HPLC-ESI-MS Metabolite Analysis Protocol

The extracts were analyzed utilizing a ThermoFinnigan LCQ-Duo ion trap mass spectrometer (ThermoElectron Corporation, Waltham, Ma, USA) with an ESI source (ThermoQuest Corporation, Austin, Tx, USA). A ThermoFinnigan HPLC system using a C18 reversed-phase column (Zorbax Eclipse XDB-C18, Rapid resolution, 4.6 × 150 mm, 3.5 µm, Agilent, Santa Clara, CA, USA) was used^{17 (link)}. Water and acetonitrile (ACN) (Sigma-Aldrich GmbH, Germany) (0.1% formic acid each) were used as a mobile phase. At 0 min, ACN was 5% and then increased to 30% in 60 min at 1 mL/min with a 1:1 split before the ESI source. Autosampler surveyor ThermoQuest was utilized to inject the extracts and the system was controlled by Xcalibur software (Xcalibur^TM 2.0.7, Thermo Fischer Scientific, Waltham, Ma, USA). The MS operated in the negative mode as reported before^{18 (link)}. The ions were detected in a full scan mode and mass range of 50–2000 m/z.