Off-line Coupling of Isotachophoresis and High Performance Liquid Chromatography for Determination of Flavonoids in Plant Extract

Abstract

The use of isotachophoretic sample pretreatment coupled with high performance liquid chromatography for the analysis of some flavonoids occurring in plant extracts of Hypericum perforatum and Crataegus sp. is described. The samples were extracted with methanol by means of sonication in low temperature. The optimal leading electrolyte was used 10 mM Cl− as a leading ion in a buffer system at apparent pH*=7.2 (adjusted by TRIS) and terminating electrolyte was 50 mM boric acid at apparent pH*=8.2 (adjusted by barium hydroxide). The ITP electrolytes contained 20% (v/v) of methanol. To improve the sample pre-treatment, a pair of discrete ITP spacers defining the trapped constituents was used. Major components presented in the extracts were separated on a Discovery C18 and Discovery RP Amid C16 columns with a gradient mobile phase consisting of methanol, acetonitrile and diluted ortho-phosphoric acid. The quantification was performed by using external standards. The recoveries of the coupled ITP-HPLC analytical procedure were in the range of 91.2–95.6%.