A rapid method for simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and ultra-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a rapid ultra-performance liquid chromatography (UPLC) method was developed for simultaneous determination of 15 Flavonoids, including hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2''-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed on Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (50 mm x 2.1mm I.D., 1.7 microm) and gradient elution of 50mM acetic acid aqueous solution and acetonitrile within 12 min. All calibration curves showed good linearity (R2>0.9997) within test ranges. The LOD and LOQ were lower than 0.13 and 0.52 ng on column, respectively. The R.S.D.s for intra- and inter-day of 15 analytes were less than 5.0% at three levels, and the recoveries were 95.0-103.7%. The validated method was successfully applied to quantitatively analyze 15 Flavonoids in different species of Epimedium. The results showed there were great variations among the contents of investigated Flavonoids. Hierarchical clustering analysis based on characteristics of 15 investigated compounds peaks in UPLC profiles showed that 37 samples were divided into 3 main clusters, which were in accordance with their Flavonoids contents. The simulative mean chromatogram of the high content cluster was generated to compare the samples from different species and/or locations of Epimedium. Four Flavonoids including epimedin A, B, C and icariin were selected as markers for quality control of the species of Epimedium used as Yinyanghuo.