# New Methods for the Comprehensive Analysis of Bioactive Compounds in _Camabis sativa_ L. (hemp)
## 2 Results and Discussion
### Identification of Non-Psychaoctive Cannabinoids and Flavonoids in Hemp Extracts  
The identification of secondary metabolites in hemp extracts was carried out by combining the information obtained from UV/DAD and MS detectors. For a complete characterisation, the experimental data were compared with the literature [3; 9; 12; 32] and with commercially available reference standards. The UV spectra were used to preliminarily assign the chromatographic peaks to a chemical class. As a matter of fact, cannabinoic acids, such as CBDA and CBGA, can be easily distinguished from their decarboxylated counterparts (CBD and CBG, respectively) by their UV/Vis spectra: CBDA and CBGA are indeed characterised by three absorption maxima (\(\lambda_{\text{max}}\)), one at 220-223 nm, a second one at 266-270 nm and a third one around 305 nm; CBD and CBG show a first \(\lambda_{\text{max}}\) at 210-215 nm and an additional one at 270 nm [9; 13]. Canniprene showed a UV/Vis spectrum comparable to that of neutral cannabinoids, with a \(\lambda_{\text{max}}\) at 204 nm and a minor one at 280 nm. Also cannflavins can be recognised on the basis of their UV/Vis spectra, since they possess a representative chromophore: indeed, the spectra  
Figure 7: **(a)** Representative HPLC-UV/DAD chromatogram of a hemp extract (sample C6) at 342 nm obtained by using the analytical method optimised for flavonoids and related compounds. For peak numbering, see Table 1. **(b)** Representative HPLC-UV/DAD chromatogram of a hemp extract (sample C6) at 210 nm obtained by using the analytical method optimised for cannabinoids. For peak numbering, see Table 1.  
of cannflavins A and B showed a first \(\lambda_{\rm max}\) at 213-215 nm, a second one at around 273-274 nm and, finally, one at 342 nm. As regards MS and MS\({}^{2}\), the experiments were carried out both in the positive and in the negative ion mode. The MS and MS\({}^{2}\) data are shown in Table 1. In particular, cannabinoic acids showed a higher intensity of the signals in the negative ion-mode, as opposed to decarboxylated compounds and canniprene, which ionised better in the positive ion mode. Conversely to cannabinoids, flavonoids (cannflavin A and B) were only detectable when positively ionised. As regards the MS data acquired in the positive ion mode, both CBDA and CBGA generated sodium adduct ions [M + Na]\({}^{+}\)[3], in addition to their pseudo-molecular ions [M + H]\({}^{+}\). CBGA was easily distinguished from CBDA, thanks to its different molecular weight. As to their MS\({}^{2}\) spectra carried out in the positive ion mode, both CBDA and CBGA showed base peaks related to the loss of water (\(-18\) u) at \(m/z\) 341 and 343, respectively. CBDA showed an additional product ion at \(m/z\) 285, attributable to the loss of a C\({}_{4}\)H\({}_{8}\) group [9; 33], which was not observed in the MS\({}^{2}\) spectrum of CBGA. Both CBG and CBD showed product ions at \(m/z\) 233 and 207 in the MS\({}^{2}\) experiments performed in the positive ion mode, although their relative intensity was different. The product ion at \(m/z\) 259 in the MS\({}^{2}\) spectrum of CBD was due to the loss of a C\({}_{4}\)H\({}_{8}\) group [9; 33], probably from the side alkyl chain, which was absent in the MS\({}^{2}\) spectrum of CBG. As to canniprene, the MS spectrum carried out in the positive ion mode showed the pseudo-molecular ion [M+H]\({}^{+}\) at \(m/z\) 343 and a base peak at \(m/z\) 287, due to the loss of a C\({}_{4}\)H\({}_{8}\) group of the side prenyl group. In the MS spectra of cannflavins A and B only the pseudo-molecular ion [M + H]\({}^{+}\) was observed. The MS\({}^{2}\) spectra of these compounds showed only one intense fragment at \(m/z\) 313, generated after the loss of the side prenyl moiety. As for the MS data obtained in the negative ion mode, both CBDA and CBGA generated product ions corresponding to the loss of water (\(-18\) u) and of CO\({}_{2}\) (\(-44\) u), together with their pseudo-molecular ions [M - H]\({}^{-}\).