Abstract
The aim of this work was to develop a supercritical fluid chromatographic method to study the applicability of this emerging technique to cannabinoid analysis and showcase its advantages. During method development, the authors focused on nine phyto-cannabinoids to assess the selectivity needed to potentially perform the quantitation of each cannabinoid. After method development, robustness studies were carried out on this method to gain more information about its qualitative behavior (in terms of critical resolutions) when varying some crucial parameters (concentration of additive, column temperature, starting gradient conditions and column batch). Once the robustness was evaluated and the parameters most affecting the selected responses were individuated, the SFC method was applied for a simulated routine use to generate quantitative results concerning the concentrations of the main cannabinoids in real cannabis samples. The samples were also analyzed by means of an ultra-high-performance liquid chromatographic method currently used in the laboratory for the same objective. Finally, the results obtained with both analytical methods were compared to evaluate their accordance. The Bland-Altman method was applied as a statistical strategy to evaluate the degree of accordance between the results generated and display the data in a difference plot. The ultra-high performance supercritical fluid chromatography quantitative results were in accordance with the ultra-high performance liquid chromatography results, demonstrating the applicability of this technique for cannabinoid analysis.
Highlights
Robustness studies for the ultrahigh performance supercritical fluid chromatography (UHPSFC) method were conducted on a mix standard solution containing the nine CNBs of interest: CBD, Δ8-the well-known ∆9tetrahydrocannabinol (THC), THC, CBC, CBN, tetrahydrocannabinolic acid (THCA-A), cannabidiolic acid (CBDA), CBG, and cannabigerolic acid (CBGA)
UHPSFC, employing a mobile phase in super/subcritical conditions, allows for chromatographic analysis to be performed with a very short analysis time (6 min vs the 18 min of the reference ultrahigh-performance liquid chromatography (UHPLC) method used for this study)
The selected final conditions allow for appropriate selectivity to potentially quantify nine phyto-cannabinoids employing a low amount of organic solvent
Summary
The main substances of interest in cannabis plants are the phytocannabinoids, among them THC, but other compounds are present in the plant material, such as terpenes, flavonoids and phenolic derivatives, which possess health-potent properties by themselves or can act synergistically with other CNBs.[4] Today, more than 90 CNBs have been determined and can be classified into 10 subclasses.[5,6] The most important substances belonging to CNBs are THC, cannabidiol (CBD), cannabinol (CBN), and to a lesser extent, cannabigerol (CBG), and cannabichromene (CBC) These compounds, and more THC and CBD used alone or in combination, present potential pharmacological properties for the treatment of different diseases, such as multiple sclerosis, cancer and chronic pain, as well as epilepsy and anxiety disorder.[2,4,7] CBD possesses some pharmacological properties but lacks psychotropic properties, unlike THC.[7,8] some countries have legalized cannabis for therapeutic use, and in 2018, the FDA approved Epidiolex® as a drug containing cannabidiol, while Sativex®, a combination of THC and CBD, is currently prescribed in several countries (but not in the USA). Some countries such as Uruguay and Canada have legalized cannabis for recreational use.[8,9]
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