*2.1. Reagents*

The chemicals employed during the development of this study have analytical reagen<sup>t</sup> grade. Purified water used for extraction and analytical experiments was obtained from a Milli-Q system from Millipore (Bedford, MA, USA). Moreover, ethanol used to obtain artichoke PLE extracts was purchased from VWR Chemicals (Radnor, PA, USA), whereas sand and cellulose filters were from Fisher Chemicals (Waltham, MA, USA). The mobile phases used for the analysis were prepared with formic acid and LC-MS-grade methanol provided from Sigma–Aldrich (Steinheim, Germany) and Fisher Chemicals (Waltham, MA, USA), respectively.

### *2.2. Plant Material*

By-products such as bracts and stems were obtained from the industrial processing of artichokes. The post-harvest processing of artichokes consists in a minimally blanching step carried out in the same day of harvesting before peeling. This processing was performed with water for 20 min at 98 ◦C. After blanching, the artichokes were pealed and the bracts and stems were separated from the artichoke hearts. These by-products were frozen at −45 ◦C and lyophilized for 24 h in a freeze-dryer (Vertis SP Scientific, Thermo Fisher (Madrid, Spain).

### *2.3. Pressurized Liquid Extraction*

PLE extracts from artichoke by-products were obtained with an accelerated solvent extractor (ASE 350, Dionex, Sunnyvale, CA, USA) applying different combinations of temperature and solvent composition (mixtures of ethanol and water in different proportions) previously applied to food by-products. These conditions were selected to cover a wide range of dielectric constant (from 19 to 59.1) [19,20].

Briefly, PLE static extractions were performed during 20 min under a pressure of 1500 psi. Thus, the tested ranges of instrumental parameters were as follow: 0–100% of aqueous ethanol mixtures as extraction solvent and 40 to 200 ◦C as extraction temperature.

The experiments were performed with extraction cells of 33 mL filled with a homogenous mixture composed of 4 g of artichoke sample plus 8 g of sand. This sample-sand ratio (1:2, w:w) allows to perform the experiments avoiding technical problems (fundamentally blockage of the lines of the PLE instrument) with the maximum amount of sample. The solvents used in each PLE experiment were previously sonicated for 15 min in order to eliminate the oxygen dissolved in the mixture, which could provoke a degradation of compounds susceptible of oxidation.

The PLE process begins with a cell heat-up step previous to the extraction in order to attain the pertinent extraction temperature. The duration of this phase is dependent of the temperature set-point and established by the instrument (lasting from 5 to 9 min). Then, the extraction step was carried out applying the corresponding conditions according to Table 1.


**Table 1.** Experimental pressurized liquid extraction (PLE) conditions.

The obtained extracts were immediately cooled at room temperature in an ice-bath.The supernatants were separated after a centrifugation step of 15 min at 4 ◦C applying

12,499 as relative centrifugal force (RCF). Then, they were completely evaporated under vacuum at 35 ◦C in a Savan SC250EXP Speed-Vac (Thermo Scientific, Leicestershire, UK). The dry extracts were stored refrigerated ( −20 ◦C) avoiding light exposure until further use.

### *2.4. HPLC-ESI-TOF-MS Analysis*

The analysis of the artichoke by-products PLE extracts was carried out by highperformance liquid chromatography (HPLC) with a diode array detector (DAD) coupled to time-of-flight mass spectrometry with an electrospray interface (ESI-TOF-MS). The instrument was an Agilent 1200-RRLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, a binary pump, an auto-sampler, a thermostatically controlled column compartment, and a DAD detector. Mass detection was performed using a micrOTOF II analyzer from Bruker Daltonik (Bremen, Germany). The extracts were dissolved in aqueous ethanol (50:50, v/v) at 5 mg/mL and analyzed in triplicate. Before the HPLC injection, the samples were filtered through a 0.45 μm syringe filters of regenerated cellulose to avoid solid particles.

The separation occurs in an Agilent Zorbax Eclipse Plus C18 column (150 mm × 4.6 mm id, 1.8 μm). The mobile phases consist of water with 0.1% formic acid (A) and methanol (B) at a flow rate of 0.8 mL/min. The analytical method used an elution gradient according to the following linear multi-step profile: 0 min, 5% B; 3 min, 50% B; 25 min, 75% B; 35 min, 100% B, 40 min, and 5% B plus 5 min of stabilization at initial conditions before the next analysis. The analyses were carried out at 25 ◦C injecting 10 μL of sample in each run.

Detection was performed within a mass range of 50–1000 *m/z* operating in negative ionization mode. In order to achieve a stable ionization, the flow rate from the HPLC was split before the connection to the ESI interface (Agilent Technologies) until 125 μL/min. Ultrapure nitrogen was used as drying and nebulizing gas with flows of 9.0 L min−<sup>1</sup> and 2.0 bar, respectively. The operating parameters applied for the ionization and ion transfer were: capillary voltage of +4.5 kV; drying gas temperature, 190 ◦C; capillary exit, −150 V; skimmer 1, −50 V; hexapole 1, −23 V; RF hexapole, 100 Vpp; and skimmer 2, −22.5 V.

In order to recalibrate the mass spectra acquired during the analysis to achieve accurate mass measurements with a precision better than 5 ppm, 10 mM sodium formate solution was used as calibrant. This mixture is automatically injected at the beginning of each run by means of a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, IL, USA) directly connected to the ESI interface, equipped with a Hamilton syringe (Reno, NV, USA). All data acquisition and processing operations were controlled with HyStar 3.2 and Data Analysis 4.0 software, respectively (Bruker Daltonics GmbH, Bremen, Germany). The software provides a list of possible elemental formulas using the Generate-Molecular Formula Editor. This information provided by the analytical platform was used for identification purposes together with the one reported in databases and bibliography.

### *2.5. Statistical Analysis*

Data were statistically treated using Origin (Version Origin Pro 8.5, Northampton, MA, USA). For these data, set one-way analysis of variance (ANOVA, Tukey's test) at a 95% confidence level (*p* ≤ 0.05) was performed to point out the differences in semiquantitative bioactive compounds contents found between PLE artichoke samples with statistical significance.
