Column switching high-performance liquid chromatography with two channels electrochemical detection for high-sensitive determination of isoflavones.

Column switching HPLC with electrochemical detection (HPLC-ED), which consists of one pre-column and two electrochemical detectors subsequent to each analytical column, called HPLC-2ED, has been developed for determining isoflavones (daidzin, genistin, daidzein, and genistein) with high sensitivity. In the present HPLC-2ED, the eluted daidzin and genistin from the pre-column were separated on an analytical column using a methanol-water-phosphoric acid mixture (30:70:0.5) as the mobile phase (MP), and daidzein and genistein were separated on another analytical column using a methanol-water-phosphoric acid mixture (50:50:0.5). The way of the elute flow from the pre-column was changed by rotating the switching valve at 17 min. The difference in retention times of genistein between isocratic HPLC-ED and HPLC-2ED was 52.2 min. The detection limit (S/N=3) per column injection (5 microL) of genistein was 0.5 pg. The sensitivity by the present method is superior to that of previously reported gradient HPLC-ED for the determination of isoflavones.