A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis

Abstract

Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ⁹-trans-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study's approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.

Figure 1

Phytocannabinoid biosynthesis and degradation routes and products according to the literature^–. The most prevalent acid components are presented for each type of phytocannabinoid.

Figure 2

Observed chromatographic and MS characteristics of phytocannabinoid standard materials. (A) Total ion current (TIC) chromatogram of the 13 available phytocannabinoid standards, and their MS/MS spectra according to (B) CBG, (C) Δ⁹-THC, (D) CBD, (E) CBC, (F) Δ⁸-THC, (G) CBL, (H) CBN, (I) CBGA, (J) Δ⁹-THCA, (K) CBDA, (L) Δ⁹-THCV, (M) CBDV, and (N) CBDVA. Fragmentation structures for (O) CBG, (P) Δ⁹-THC, (Q) CBD, (R) CBC, (S) Δ⁸-THC, (T) CBL, and (U) CBN were determined according to the observed MS/MS spectra of each phytocannabinoid. Values of m/z in this figure are presented as nominal masses to improve interpretation of spectra. Accurate masses for the main fragments appear in Supplementary Figs S3–S9. The blue and green highlights mark identical m/z fragments for the neutral and acid phytocannabinoids.

Figure 3

LC/MS TIC of (A) untreated and (B) decarboxylated high-Δ⁹-THCA; (C) untreated and (D) decarboxylated high-CBDA; and (E) partly decarboxylated high-CBGA Cannabis strains injected in the prepared concentration, with annotated phytocannabinoids identified according to analytical standards on the samples with the highest expression.

Figure 4

Phytocannabinoid profiling of 36 different medical Cannabis strains. The LC/MS concentrations of each phytocannabinoid were normalized to the highest value and compared per phytocannabinoid in a heat map. Strains were arranged according to increasing Δ⁹-THCA content (first line) and phytocannabinoids by subclasses.

Figure 5

Phytocannabinoid profiling of a specific high-CBD Cannabis strain that is used to treat children with epilepsy. (A) LC/MS differential analysis of four samples from the same strain with the same genetics, planted and harvested in the same way and at the same time, but grown at four different greenhouses. Two samples were extracted from each plant and prepared in three dilutions (1:9, 1:99, and 1:999  v/v Cannabis extract to ethanol). Samples were injected to the LC/MS in duplicates. The average LC-MS concentrations of each phytocannabinoid were normalized to the highest value and compared per phytocannabinoid in a heat map. Strains were arranged according to increasing Δ⁹-THCA content (first line) and phytocannabinoids were arranged by subclasses. Quantitative comparison of the absolute concentrations of (B) CBDA, (C) Δ⁹-THCA, (D) CBGA, (E) CBCA, (F) CBDVA, (G) Δ⁹-THCVA, (H) CBGVA and (I) CBCVA, show significant differences between Cannabis samples from the same strain as a result of growing conditions. All values are reported as mean ± SD (one-way ANOVA, Tukey HSD post hoc test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6

Effect of equally high CBD Cannabis strain extracts on pentylenetetrazol (PTZ)-induced convulsions. (A) Comparison of the LC/MS concentrations of the five Cannabis extracts that were used to evaluate the anticonvulsant effect of Cannabis extracts using the PTZ-induced convulsion test in mice. Values were color-coded according to order of magnitude of concentrations. All strains contained similar CBD contents (50% w/w) but differed in the concentrations and ratios of other phytocannabinoids. (B) The analyzed Cannabis extracts were found to reduce PTZ-induced incidence of tonic-clonic seizures and mortality. (C) Both Cann4 and Cann5 extracts significantly increased latency to first tonic-clonic seizures (F_5,103 = 4.4, p = 0.001). Values are reported as mean ± SEM, *p < 0.05, ***p < 0.001 vs. a control group. Percent of protection against incidence of tonic seizures and subsequent death to PTZ were compared among groups using the χ² test (p > 0.05 for both seizure protection and mortality protection).