Differentiation of CBD and Δ⁹-THC isomers using copper-ion complexation and electrospray ionization-tandem mass...

The differentiation of illicit marijuana and legal hemp, based on the 0.3 % Δ⁹-tetrahydrocannabinol (Δ⁹-THC) threshold outlined in the 2018 Agricultural Improvement Act, impacted the seized drug community and the commercial hemp industry. Previously, the presence of Δ⁹-THC was sufficient for the identification of illicit marijuana; however, current legislation now requires quantitative or semi-quantitative analyses. Cannabidiol (CBD) and Δ⁹-THC are the primary cannabinoids in hemp and marijuana, respectively, but the presence of additional cannabinoids, such as Δ⁹-THC isomers, creates a significant analytical challenge. This study investigates copper (Cu)-ion complexation as a novel approach for differentiating CBD and Δ⁹-THC isomers.

The binding of Cu⁺ ions to cannabinoids generates characteristic ions in the full scan mass spectra due to preferential binding affinities and distinct oxidation products from Cu-catalyzed O₂ activation. Thirteen Cu-cannabinoid complexes were analyzed to identify characteristic ions enabling isomer differentiation. Common cannabinoids such as Δ⁹-THC, CBD, Δ⁹-tetrahydrocannabinolic acid (Δ⁹-THCA), and cannabidiolic acid (CBDA) were differentiated in full scan due to characteristic ions at nominal m/z 416, m/z 480, m/z 565, and m/z 524, respectively. Additionally, Δ⁹-THC was differentiated from Δ⁸-tetrahydrocannabinol (Δ⁸-THC) based on the absence of the precursor ion at nominal m/z 416 for Δ⁸-THC. The Cu-ion complexation method was successfully applied to the analysis of 10 simulant mixtures and fortified cannabis extracts, although competition for Cu⁺ ions in solution in authentic extracts increased the difficulty of cannabinoid identification.

This study demonstrates the potential of a novel Cu-ion complexation approach for differentiating CBD and Δ⁹-THC isomers using electrospray ionization-tandem mass spectrometry. Given the challenges associated with the increasing prevalence of Δ⁹-THC isomers in cannabis extracts and the limitations associated with current analytical techniques for cannabis testing, this study demonstrates a novel alternative approach for differentiating Δ⁹-THC isomers, which is essential for the differentiation of legal hemp and illicit marijuana.