An HPLC system, consisting of Surveyor Autosampler, Surveyor LC pump, Surveyor Photo Diode Array (PDA) detector (Thermo Finnigan, Waltham, MA, USA), and a reversed-phase column (ZORBAX 300SB-C18, 2.1 × 150 mm, 3.5 μm; Agilent, Santa Clara, CA, USA) was used to separate the metabolites. 10 μL of samples were injected for the analysis throughout. The gradient profile was 8 % B for 2 min, increased to 20 % B in 38 min, then to 100 % B in 12 min and maintained for 10 min, and decreased to 8 % B in 2 min and maintained for 10 min (A = 0.1 % formic acid in water, B = 100 % acetonitrile). The flow rate was 0.15 mL/min. The acquisition time was 55 min and delay time was 5 min per spectrum. The separation was monitored at 325 nm.
An ion trap mass spectrometer (LCQ DECA XP MAX) coupled with an ESI source (Thermo Finnigan) was used to identify the metabolites. The MS parameters were the following: sheath gas (nitrogen) flow rate, 40 arb; aux/sweep gas (nitrogen) flow rate, 10 arb; spray voltage, 4.5 kV; capillary temperature, 320 °C. Collision energy and other tune parameters were optimized for dissociation of parent ions into product ions for each metabolite. The mass spectrometer was acquired in data-dependent MS/MS mode: each full MS scan (in the range 100–220 m/z) was followed by three MS/MS of selected ions.
An ion trap mass spectrometer (LCQ DECA XP MAX) coupled with an ESI source (Thermo Finnigan) was used to identify the metabolites. The MS parameters were the following: sheath gas (nitrogen) flow rate, 40 arb; aux/sweep gas (nitrogen) flow rate, 10 arb; spray voltage, 4.5 kV; capillary temperature, 320 °C. Collision energy and other tune parameters were optimized for dissociation of parent ions into product ions for each metabolite. The mass spectrometer was acquired in data-dependent MS/MS mode: each full MS scan (in the range 100–220 m/z) was followed by three MS/MS of selected ions.