Abstract: Icariin and epimedins A, B, C are a series of active flavonol glycoside in Epimedium brevicornum Maxim. A pilot-scale preparative high performance liquid chromatographic (HPLC) method was developed to purify the four flavonol glycosides as reference standards from the crude extract of Epimedium brevicornum Maxim. After the crude extract containing ~20% flavonols was enriched using macropore resin, the obtained target fractions were subjected to pilot-scale preparative HPLC purification. With the aid of a self-packed pilot-scale preparative column (220 mm×77 mm, 10 μm), the 4 target compounds were separated well within 35 min in a single chromatographic run by the elution with acetonitrile-water (26:74 or 30:70, v/v). By repetitive injection of the enriched target fraction onto the preparative column, 33 g icariin as well as 4.6 g epimedin C, 3.7 g epimedin B, and 0.6 g epimedin A were obtained from 300 g crude extract. The purities of all products were greater than 98%. This pilot-scale preparative HPLC technique and the two step separation technology for 4 target compounds are quite useful for the production of the reference standard series with good purity like icariin, epimedins A, B, C standards due to its high performance, rapid separation and more amounts of products obtained.

Key words: epimedins A, B, C, flavonol glycosides, high performance liquid chromatography （HPLC）, reference standard series, Epimedium brevicornum Maxim, icariin, pilot-scale preparation