Development of gas and liquid chromatographic methods for the separation and quantification of 11 cannabinoids

New research into the medicinal properties of compounds within Cannabis is changing the way this convert plant material is used, both in Western Australia and around the world. The abuse of Cannabis as an illicit and recreational drug has been the focus of research for many decades, however this focus is shifting towards potential therapeutic benefits, such as pain relief and anti-seizure applications. Cannabis contains many compounds of pharmacological relevance, but the group of most interest is the cannabinoids. These include the main psychoactive component of Cannabis, Δ 9 tetrahydrocannabinol (THC), which is the focus of legislation in Australia, with regulation for manufacture of hemp materials restricting content to less than 0.3% w/w. There are numerous other cannabinoids that may be of benefit for medicinal applications, as they appear not to exhibit the negative psychoactive effects of THC. In order for them to be better understood and developed, new analytical techniques are needed to identify and quantify these other cannabinoids. The aim of this study was to utilise current instrumentation to develop and optimise a method to separate and quantify 11 common cannabinoids. The target cannabinoids were: Δ 8 and Δ 9THC, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidivarin (CBDV), tetrahydrocannabivarin (THCV, cannabigerolic acid (CBGA), cannabidiolic acid (CBDA) and Δ9 tetrahydrocannabinolic acid A (THCA-A). The three instrumental techniques explored were gas chromatography/mass spectrometry (GC/MS), liquid chromatography/photodiode array (LC/PDA) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). GC/MS is well-established for both cannabinoid analysis and Δ 9THC quantification. The method optimised in this study utilized previous work, with a focus on neutral cannabinoids. After optimisation of ramp rates and columns for GC/MS, 8 neutral cannabinoids were separated and quantified. Peak signals and retention times varied between analyses, meaning that identification was challenging. LC/PDA provided some advantages to GC/MS, as the cannabinoids were more stable using this ‘softer’ technique. LC/PDA method optimisation focused on flow rate, buffers and gradients, with all 11 cannabinoids separated and quantified. Retention times variability was observed, but this was found to be due to instrumental problems. The key disadvantage of this method was the inability to discriminate between cannabinoids based solely on their UV spectra. Oil and plant material samples were successfully analysed via this method and found to contain a range of cannabinoids at various concentrations. The data was compared with previous methods and found to be equivalent or better. Full validation of the LC/PDA method could not be completed, but data to date is promising. LC/MS/MS was able to individually detect and identify several cannabinoids, with 7 peaks observed in the mixed standard. This demonstrated issues with co-elution, in addition to higher noise levels for this method. LC/MS/MS combined the best of both GC/MS and LC/PDA, using a ‘softer’ chromatographic technique (LC) with a detection method with greater discriminatory power (MS). The ability to separate and analyse all 11 cannabinoids simultaneously would be an advantage to the medicinal Cannabis industry, as a commercial method that has high throughput, and is time and material efficient would provide essential information on composition of products. It would also allow us to identify products that may produce a desired or undesired therapeutic effect. Additionally, this research aids monitoring of the products for legislative purposes. When fully developed and optimised, the method could be implemented in a commercial setting to quantify cannabinoids in Cannabis and Cannabis products such as oils