After being sonicated for
60 min, the methanol solution was concentrated under reduced
pressure at a temperature below 40 C until its volume was reduced
to 2.0 ml. The extract was then subjected to separation by
a 3 100 mm Zorbax Eclipse column (Agilent Technologies) with
a particle size of 3.5 lm. The mobile phase consisted of formic
acid:de-ionised water (0.5:99.5) (solvent A) and methanol (solvent
B), with gradient elution started at 90:10 (A:B), maintained for
10 min. Its composition was changed to 75:25 over 60 min. The
flow rate was 0.4 ml/min. The total HPLC effluent was delivered
into a Z-SprayTM ES source of a Micromass Q-TOF 2TM hybrid
quadrupole/time of flight mass spectrometer (Micromass,
Manchester, UK), which was operated in positive ionisation mode