HPLC analysis of phenolic compounds

The identification of individual compounds were performed in a LCQ Deca XP Max LC-MSⁿ system (Thermo Finnigan, CA) equipped with an autosampler, a Surveyor 2000 quaternary pump and a UV 2000 PDA detector, using a 150 × 2.00 mm Synergi 4 µ Hydro RP 80 A column (Phenomenex, Torrance, CA) and a guard column of the same chemistry. The LC-MSⁿ system with a Z-spray ESI source was run by Xcalibur software, version 1.3 (Thermo Finnigan-Surveyor, San Jose, CA, USA). The mobile phase flow rate was set at 0.25 mL/min, while the elution gradients were performed with solvent A, consisting of acetonitrile/methanol (1:1) (containing 0.5% formic acid); and solvent B, consisting of water (containing 0.5% formic acid). The applied elution conditions were: 0–2 min, 2% A, 98% B; 3–5 min, 5% A, 95% B; 5–7 min, 25% A, 75% B; 7–12 min, 55% A, 45% B; 12–24 min, 55%A-80%A, 24–27 min held isocratic at 80%A, 28–30 min 90% A, 10% B; 31–33 min held isocratic, 100% A; 34–40 min, 2% A, 98% B, to the starting condition. The chromatograms were monitored at 280 nm, and complete spectral data were recorded in the range 200–600 nm. ESI was performed in the negative ionization mode, nitrogen was used as sheath gas with a flow of 59 arbitrary units, and He gas was used as dampening gas. The capillary voltage, −4.17 V; spray voltage, 5 kV; capillary temperature, 275 °C; and tube lens voltage at −55V. Collision energies of 30% were used for the MSⁿ analysis.

Chromatographic separation and quantification was performed on same LC-MSⁿ system. The elution gradient was formed with solvent A [0.5% formic acid -water] and solvent B [0.5% formic acid in acetonitrile]. A linear gradient was set up with A and B: 0 min 98% A, 10 min 75% A, 20 min 75% A, 30 min 25% A, 35 min 0% A, 38 min 98% A. The flow rate was 200 µl/min. The injection volume was 10 µl. Retention time and spectral profile were used for identification detected by a photodiode array detector (PDA) scanning between 190–600 nm, the quantification was done by comparison with external standards obtained from Sigma-Aldrich (St. Luis, MO).