Dionex ultimate 3000 series instrument

The HPLC analysis was carried out on a Dionex ultimate 3000 series instrument (Thermo Scientific Inc.) coupled to a binary pump, an autosampler and a column compartment (kept at 20 • C). Separation was achieved on a Phenomenex Gemini C 18 column (5 m, 250 × 3.0 mm i.d.) using a mobile phase composed by CH 3 CN (A) and water/formic acid (0.1%, v/v) at a flow rate of 0.4 mL min -1 . The following gradient program was used: 20% A (0 min), 25% A (10 min), 25% A (20 min), 50% A (40 min), 100% A (42-47 min) and 20% A (49-55 min).
For HPLC-ESI-MS n analysis, a Bruker Esquire model 6000 ion trap mass spectrometer (Bremen, Germany) with an ESI source was used. MS n analysis was performed in negative and positive mode and scan range was set at m/z 100-1000 with speed of 13,000 Da/s. The conditions of ESI were as follows: drying and nebulizer gas (N 2 ) flow rate and pressure, 10 mL min -1 and 50 psi; capillary temperature, 325 • C; capillary voltage, 4.5 keV; collision gas (He) pressure and energy, 1 × 10 -5 mbar and 40 eV. The acquisition of MS n data was made with the auto MS n mode, selecting an isolation width of 4.0 m/z, and a fragmentation amplitude of 1.0 V (MS n up to MS 4 ). Samples were filtered through 0.45 m PTFE membrane filters, and 10 L were injected.

The HPLC-ESI-MS n analysis was performed on a Dionex ultimate 3000 series instrument (Thermo Scientific Inc.) equipped with a binary pump, an autosampler and a column compartment (kept at 30 C) and coupled with a Bruker Esquire model 6000 ion trap mass spectrometer with an ESI source (Bremen, Germany). Analysis was carried out on a Phenomenex Gemini C 18 column (5 mm, 250 Â 3.0 mm i.d.) using the same conditions as previously (Gouveia and Castilho, 2012c) . Extract solutions (5 mg mL À1 ) were prepared by dissolving the dry extract (DE) in the initial HPLC mobile phase. After filtration through 0.45 mm PTFE membrane filters, 5 mL was injected.