3.3. LC-MS Analysis: Phenolic Profile of the Extracts

The LC-MS analysis was conducted using an ABSciex API 3000 triple quadrupole mass spectrometer (AB Sciex LLC, Framingham, MA, USA) coupled with an Agilent 1100 HPLC system with binary pump and autosampler (Agilent Technologies, Inc., Santa Clara, CA, USA). Acquisition and data reduction were performed using Analyst 1.6.2 software (AB Sciex LLC, Framingham, MA, USA).

The HPLC separation was carried out on an Agilent Phenyl Column (3 × 100 mm; 2.7 μm), and the eluents were (A) acidified water (at pH2.5 adjusted with HCOOH) and (B) acetonitrile/water (90/10, at pH2.5 adjusted with HCOOH). A gradient solvent system was used as follows: 0–3 min, 5% B; 3–18 min, 5–40% B; 18–28 min, 40% B; 28–38 min, 40–80% B; 38–43 min, 80% B, 43–45 min, 80–5% B, at a flow rate of 0.4mLmin⁻¹.The MS analysis was carried out under the following experimental conditions: Atmospheric Pressure Chemical Ionization (APCI) using the heated nebulizer interface; Needle Current (NC), −5 µA; Nebulizer Gas (air), 10 (arbitrary units); Auxiliary Gas (air), 3 L min⁻¹; Auxiliary Gas Temperature (TEM), 550°C; Curtain Gas (CUR, nitrogen), 6 (arbitrary units); Collision Gas (CAD, Nitrogen), 9 (arbitrary units, corresponding to 2.6 × 10⁻⁵ Torr collision cell pressure).

The identification of the different phenolic components was performed using a targeted approach, using a multiple reaction monitoring (MRM) method, optimized with standards for 19 target compounds (chosen based on previous studies of polyphenolic composition of Hibiscus spp. [25,35]): two flavan-3-ols (catechin and epicatechin), seven flavonols (quercetagetin-7-O-glucoside, rutin, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, kaempferol-7-O-glucoside, kaempferol-3-O-glucoside, and quercetin), one cinnamate ester (chlorogenic acid), two hydroxycinnamic acids (p-coumaric and trans-ferulic acids), two dihydrochalcones (phloridzin and phloretin), one oxyflavone (tiliroside), and four anthocyanins (myrtillin, kuromanin, peonidin-3-O-glucoside, and oenin). The retention time and the relative MRM transitions (quantifier and qualifier) were reported in the Supplementary Table S1. Moreover, additional tentative identifications have been suggested using an untargeted approach, scanning the quadrupole from m/z 100 to 1000 Da.