3.4. HPLC Condition

Chromatographic determination was performed on VWR Hitachi Chromaster 600 chromatograph (Merck) with a pump (5160), a degasser, a thermostat (5310), an autosampler (5260), a DAD detector (5430) and EZChrom Elite software. The extracts were analyzed on C18 reversed-phase column LiChrospher 100 (Merck) (25 cm × 4.0 mm i.d., 5 μm particle size), at a temperature of 30 °C. A 20 µL portion of the sample was injected. A mixture of acetonitrile (A) and water (B) with 0.025% of trifluoroacetic acid was used as a mobile phase. The gradient program was as follows: 0–10 min 20% A, 80% B; 10–30 min 25% A, 75% B; 30–60 min 35% A, 65% B. The flow rate of the eluent was 1.5 mL·min⁻¹. The data were collected in a wavelength range from 200 to 400 nm. The chromatographic fractions eluted at a retention time characteristic for the investigated isoflavones were collected using a Foxy R1 fraction collector (Teledyne Isco, Lincoln, NE, USA) and analyzed by direct-injection mass spectrometry with electrospray ionization (micrOTOF-Q II, Bruker Daltonics, Bremen, Germany) using Compass DataAnalysis software Version 4.1.