# New Methods for the Comprehensive Analysis of Bioactive Compounds in _Camabis sativa_ L. (hemp)
## 1 Introduction
As to canniprene, it has been demonstrated to exert an anti-inflammatory activity as well, by inhibiting the production of pro-inflammatory eicosanoids [8]. Figure 1: Chemical structures of main non-psychoactive cannabinoids in hemp infloresances. As regards the other compounds present in hemp, terpenes are responsible for the characteristic aroma of the plant. The main volatiles detected in the aerial parts of the plant include both mono- and sesquiterpenes [14; 15; 17; 27; 28; 29], with \(\beta\)-myrcene and \(\beta\)-caryophyllene as the most representative compounds (Figure 3), respectively. As regards monoterpenes, \(\beta\)-myrcene is known to possess anti-inflammatory, analgesic, and anxiolytic properties [17]. As for sesquiterpenes, \(\beta\)-caryophyllene was found to be an anti-inflammatory agent and to exert a gastric cytoprotector activity; it has been demonstrated also to be able to bind to the cannabinoid receptors type 2 (CB\({}_{2}\)) and, in this context, it can be considered as a phytocannabinoid [17]. Some biological activities of cannabinoids are known to be enhanced by the presence of other secondary metabolites in _C. sativa_ extracts, such as in cases of sleeping disorders and anxiety [30]. This effect has been attributed to a strict interaction between cannabinoids and terpenes, resulting in a synergistic action [30]. As an example, terpenes are able to increase blood-brain barrier permeability and they can also interact with neurotransmitter receptors, thus contributing to cannabinoid-mediated analgesic and psychotic effects [17]. Finally, also flavonoids may modulate the pharmacokinetics of some cannabinoids, by means of the inhibition of hepatic P450 enzymes [17]. With this in view, the development and application of advanced analytical methods for a comprehensive analysis of _C. sativa_ extracts plays a pivotal role in order to have a reliable evaluation of their chemical composition both for a higher reproducibility of biological assays and also to guarantee the efficacy and safety in their pharmaceutical use. In the light of all the above, the present study was aimed at the first multi-component analysis of the bioactive compounds present in hemp female inflorescences of different varieties, including cannabinoids, hemp-specific phenolics, and volatile compounds. Innovative HPLC and GC methods were developed and applied in this study to the chemical classes of compounds cited above. In particular, the profiling of non-psychoactive cannabinoids in hemp extracts was carried out by means of HPLC with UV/DAD, ESI-MS, and ESI-MS\({}^{2}\) detection. A new RP-HPLC-UV/DAD, ESI-MS, and ESI-MS\({}^{2}\) method, together with a selective extraction protocol, was developed as well and applied for the determination of hemp phenolics (including cannflavin A, cannflavin B, and canniprene). The study on _C. sativa_ volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. The analytical methods developed in this study represent reliable and useful tools for a complete characterization of hemp plant material and extracts to be used in the pharmaceutical and nutraceuticaltical fields. Figure 3: Chemical structures of main terpen in hemp inflorescences. Figure 2: Chemical structures of main flavonoids and related compounds in hemp inflorescences.