## 2 Materials and Methods
### Chromatography
#### 2.4.3 UHPLC-DAD/MS/MS  
After the chemical characterization of the extracts, seven promising samples, particularly rich in target bioactive compounds (Table 2) were selected, and further analyzed (as described below) to further inspect the phytochemical profiles. Samples VE45E and VE45F, as well as samples VN45E and VN45F, were treated as duplicates.  
Each extract reported in Table 2 was 5-fold diluted with a mixture of water/methanol 1:1 (\(v/v\)) acidified with 0.1% formic acid. After vortexing, each extract was centrifuged at 7378 g for 5 min at 10 \({}^{\circ}\)C, before UHPLC-DAD-MS/MS analysis.  
For the mobile phase, a binary system of water (phase A) and acetonitrile (phase B) was used, both acidified with 0.01% formic acid. The starting gradient consisted of 99% phase A, maintained for 0.5 min and then decreased to 85% at 3 min. From 3 to 6 min phase A was reduced to 50%, then dropped rapidly to 5% at 9 min to clean the column. At 11 min the starting gradient at 99% of A was restored and maintained until 14 min to guarantee a complete equilibration of the column for the next analysis. The mobile phase pumped at 0.4 mL/min. The column compartment was set at 40 \({}^{\circ}\)C, while the autosampler temperature was set at 10 \({}^{\circ}\)C. An Acquity UPLC HSS T3 (2.1 \(\times\) 100 mm) equipped with a VanGuard Acquity UPLC HSS T3 (2.1 \(\times\) 5 mm) guard column was used as the chromatographic column (Waters, Milford, MA, USA). The photodiode array detector scanned in a range of 220-500 nm. The injection volume was 2 \(\mu\)L.  
Different spectrometric modalities were used to characterize the phenolic components in the various extracts. Initially, extracts 29, 63, 65, and VN45E were analyzed in MS Scan mode both in ESI+ and ESI\(-\), scanning from \(m/z\) 100 to 1500. In negative mode, the capillary voltage was set to 2.3 kV, the cone voltage was 60 V, with the source and desolvation T set at 150 \({}^{\circ}\)C and 600 \({}^{\circ}\)C, respectively. Nitrogen has been used as nebulizer gas at P equal to 7 bar. In positive mode, 1 kV was applied on the capillary with a cone voltage set at 80 V, while the other MS parameters were the same used for negative mode. Ultra-pure argon has been used as collision gas. Following the MS Scan analyses, both the most abundant ions and the ions inherent in chromatographic peaks with increased/decreased ion abundance between the various samples were fragmented in MS/MS mode (collision energy set to 30 eV). This led to obtaining the peak identification or at least to ascribe each chromatographic peak with a specific (poly)phenolic subclass. In addition, the extracts 29, 63, 65, and VN45E were analyzed in ESI by monitoring the precursor ions of the fragment ions with \(m/z\) 163, 179, and 193, corresponding to coumaric, caffeic, and ferulic acids, respectively. This analytical approach was used since these hydroxycinamic acids are particularly abundant in _Verbascum_ spp. as glycosides and as esters of iridoids and phenylethanoloids [34; 35].  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample Code** & **Biomass** & **Pretreatment** \\ \hline VE45E & _Verbascum nigrum_ & EH + HA \\ VE45F & _Verbascum nigrum_ & EH + HA \\
63 & _Verbascum nigrum_ & EH + HA \\
65 & _Verbascum nigrum_ & EH \\ VN45E & _Verbascum nigrum_ & H + HA \\ VN45F & _Verbascum nigrum_ & H + HA \\
29 & _Verbascum nigrum_ & HA \\ \hline \hline \end{tabular}
\end{table}
Table 2: Samples of _V. nigrum_ investigated to comparative analysis.  
As a result of the comparative analyses mentioned above, a final method in Multiple Reaction Monitoring (MRM) mode was developed, which is the most sensitive and selective method to perform a comparison of the main phenolic compounds in the extracts reported in Table 2. Each diluted extract was analyzed twice.