The HPLC analyses were performed using a Jasco series liquid
chromatograph, according to the method described by Lopes da
Silva et al. (2007), with a slight modification. Separation was
achieved using a Sun Fire C18250 mm 4.6 mm column (Waters)
at a temperature of 35 8C. The solvents, (A) 0.1% trifluoroacetic acid
in water and (B) HPLC-grade acetonitrile, were used, establishing
the following gradient using a flow rate of 1 mL/min: 0–40% B for
24 min, isocratic 40% B for 6 min, 40–0% B for 5 min and isocratic
0% B for 5 min.