# New Methods for the Comprehensive Analysis of Bioactive Compounds in _Camabis sativa_ L. (hemp)
## 2 Results and Discussion  
### Extraction of Non-Psychoactive Cannabinoids and Flavonoids from Hemp Inflorescences  
The development and application of a suitable extraction procedure covers a crucial role to achieve a reliable characterization of a plant material. For what concerns cannabinoids, a simple dynamic maceration (DM) at room temperature was applied by using ethanol (EtOH) as the extraction solvent, which has been demonstrated to be the best solvent for the extraction of these bioactive compounds [9]. EtOH is indeed a good solvent for the extraction of many phenolics from plant material [31]. As a matter of fact, cannflavins A and B were detected in hemp ethanolic extracts as well, but, conversely to cannabinoids, their content in the infloresences was extremely low [8; 10; 25]; in addition, the concomitant presence of cannabinoids in the same extract made them difficult to be quantified. For this reason, two different protocols were optimised and applied in this work for the extraction of non-psychoactive cannabinoids and flavonoids from hemp. Starting with the extraction conditions previously optimised for cannabinoids, the method was modified as follows. First, the solvent was properly chosen in order to have a good yield of hemp flavonoids from the plant material; to do this EtOH, acetone and ethyl acetate (EtOAcc) were tested. The extracts were also concentrated 25:1 before HPLC analysis, in order to increase the intensity of the UV/DAD signal. Of the solvents tested, acetone resulted in being the best one and it was therefore selected for use in the subsequent steps (Figure 4). In order to reduce any possible interference from cannabinoids in the concentrated extract, a sample pre-treatment was considered necessary. Indeed, the amount of cannabinoids in hemp infloresences is much higher than that of flavonoids; thus, the peaks related to these compounds in the extracts may interfere with the quantification of other constituents at 210 nm. Conversely to other phenolics, cannabinoids can dissolve also in non-polar solvents (i.g. \(n\)-hexane, chloroform, dichloromethane (DCM)) [9]; hence, a sample pre-treatment by means of DM repeated three times with a non-polar solvent, including \(n\)-hexane and its combination with DCM, ethyl ether and toluene, was tested to be suitable to remove cannabinoids. After this treatment, the residue was extracted three times with acetone by means of DM. As shown in Figure 5, \(n\)-hexane alone resulted in being the most suitable solvent for the sample pre-maceration in comparison with other non-polar solvents tested. Indeed, its combination with DCM or ethyl ether or toluene, led to a decrease in the final amount of flavonoids extracted from hemp. Figure 4: HPLC-UV/DAD chromatograms of a hemp extract (sample C6) in acetone (blue line), EtOAc (red line) and EtOH (green line), recorded at 342 nm. For peak numbering, see Table 1.  
Finally, in order to facilitate the removal of cannabinoids, a decarboxylation of the plant material, prior to the pre-maceration with \(n\)-hexane, was also taken into account in order to convert cannabinoic acids into their neutral counterparts, which are more soluble in this solvent. In particular, the plant material was heated at 120 \({}^{\circ}\)C for 1 h or at 80 \({}^{\circ}\)C for 2 h, respectively. After this decarboxylation step, the pre-maceration process with \(n\)-hexane and the extraction with acetone previously optimised were applied. As shown in Figure 6, the peak areas related to flavonoids decreased after the heating process and the removal of cannabinoids was not significantly increased; therefore, it was not further pursued. In the light of all the above, a two-step extraction, based on an initial pre-maceration of the plant material with \(n\)-hexane and on the subsequent extraction with acetone, was selected as the best protocol for hemp flavonoids. The optimized extraction techniques, based on dynamic maceration at room temperature, did not cause the degradation of the compounds of interest or the formation of artefacts.