more volatile and labile form for better separation, and higher sensitivity of results. This common practice consists of comparing underivatized, and derivatized cannabinoids, and identifying the differences in retention time in any given chromatogram.

For example, Dr. Allegra Leghissa informs us that Δ-9-THC (natural form) would elute in 19.5 minutes, while its acetylated/derivatized form will elute in 17.5 minutes. This is due to the fact that acetylation prevents the degradation of the carbonyl group through steric means, lowering the boiling point. The alteration in boiling point will correlate with the potency and presence of a given compound.

Derivatization in GC-MS can also be used to elucidate the potency of two other cannabinoid variants: cannabichromene (CBC) and cannabidiol (CBD). When underivatized, the co-eluting molecules will give rise to one peak, which cannot be well interpreted.

However, given that each structure has a different number of acetylation sites, derivatization will clarify structural differences within the molecule, and further differentiation within the resulting chromatograph. This is imperative within the marijuana industry. For instance, if someone were to purchase a CBD oil, he or she will want to make sure that, allegedly, there is no other trace of cannabinoids in the extract.

Analysis of Cannabinoids using GC-VUV (Vacuum ultraviolet detector)

While the superlative nature of GC has already been established, the different variants of detector that one can fit to this apparatus is still up for interpretation. A flame ionization detector, for example, provides very sensitive results for quantitative assays, though the differentiation of different terpenes is unfavorable because the distinctions between each analyte are solely based on retention indices. The same problem results in mass spectrometry, great for quantitative analysis, though it bases all results on predetermined standards and retention indices.

GC-VUV however, though not as sensitive as the previous two apparatus, relies on the absorbance spectrum rather than predetermined standards. Each molecule, whether they are terpene or cannabinoid, isomer or not, will have a unique absorbance spectrum. This causes the distinctions between all different analytes to be very easily interpretable. It is complementary to mass spectrometry because isobaric compounds are fragmented similarly while addressing the issues of co-elution and superimposing peaks. This is done by the addition of two eluting peaks, and subtracting the coeluting peak post-run, yielding promising results.

Further Reading

• All Cannabis Analysis and Testing Content
• Recent Advancements in Cannabis Analysis
• Cannabinoids and Obesity
• Microbial Testing in Cannabis
• What is Cannabinomics?