# New Methods for the Comprehensive Analysis of Bioactive Compounds in _Camabis sativa_ L. (hemp)
## 2 Results and Discussion
### Extraction of Volatile Compounds from Hemp by HS-SPME  
The terpene composition of hemp infloresences has been found to enhance the biological activity of hemp-based products [30]; in this perspective, the development of a fast and reliable procedure for the extraction of the volatile compounds from the plant material is highly recommended for its complete characterisation. \begin{table}
\begin{tabular}{c c c c c c} \hline
**Compound** & **C1** & **C2** & **C3** & **C4** & **C5** & **C6** \\ \hline CBDA & 22.6 \(\pm\) 2.5 & 4.1 \(\pm\) 1.7 & 36.4 \(\pm\) 1.0 & 4.1 \(\pm\) 0.1 & 3.7 \(\pm\) 0.2 & 17.6 \(\pm\) 0.4 \\ CBGA & 0.4 b  & 0.2 \(\pm\) 0.1 & 0.6 b  & 0.2 b  & \(\sim\)LQO & 0.1 b  \\ CBG & \(<\)LQO & 0.3 \(\pm\) 0.1 & \(<\)LQO & 0.3 b  & \(<\)LQO & \(<\)LQO \\ CBD & 1.9 \(\pm\) 0.2 & 5.9 \(\pm\) 0.4 & 6.3 \(\pm\) 0.3 & 5.9 \(\pm\) 0.1 & 8.6 \(\pm\) 0.5 & 6.4 \(\pm\) 0.4 \\ \hline \end{tabular} Experimental conditions as in Section 3.4. \({}^{\text{a}}\) Data are expressed as mean (\(n=6\)) \(\pm\) SD. \({}^{\text{b}}\) SD \(<\) 0.05.  
\end{table} 
Table 2: Amount (mg/g) of the main non-psychoactive cannabinoids in hemp infloresences \({}^{\text{a}}\). \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Compound** & **C1** & **C2** & **C3** & **C4** & **C5** & **C6** \\ \hline CFL-A & 163.4 \(\pm\) 2.4 & 109.4 \(\pm\) 5.7 & 318.9 \(\pm\) 5.8 & 59.9 \(\pm\) 3.4 & 89.5 \(\pm\) 6.7 & 140.6 \(\pm\) 12.5 \\ CFL-B & 100.9 \(\pm\) 1.7 & 21.0 \(\pm\) 2.6 & 412.2 \(\pm\) 4.0 & 25.3 \(\pm\) 0.2 & 61.4 \(\pm\) 5.7 & 75.9 \(\pm\) 6.6 \\ Canniprene & \(<\)LOD & \(<\)LOD & \(<\)LOD & \(<\)LOD & \(<\)LOD & \(<\)LOD \\ \hline \end{tabular} Experimental conditions as in Section 3.4. \({}^{\text{a}}\) Data are expressed as mean (\(n=6\)) \(\pm\) SD. For sample C6, \(n=18\).  
\end{table} 
Table 3: Amount (mg/g) of the main flavonoids and related compounds in hemp infloresences \({}^{\text{a}}\). HS-SPME is being increasingly selected as the technique of choice in the ambit of the extraction of volatile compounds in the field of natural product research [14; 15; 27; 28]. In this study, a new HS-SPME procedure was optimised in order to achieve a reliable characterisation of the volatiles from hemp infloresances. To do this, sample C3 was used for the optimisation of the HS-SPME conditions, as the essential oil from the same variety was also available. Firstly, the performance of two different SPME fibres was assessed, including both a polydimethylsiloxane (PDMS) and a divinylbenzene-carboxen-polydimethylsiloxane (DVB/CAR/PDMS) fibre. The representative GC-FID chromatograms of the HS profile of hemp infloresances obtained by using the PDMS and the DVB/CAR/PDMS fibres are shown in Figure 8. In order to compare the fibre performance, the same temperature, equilibrium time, and extraction time were employed. The extraction efficiency of volatile compounds was found to be higher when the DVB/CAR/PDMS fibre was used; this fibre was therefore selected for this work. For the outcome of the overall extraction procedure, it is also important to properly set the extraction temperature, together with the equilibrium time and the extraction time. On the basis of previous data available in the literature [14; 15; 27; 28], temperatures of 40, 60, and 80 \({}^{\circ}\)C, equilibrium times of 20 and 30 min and extraction times of 10 and 20 min were evaluated in this study. As concerns the effect of temperature, a lower temperature was found to be much better, since an increase in the extraction temperature led to a loss in monoterenes with respect to the sesquiterpenes. In general, the chromatographic profile of hemp volatile compounds at 40 \({}^{\circ}\)C was characterised by higher peak areas (Figure 9); therefore, it was finally selected as the optimal extraction temperature.