Confusing cannabinoids

Among the world’s oldest cultivated plants, multi-purpose Cannabis sativa has been used medicinally for thousands of years across various cultures.^1,2 Parke-Davis, a US company once hailed as ‘the largest pharmaceutical company in the world’ advertised an extract of cannabis ‘grown in our own botanical gardens’ in the 1930s. The same decade saw cannabis prohibition laws spread across the US, which turned the one-time staple crop into an outlawed plant. This status shift was called necessary by proponents citing safety concerns, while other scholars detail the role bigotry played in criminalisation.³ Today, research into cannabis-derived treatment is again seeing tremendous growth despite a myriad of complicated and ever-changing laws worldwide regulating cannabis, its cannabinoids and related products. One cannabinoid has become the chemical dividing line between the legal and illegal ⎼ Δ⁹-tetrahydrocannabinol (THC).

THC is a psychoactive substance and tightly controlled. In 2018, the US Agricultural Improvement Act designated cannabis with less than 0.3% dry weight concentration THC to be hemp, which is legal throughout the US. A THC concentration over 0.3% earns the designation of marijuana, a federally controlled substance but legal for recreational and/or medical use in several states. The EU currently uses a less than 0.2% dry weight concentration THC limit to designate hemp, but the 2023 Common Agricultural Policy will boost that to 0.3%.

Another cannabinoid – cannabidiol (CBD) – has a far more relaxed status than THC. CBD has been traditionally considered non-psychoactive though, like THC, it is pharmacologically active. A blunt simplification is that THC can get a person high while CBD cannot. 

Sharing a source with THC means sharing the scrutiny and regulations levelled at THC

Research and development exploring THC and CBD (along with cannabis or cannabis derived products) as treatment drugs for an array of conditions has seen tremendous growth, though the number of approved drugs is small. Beyond approved pharmaceuticals, CBD is popping up in ‘shampoos, lattes, body oils, gummy bears and dog treats’ and is sold online, at farmers markets, local shops, and in major retail outlets. The more widespread legal use of CBD does not mean products have no oversight worldwide. Sharing a source with THC means sharing the scrutiny and regulations levelled at THC. CBD sourced from a plant deemed marijuana rather than hemp can be illegal. CBD products with ‘too much’ THC can be illegal. A CBD product’s legality can often come down to how much THC is present at some stage of growth or production, necessitating reliable qualitative and quantitative analysis.

Given that CBD products are often complex matrices like foodstuffs, beauty products and e-liquids for vaping, separation techniques are an analytical go-to. Gas chromatography–mass spectrometry (GC–MS) is a popular choice for evaluating cannabis-related samples, as it can resolve complicated mixtures and provide sensitive, selective and speedy analysis. A drawback is that thermally sensitive compounds may fall apart, miring measurements in complications. Decomposition products may artificially inflate determinations of one targeted compound while deflating another. In forensic chemistry, this may land products and persons on the wrong side of the law.

In Japan, any amount of Δ⁹-THC in CBD products is prohibited.⁴ Like other forensic analysts concerned CBD could thermally decompose to Δ⁹-THC during GC–MS analysis, Kenji Tsujikawa and colleagues at Japan’s National Research Institute of Police Science set about probing this phenomenon.⁴ Their initial evaluation of a methanol solution of a CBD e-liquid via GC–MS used splitless mode injection at an injector temperature of 250°C. Splitless mode is used for trace and quantitative analysis as the entire injected sample is directed to the column.⁵ Methanol is often a solvent of choice for this type of analysis – especially because acid induced intramolecular cyclisation of CBD to Δ⁸-THC and Δ⁹-THC is well-documented^4,6 – and 250°C is recommended as ‘a good initial inlet temperature’.

The researchers identified a Δ⁹-THC peak at these initial conditions, but no Δ⁹-THC peak was observed at an injector temperature of 200°C despite both analyses using the same column oven temperature. It appeared CBD was thermally decomposing during GC-MS analysis, though not on-column. Sharp peaks further pointed to the injector temperature being the culprit.

To explore contributing and mitigating factors to CBD’s decomposition, Tsujikawa and team examined the effects of a range of injector temperatures on splitless mode, and identified an injector temperature of 200°C as offering a ‘balance between the sensitivity and prevention of thermal decomposition’.⁴ Split mode injection at 250°C saw no thermal decomposition, likely because samples in this mode spend less time in the injector. If trace analysis is not required, split mode is a decomposition work-around. However, if tracking miniscule quantities of THC is required, splitless mode would be the go-to.

The researchers then probed the impact of the injector’s glass lining on CBD decomposition. In splitless mode at an injector temperature of 250°C, the peak intensity of Δ⁹-THC was ‘remarkably increased’⁴ with a used liner compared to a new liner, with Δ⁸-iso-THC and Δ⁸-THC also popping up. As these compounds can be produced from CBD or Δ⁹-THC under acidic conditions, this indicated that the surface of the used glass liner had deteriorated, exposing acidic silanol groups.⁷ With acidic conditions promoting the decomposition of CBD, the researchers evaluated the impact of adding varying amounts of the basic compounds methylamine and trimethylamine over a range of temperatures. This did not eliminate CBD decomposition, but some mitigation was observed, particularly when using low concentrations of methylamine.

To combat CBD decomposition during GC–MS trace analysis, Tsujikawa and colleagues recommend dissolving samples in methylamine-added solvent, a new glass liner, an injector temperature of 200 °C, and vigilance. Being mindful of thermal decomposition of CBD can guard against mendacious measurements.