Esi interface

The HPLC-ESI-MS/MS analyses were performed using a 1100 Series Agilent Technologies LC/MSD system equipped with a diode array detector coupled to a quadrupole mass spectrometer with an ESI interface (Agilent Technologies, Mississagua, Ont.). The reverse-phase separation was performed on a Vydac C18 column (5 μm; 250 mm × 4.6 mm; Grace, Hespedia, CA.). The compounds were separated using eluent (A): 4.5% aqueous formic acid and eluent (B): acetonitrile diluted to 80% with 4.5% formic acid in water. The gradient was as follows: 7 min 15% B; 15 min 20% B; 16 min 100% B and in 24 min returned to 100% of eluent A. MS parameters were as follows: capillary voltage 4000 V; drying gas temperature 350 o C; nitrogen fl ow 12 L/min; nebulizer pressure 60 psi. The instrument was scanned positive and negative ions in the range from 100 to 1500 m/z at a rate of 2.0 s/cycle. Compounds identifi cation is based on spectra library developed in laboratory and published data (Gil-Izquierdo and Mallenthin, 2001) .

Analysis was firstly carried out using a HP-1100 liquid chromatograph equipped with a DAD detector and a HP1100 MSD API-electrospray (Agilent-Technologies, Palo Alto, CA) operating in positive and negative ionization mode under the following conditions: gas temperature 350 C, nitrogen flow rate 10 L/min, nebulizer pressure 35 psi, quadrupole temperature 30 C, capillary voltage 4000 V, and applied fragmentors in the range 50-250 V.
Further identification and characterization of the phenolic compounds was performed by a TOF-MS with an ESI interface (Agilent Technologies) under the following conditions: gas temperature 300 C, nitrogen flow rate 12 L/min, nebulizer pressure 20 psi, capillary voltage 3800 V, and applied fragmentors in the range 80-300 V.
The column was a Synergi Max RP 80 A (4 mm; 150 mm  3 mm i.d.) from Phenomenex (Castel-Maggiore, BO, Italy). The mobile phases were: (A) acidified water (pH 2.0) and (B) acetonitrile. The following multistep linear gradient was applied: from 95% to 78% A in 8 min, 4 min to reach 74% A, then 13 min to arrive at 65% A, and finally 3 min to reach 100% B with a final plateau of 4 min. The total time of analysis was 32 min, flow rate was 0.4 mL/min, and oven temperature was 26 AE 0.5 C, as described in our previous study (Ieri et al., 2011) (link).