2.5.1. HPLC determination conditions for isoflavones
The HPLC conditions were adopted to the method developed by
Rostagno, Villares, Guillamón, García-Lafuente, and Martinez(2009), with some modification in the chromatographic conditions
to achieve good isoflavone separation. Separations were performed
in an ODS C18 column (4.6 mm 150 mm, 3.9 lm) fitted with a
guard column. The mobile phase was composed of trifluoroacetic
acid (TFA) 0.1% in water (A) and TFA 0.1% in acetonitrile (B). The
elution was performed with a linear gradient from 100% to 58%
(v/v) of eluent A in 24 min, holding these conditions for 10 min
and returning to 100% in 10 min. The chromatographic analysis
was performed at 35 C, the flow rate was 1.0 mL/min and detection
wavelength was 254 nm. Injections of 5lL were effected with
the automatic injector. Twelve standards of soy isoflavones were
dissolved in absolute HPLC-grade methanol and the concentration
of each standard was from 15 to 50 lg/mL.