# Phenolic and Volatile Composition of a Dry Spearmint (_Mentha spicata_ L.) Extract
## 3 Materials and Methods
### Characterization and Quantification of Phenolic Fraction by UHPLC-ESI-MS\({}^{n}\)  
The extraction of phenolic compounds was performed on 200 mg of spearmint extract by adding 1 mL of 80% aqueous methanol acidified with formic acid (1%), according to [PERSON] et al. (2015) [50]. The solution was shaken in an ultrasonic bath at room temperature for 25 min. The mixture was then centrifuged at 10,480 \(g\) for 5 min at room temperature. In order to obtain an exhaustive extraction of the phenolic fraction, two additional extractions were performed on the same sample. The three supernatants were pooled before UHPLC-ESI-MS\({}^{n}\) analyses. Each sample was extracted in quadruplicate. Methanolic extracts of spearmint were analyzed using an Accela UHPLC 1250 equipped with a linear ion trap-mass spectrometer (MS) (LTQ XL, Thermo Fisher Scientific Inc., San Jose, CA, USA) fitted with a heated-electrospray ionization probe (H-ESI-II; Thermo Fisher Scientific Inc.). Separations were performed using a BlueOrchid C18 column (50 \(\times\) 2 mm, 1.8 \(\upmu\)m particle size, Knauer, Berlin, Germany). The total volume injected was 5 \(\upmu\)L and the column oven temperature was 30 \({}^{\circ}\)C. Two MS experiments in negative mode were performed according to a previous protocol [51]. Optimal parameters for epicatechin analysis (Experimental Conditions 1) were carried out using the following conditions. The MS was operated using a capillary temperature equal to 275 \({}^{\circ}\)C, while the source heater temperature was set to 200 \({}^{\circ}\)C. The sheath gas flow was operated at 40 units, while both auxiliary and sweep gas were set to 5 units. The source voltage was 4 kV. The capillary and tube lens voltages were \(-42\) and \(-118\) V, respectively. Elution was performed at a flow rate of 0.3 mL/min. The gradient started with 99% of 0.1% aqueous formic acid, keeping isocratic conditions for 2 min, followed by a 10 min linear gradient of acetonitrile in 0.1% formic acid which started at 1% and was increased to 40%. The acidified acetonitrile was increased to 80% between minutes 12 and 13 min, and maintained for 3 min, followed by 4 min at the starting conditions to re-equilibrate the column. Analyses were carried out using full scan, data-dependent MS\({}^{3}\) scanning from \(m/z\) 100-1500, with collision-induced dissociation (CID) equal to 30 (arbitrary units). Pure helium gas was used for CID. The second experimental framework utilized MS with conditions optimized for rosmarinic acid analysis (Experimental Conditions 2). The capillary temperature was set to 275 \({}^{\circ}\)C, while the source heater temperature was 50 \({}^{\circ}\)C. The sheath gas flow was operated at 40 units, while auxiliary and sweep gas were set to 5 and 0 units, respectively. The source voltage was operated at 4 kV. The capillary and tube lens voltages were \(-26\) and \(-78\) V, respectively. Analyses were carried out using full scan, data-dependent MS\({}^{3}\) scanning from \(m/z\) 100-1500, with CID equal to 30 (arbitrary units). The chromatographic conditions were identical to those used for the preliminary phenolic analyses. Quantification was performed using selected ion monitoring mode (SIM) by selecting the relative base peak at the corresponding mass to charge ratio (\(m/z\)) under Experimental Conditions 2, based on rosmarinic acid. Different dilutions of the extract in 0.1% aqueous formic acid (dilution factors ranging from 10-1000) were used to avoid signal saturation and quantify within the linearity range of the reference compounds. Volatile Extraction and Characterization by Head Space Solid Phase Microextraction (HS-SPME) Coupled with GC-MS Technique  
The volatile fraction of the spearmint sample was characterized following the protocol of [PERSON] et al. (2012) [34] with slight modifications. Briefly, 100 mg of spearmint extract were placed in a 30 mL glass vial. For each SPME analysis, 100 \(\upmu\)L of an aqueous toluene standard solution (348 mg/L) were added to the sample.