# Influence of Altitude on Phytochemical Composition of Hemp Inflorescence: A Metabolomic Approach
## 2 Results and Discussion
### Quantification of Cannabinoids by HPLC-Q-Exactive-Orbitrap(r)-MS Analysis

Table 2: Results regarding the bioaccumulation of the main cannabinoids in the infloresescences of mountain Kompolti samples and corresponding plains clones (ug/g, mean of four biological samples \(\pm\) SD). HPLC-Q-Exactive-Orbitrap(r)-MS Untargeted Metabolomics Approach: Phytocannabinoids Profiling and Identification of Polyphenic Structures  
Metabolomic fingerprinting of the inflorescences that can be used for pharmacological/nutraceuticalul purposes is important to evaluate a plant metabolite quality and variability. Chromatographic/HRMS fingerprints have been used in the modelling and prediction of pharmacological activities of many medicinal plants [34], but only sporadically for cannabis. Also in this study, as performed in our recently published work [13] the compounds that characterize hemp inflorescences were identified by HPLC-Q-Exactive-Orbitrap(r)-MS untargeted metabolomics approach that consist of chromatographic separation, HRMS acquisition, and post-analysis data elaboration applying the Compound Discoverer software. Unlike in the above-mentioned paper, here we analyzed the samples separately, using two types of ionization: both positive and negative (Table 3) that enabled a more profound approach. The negative mode revealed the presence of about 80 compounds, whereas the positive mode individuated more than 190. By introducing the detection in negative polarity, it was possible to identify compounds not detected previously [13]. For example, CBGA methyl ester (CBGMA) was not detected in positive ionization, but its presence was clearly confirmed by fragmentation pattern obtained in negative mode (Figure 1). This strategy consequently allowed for an in-depth cluster analysis that clearly demonstrated the differences between the samples coming from the mountains and those cultivated in the plains (Figure 2). Two acquisition modes showed to be complementary in the statistical evaluation of differences between two samples group. As regards the data obtained, it was possible to detect other minor secondary metabolites already identified in other cannabis species. The CBD-family remains to be most abundant (Table 3), enriched by the presence of two sesquiterpene CBDA esters. This CBDA-terpene (inter)reaction needs further elucidation, especially whether it depends on climate/environmental conditions [8; 9; 13]. In any case, special attention must be paid to cannaflavins that belong to the class of premyIflavonoids. This flavins are secondary metabolites exclusive to the _Cannabis_ genus and were detected in both environments. Their notable presence in mountain samples points towards alternations in their synthesis [16]. The higher levels detected in the mountain samples may possibly be a consequence of the lower average temperature, combined with high solar radiation experienced at the beginning of plant flowering. In fact, it was reported that different classes of flavonoids are involved in plant protection mechanisms, specifically for their radical scavenger activity and screening ability against short wavelength UV-B light [35]. Furthermore, in the Kompolti inflorescence, the remarkable presence of a signal with m/z value at 433.14931 was identified and leads towards the recognition of a particular flavonoid: 3-methoxyinobiletin (3,3',4',5,6,7,8-heptamethoxyflavone) (Figure 3). This compound has not been reported for any variety of hemp so far. Furthermore, it belongs to the class of polymethoxyflavones that have anti-inflammatory and anti-carcinogenic activities and occur as \"novel nutraceuticalul compounds\" [36]. In order to perform its absolute identification, it is necessary to isolate it from the inflorescence and define it with a more detailed analytical approach, also including NMR analysis. Our metabolomics mapping identified two important phytohormones: salicylic and abscisic acid that have not been reported for cannabinoids inflorescences so far. Their presence was confirmed in our mountain samples, but their amount turned to be more than 10 times higher in lowland specimens.