Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
J Chromatogr A. 2010 Apr 23;1217(17):2986-9. doi: 10.1016/j.chroma.2010.02.050. Epub 2010 Mar 1.
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.
柱切换高效液相色谱电化学检测法（HPLC-ED），由一个前置柱和两个电化学检测器组成，分别位于分析柱之后，称为 HPLC-2ED，已被开发用于高灵敏度测定异黄酮（大豆苷、大豆苷元、染料木素和染料木苷）。在本 HPLC-2ED 中，从前置柱洗脱的大豆苷和大豆苷元在分析柱上用甲醇-水-磷酸混合物（30:70:0.5）作为流动相（MP）分离，而大豆黄素和染料木素则在另一个分析柱上用甲醇-水-磷酸混合物（50:50:0.5）分离。在 17 分钟时通过旋转切换阀改变洗脱液从前置柱的流出方式。等度 HPLC-ED 和 HPLC-2ED 之间染料木素保留时间的差异为 52.2 分钟。每个柱注入（5 μL）的染料木素的检测限（S/N=3）为 0.5 pg。与先前报道的用于测定异黄酮的梯度 HPLC-ED 相比，本方法的灵敏度更高。
