Simultaneous determination of the predominant hyperforins and hypericins in St. John's Wort (Hypericum perforatum L.) by liquid chromatography

Hypericin and hyperforin are believed to be among the active constituents i n common St. John's wort (Hypericum perforatum L.). Presently, dietary supp lements are generally standardized to contain specified levels of hypericin and hyperforin, and the related compounds, pseudohypericin and adhyperfori n. A rapid method was developed for simultaneous determination of these 4 a ctive constituents by liquid chromatography (LC). A 1 g portion of dried, f inely ground leaf/flower sample is extracted with 20 mL methanol for 2 h, A 0.6 mt aliquot of the crude extract is combined with 5.4 mL acetonitrile-m ethanol (9 + 1) and passed through a mixed solid-phase cleanup column. The eluate is examined by LC for hyperforin, adhyperforin, hypericin, and pseud ohypericin on a Hypersil reversed-phase column by using simultaneous ultrav iolet (284 nm) and fluorescence detection (excitation, 470 nm; emission, 59 0 nm), The compounds are easily separated isocratically within 8 min with a mobile phase of acetonitrile-aqueous 0.1M triethylammonium acetate (8 + 2) . Average recoveries of hyperforin and adhyperforin were 101.9 and 98.4%, r espectively, for 3 sample mixtures containing concentrations ranging from a pproximately 0.2 to 1.5% combined hyperforins per gram dry weight. Average relative standard deviation (RSD) values for hyperforin and adhyperforin fo r all 3 mixtures were 18.9 and 18.0%, respectively, Average recoveries of h ypericin and pseudohypericin were 88.6 and 93.3% respectively, from 3 sampl e mixtures containing concentrations ranging from approximately 0.2 to 0.4% combined hypericins per gram dry weight, Average RSD values for hypericin and pseudohypericin for all 3 mixtures were 3.8 and 4.2%, respectively.