Identification of 69 magnesium-free chlorophyll derivatives in decarboxylated extracts of medical Cannabis chemovars using ESI-LC-HRMS/MS

The Cannabis plant contains many groups of bioactive metabolites with potential pharmacological properties. These properties have mainly been attributed to cannabinoids and terpenoids, with other groups receiving little attention. The usefulness of the abundant group of chlorophyll derivatives (CDs) was illustrated in various applications, but little is known regarding their presence and significance in Cannabis. We hypothesized that the heating accompanying the process extract preparation would result in a pool of CDs that have lost the central magnesium (Mg) ion. Herein, we introduce a liquid chromatography-tandem mass spectrometry approach for separating, identifying, and quantifying Mg-free CDs in Cannabis extracts after decarboxylation. We assessed 14 Cannabis cultivars representing the four types of chemovars and identified 69 distinct Mg-free CDs. These were separated into five families: Pheophorbide a, Pheophorbide b, Pheophytin a, Pheophytin b, and Purpurin 18. The structure of the Mg-free CDs was determined through their MS/MS fragmentation spectra as phytylated derivatives compared to dephytylated ones, and then further to pyroderivatives, alkylated and oxidized CDs. Substantial variation was found between the four different chemovar types and within different cultivars of the same chemovar, with the family of Pheophytin a, the most prevalent in all extracts. Type III chemovar high-cannabidiol plants contained significantly higher amounts of Mg-free CDs. These differences in abundance may result in variations in the therapeutic effects of plants that are considered similar according to their cannabinoid profiles.