Investigating the Protonated Cannabinoid Dimer Detected in the Forensic Analysis of <i>Cannabis</i> by DART-MS: A Combined Mass Spectrometry and Computational Study

Direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) enables the rapid chemical profiling of <i>Cannabis</i> samples. This approach reveals a prominent signal at nominal <i>m</i>/<i>z</i> 315, characteristic of protonated isomeric cannabinoids such as Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), cannabichromene (CBC), Δ⁸-THC, and cannabicitran (CBT). Interestingly, the spectra of standards of these cannabinoids also display a peak at nominal <i>m</i>/<i>z</i> 629, corresponding to the protonated dimer species [2M + H]⁺. To elucidate the likely structures of these ions, density functional theory calculations were performed for protonated homo- and heterodimer combinations of the five cannabinoids. The calculations indicated that CBDH⁺••CBD, Δ⁹-THCH⁺••Δ⁹-THC, and Δ⁸-THCH⁺••CBC are the most stable. Population analysis calculations further revealed a temperature dependence, with Δ⁸-THCH⁺••CBC being the dominant species below 240 K, while CBDH⁺••CBD becomes the most abundant in the 240-800 K range. These findings imply that at a DART gas temperature of 623 K, the peak at <i>m</i>/<i>z</i> 629 detected in hemp, where CBD levels are high, is primarily attributable to CBDH⁺••CBD. Conversely, the population analysis studies showed that marijuana, which contains negligible CBD, would not be expected to exhibit a mass at <i>m</i>/<i>z</i> 629. Experimental DART-HRMS and field desorption (FD)-MS measurements of hemp and marijuana validated this prediction. The results support the conclusion that the peak at <i>m</i>/<i>z</i> 629 specifically observed in hemp samples is representative of the CBDH⁺••CBD adduct, as opposed to a natural product molecular marker of hemp, and it provides a rapid and reliable mass by which to distinguish hemp from marijuana by DART-HRMS.