Δ9-Tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), along with their carboxylated precursors Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and cannabidiolic acid (CBDA) are the best-known phytocannabinoids in Cannabis sativa L.. The four compounds possess two stereogenic centres, thus opening to the potential existence as four stereoisomers: (-)-trans-, (+)-trans-, (-)-cis-, and (+)-cis- isomer. The cis isomers of CBD have never been reported to occur naturally in cannabis, while the cis isomers of Δ9-THC have been recently found as a scalemic mixture in amounts as high as the (-)-trans counterpart in fiber-type cannabis plants. The (-)-trans isomer of CBD is the one found in the plant, while no record can be found on the existence of the (+)-trans isomer. While it has been established that the (-)-trans isomer of Δ9-THC occurs naturally, trace amounts of the (+)-trans isomer have been found in the medicinal cannabis variety Bedrocan by Mazzoccanti et al. by supercritical fluid chromatography (SFC) exploiting the inverted chirality column approach (ICCA). With particular attention to trans-CBD and trans-Δ9-THC, the (+)- and (-)- isomers proved to have contrasting biological activities, especially in terms of binding affinity for CB1 and CB2 receptors [15, 16]. Therefore, it is extremely important to evaluate the stereoisomeric composition of the phytocannabinoids especially in medicinal cannabis extracts. To the best of the authors’ knowledge, no analytical method has been published on the chiral separation of the phytocannabinoid carboxylated precursors, which instead are the real picture of what the plant produces enzymatically. Since no analytical standard is commercially available for the (+)-trans stereoisomers of the four compounds, a stereoselective synthesis was performed in order to develop and optimize a chiral HPLC method coupled to a diode array detector (DAD) and a high-resolution mass spectrometer (HRMS) for the separation of all stereoisomers. The method was then applied to evaluate their chiral composition in the Italian medicinal cannabis variety FM2. In particular, trans-Δ9-THCA and trans-CBDA were isolated by achiral reversed phase (RP) HPLC-DAD-HRMS from the crude extract and their stereoisomeric composition was evaluated by chiral HPLC-DAD. Then the two fractions were decarboxylated and analysed again by chiral HPLC-DAD-HRMS to investigate a possible stereoconversion during the decarboxylation step.

Enantioseparation of chiral phytocannabinoids in medicinal cannabis

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