Hp 9000 workstation

The quantitative analyses were carried out using a HP 1100 L liquid chromatograph equipped with a DAD detector and managed by a HP 9000 workstation (Agilent Technologies). A 150 mm × 4.6 mm i.d., 5 μm Zorbax Eclipse XDB, RP18 column was used. 20 μL of each sample were injected. Chromatography was carried out in gradient mode using a flow rate of 0.6 mL/min. The mobile phase was (A) formic acid/water pH 3.2 and (B) CH₃CN. The multi-step linear solvent gradient used was: 0–5 min 15–20% B; 5–7 min 20–30% B; 7–10 min 30–40% B; 10–15 min 40–50% B; 15–20 min 50–80% B; 20–25 min 80–15% B; post time 10 min; oven temperature 30 °C, flux: 0.6 mL/min. The UV/Vis spectra were recorded in the range 200–700 nm and the chromatograms were acquired at 210, 260, 280, 350 nm.
For qualitative analysis, MS experiments were conducted using a LTQ equipped with an ESI interface (Finnigan LTQ, Thermofisher Scientific, Waltham, MA). Mass spectrometry and electrospray operating parameters were optimized for negative polarity. The following final settings were used: sheath gas flow rate (arb): 30, aux gas flow rate (arb): 5, sweep gas rate (arb): 5, capillary temp (°C): 290.00, capillary voltage (V): 16.93, tube lents (V): −99.72.

Quali-quantitative analyses of carotenoids and polyphenols were carried out using an HP 1100 liquid chromatography equipped with a DAD detector and managed by an HP 9000 workstation (Agilent Technologies, Palo Alto, CA, USA) and linked to a mass spectrometer with an API/electrospray interface (Agilent Technologies). The mass spectrometer operating conditions were as follows: gas temperature, 350 °C; nitrogen flow rate, 11.0 L/min; nebulizer pressure, 40 psi; quadrupole temperature, 100 °C; and capillary voltage, 4000 V. The mass spectrometer was operated in positive and negative modes at 80–180 eV.
Compounds were separated using a 250 × 4.6 mm i.d, 5 µm LUNA C18 column (Phenomenex, Torrance, CA, USA). UV/Vis spectra were recorded in the 190–600 nm range and the chromatograms were acquired at 250, 280, 330, 350 and 450 nm. The samples were analyzed by gradient elution at a flow rate of 0.8 mL/min. The mobile phase for carotenoids was a multistep linear solvent gradient system (solvent A: acetone, solvent B: H₂O, pH 3.2 by formic acid), starting from 80% acetone up to 100% in 30 min. polyphenols were eluted using the following gradient: from 90% H₂O (adjusted to pH 3.2 by formic acid) to 100% CH₃CN in 40 min. All solvents used were of HPLC grade purity (BDH Laboratory Supplies, Poole, UK).

Analyses of flavonols, hydroxycinnamic acids, and coumarins were carried out using an HP 1100 L liquid chromatograph equipped with a DAD detector and managed by an HP 9000 workstation (Agilent Technologies, Palo Alto, CA, USA). Compounds were separated by using a 250 × 4.6 mm i.d. 5 μm LUNA C18 column (Phenomenex, USA). UV/Vis spectra were recorded in the 190-600 nm range, and the chromatograms were acquired at 250, 280, 330, and 350 nm. The samples were analyzed by gradient elution at a flow rate of 0.8 ml/min. The mobile phase was a multistep linear solvent gradient system, starting from 95% H₂O (adjusted to pH 2 by HCOOH) up to 100% CH₃CN in 53 minutes. The chemical reagents used were HPLC grade, acetonitrile (CH₃CN) HPLC grade, ethanol (EtOH) HPLC grade, Folin-Ciocalteu reagent, and sodium carbonate (Na₂CO₃); all were purchased from Sigma-Aldrich (St. Louis, Mo, USA).