Comprehensive two-dimensional liquid chromatographic analysis of Cannabis phenolics and first evidence of flavoalkaloids in Cannabis

Cannabis contains a large number of diverse metabolites. Compared to the well-studied cannabinoids and terpenoids, characterization of the phenolic composition of Cannabis has received little attention. With studies reporting synergistic effects in Cannabis, the importance of investigating polyphenols in Cannabis is becoming more apparent. We report an on-line comprehensive 2D HILIC × RP-LC method hyphenated to high resolution (HR) MS for the detailed characterization of polar phenolics in Cannabis inflorescence and leaf samples of three commercial strains. Optimal HILIC × RP-LC conditions were derived using an automated method optimization program, and provided excellent separation performance (peak capacity > 3000) and high orthogonality. Diode array and HR-MS data enabled tentative identification of 79 compounds, comprising mainly flavone and (hydroxy)cinnamic acid derivatives. Compound assignment was facilitated by the group-type separation obtained between the phenolic acids and flavonoids, as well as the structured elution patterns for the flavone glycosides. The presence of three C-glycosylated flavones, as well as several of their O-glycosylated derivatives, distinguished one of the strains from the other two. In the same strain, flavoalkaloids were detected, mainly in the leaf extracts. The structures of the alkaloid moieties could not be unambiguously assigned based on the available HR-MS data, but four classes of flavoalkaloids comprising 16 flavone derivates could be tentatively identified. This is the first time that this relatively rare and chemically interesting class of compounds has been detected in Cannabis. These findings highlight the diversity of Cannabis, and the utility of HILIC × RP-LC-HR-MS for the in-depth study of its phenolic composition.