Differentiation of cannabis types based on cannabinoid content

Abstract

Cannabis is the most widely used drug. In recent years, interest in cannabis has grown significantly. From 2009 to 2019, seizures of marijuana increased by 72%, while hashish decreased by 9%. The cannabis products available in Europe today include those with a high content of Δ 9 -tetrahydrocannabinol - Δ 9 -THC (much higher than a few years ago), as well as many products containing hemp extracts, with a low Δ 9 -THC content, which are sold on the legal commercial market. The popularity of products with low Δ 9 -THC content has become a challenge for forensic laboratories. Cannabis identification is based on the quantification of the total percentage content of Δ 9 -THC and Δ 9 -THC-2-carboxylic acid (Δ 9 -THCA-A) in dry plant material. The varieties with the content of these compounds >0.20% are classified as narcotic type cannabis in Poland. This distinction is often only possible by quantitative chemical analysis of the leaves and inflorescences of mature, non-pollinated and unfertilized female cannabis varieties. In some cases, chemical analyzes related to the content of Δ 9 -THC and Δ 9 -THCA-A may not be sufficient, e.g., when testing early growth stage plants, the stems of cannabis plants, or the plant material of male narcotic varieties used in seed production (due to the low content of the mentioned compounds). Therefore, it is necessary to look for other markers (in the form of characteristic cannabinoids) that could indicate whether it is fiber or narcotic type of cannabis. This research is a part of a broader project that mainly aims to create a genetic database for the identification of cannabis varieties (2020-2022). This project (DOB-BIO10/02/01/2019) is funded by the National Center for Research and Development as part of competition No. 10/2019 for funding research and development work for national defense and security. Samples of narcotic type cannabis and hemp plants, grown from certified or commercially available seeds in special chambers, were analyzed by means of ultra- performance liquid chromatography with spectrophotometric detection (UPLC-PDA). Cultivated cannabis plants were cut at various stages of growth in order to check for variability in the composition of individual cannabinoids during plant growing. Various plant fragments were analyzed: inflorescences, leaves, stems, and sometimes the whole plants (in the case of young specimens). Relative areas of 18 cannabinoids (in relation to the internal standard) were analyzed, as well as the concentrations of four key compounds: Δ 9 -THC, Δ 9 -THCA-A, CBD and CBDA. The chemical analyzes showed a strong relationship between the cannabinoid profile and a specific plant variety, and also indicated that the content of individual cannabinoids depends on the part of the plant being tested (usually the highest in the inflorescence samples, lower in the leaves, and the lowest in the plant stems). Two markers were tested in the study, i.e. the ratio of the sum of the concentrations of Δ 9 -THC and Δ 9 -THCA-A to the total concentration of CBD and CBDA, as well as the ratio of the sum of the content of Δ 9 -THC and CBN to the content of CBD (the latter recommended by UNODC). Chemical testing of the sum of Δ 9 -THC and Δ 9 -THCA-A is insufficient to determine the variety of cannabis plants. Therefore, two markers composed of Δ 9 -THC, Δ 9 -THCA-A, CBD, CBDA, and CBN were tested in the study to distinguish cannabis from hemp plants. The results are promising, however in some cases they may indicate the need for genetic testing of cannabis.