Kinetex c18 100

The use of an HPLC with a diode array detector and electro spray ionization coupled with a mass spectroscopy (DAD-ESI/MS) was used to identify and characterize malvidin-3-O-glucoside, vitisin B, malvidin-3-O-glucoside acyl derivatives and polymeric pigments with malvidin-3-O-glucoside; the HPLC was a Agilent Technologies™ 1100 (Palo Alto, CA, USA) chromatograph with a column RP KineteX C18 100Å (100 × 4.6 mm; 2.6 μm) (PhenomeneX, Torrance, CA, USA). The eluents used comprised eluent A (water/formic acid 95:5 v/v) and eluent B (methanol/formic acid 95:5 v/v), with the following gradient of eluent B (0.8 mL/min): from 20% to 50% from 0 min to 27 min; 50% from 27 min to 28 min, and finally, from 50% to 20% from 28 min to 29 min until a steady state was reached. According to Loira et al. [7 (link)], malvidin-3-O-glucoside has been used as an eXternal standard at a wavelength of 525 nm for the quantification of all pigments, while the identification was carried out with mass spectrometry positive scanning from 100 to 1500 m/z from 0 min to 23 min. The detection limit was set to 0.1 mg/L [41 (link)].

The HPLC analyses were performed according to Rathi et al., 2019 [36 (link)], with some modifications, as described below. The quantification was conducted on Waters HPLC system, consisting of Waters 1525 Binary pumps with Waters 2487 Dual λ Absorbance Detector (Waters, Milford, MA, USA), equipped with a reverse-phase Kinetex^® C₁₈, 100 Å (150 × 4.6 mm, 5 µm) core-shell column (Phenomenex, Torrance, CA, USA), operating at 26 °C. The mobile phase consisted of water (solvent A) and acetonitrile (solvent B), with a flow rate 0.5 mL/min and gradient elution as follows: 0–2 min 99% A; 2–6 min decreased to 40% A; 6–15 min gradually increased back to 99% A arbutin was detected at 285 nm. Six solutions of the arbutin, with different concentrations ranging from 25 to 200 μg/mL dissolved in methanol, were used to construct a linear calibration curve.

A chemical profile was obtained using ultra-high-performance liquid chromatography coupled with photodiode array detection (UPLC-PAD) on an ACQUITY UPLC system (Waters, Milford, MA, USA), which consisted of a photodiode array detector, quaternary pump, degasser, column oven, and autosampler. The separation of metabolites was performed using Phenomenex^® Kinetex C18 100 Å (75 mm × 2.1 mm; 2.6 µm). The mobile phase was formic acid 0.1% (component A) and acetonitrile (component B) as follows: 0–8 min, 16% B; 8–10 min, 16–20% B; 10–12 min, 20% B; 12–15 min, 20–22% B; 15–18 min, 22–27% B; 18–20 min, 27–30% B; 20–23 min, 30–35% B; 23–28 min, 35% B; 28–34 min, 35–90% B; and then back to 16% B in 1 min [52 (link)]. The samples were injected automatically at 3 µL. The column temperature was maintained at 30 °C. The flow rate was set at 0.4 mL/min. The photo diode array (PDA) detection was set at 340 nm. Ultra-high-performance liquid chromatography–electrospray ionization–quadrupole time-of-flight–mass spectrometry (UHPLC-ESI-QToF-MS) analysis was performed on Nexera LCMS 9030 Shimadzu Scientific-Instruments (Columbia, MD, USA) equipment. The chromatographic conditions were the same as those described previously: mass spectrum detection for negative ion mode. The capillary potentials were set at +3 kV, drying gas temperature 250 °C, and the flow rate of drying gas 350 L/min.

A chemical profile was obtained using ultra-high-performance liquid chromatography coupled with photodiode array detection (UPLC-PAD) on an ACQUITY UPLC system (Waters, Milford, MA, USA), which consisted of a photodiode array detector, quaternary pump, degasser, column oven, and autosampler. The separation of metabolites was performed using Phenomenex^® Kinetex C18 100 Å (75 mm × 2.1 mm; 2.6 µm). The mobile phase was formic acid 0.1% (component A) and acetonitrile (component B) as follows: 0–8 min, 16% B; 8–10 min, 16–20% B; 10–12 min, 20% B; 12–15 min, 20–22% B; 15–18 min, 22–27% B; 18–20 min, 27–30% B; 20–23 min, 30–35% B; 23–28 min, 35% B; 28–34 min, 35–90% B; and then back to 16% B in 1 min [52 (link)]. The samples were injected automatically at 3 µL. The column temperature was maintained at 30 °C. The flow rate was set at 0.4 mL/min. The photo diode array (PDA) detection was set at 340 nm. Ultra-high-performance liquid chromatography–electrospray ionization–quadrupole time-of-flight–mass spectrometry (UHPLC-ESI-QToF-MS) analysis was performed on Nexera LCMS 9030 Shimadzu Scientific-Instruments (Columbia, MD, USA) equipment. The chromatographic conditions were the same as those described previously: mass spectrum detection for negative ion mode. The capillary potentials were set at +3 kV, drying gas temperature 250 °C, and the flow rate of drying gas 350 L/min.