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 in common St. John's wort (Hypericum perforatum L.). Presently, dietary supplements are generally standardized to contain specified levels of hypericin and hyperforin, and the related compounds, pseudohypericin and adhyperforin. A rapid method was developed for simultaneous determination of these 4 active constituents by liquid chromatography (LC). A 1 g portion of dried, finely ground leaf/flower sample is extracted with 20 mL methanol for 2 h. A 0.6 mL aliquot of the crude extract is combined with 5.4 mL acetonitrile–methanol (9 + 1) and passed through a mixed solid-phase cleanup column. The eluate is examined by LC for hyperforin, adhyperforin, hypericin, and pseudohypericin on a Hypersil reversed-phase column by using simultaneous ultraviolet (284 nm) and fluorescence detection (excitation, 470 nm; emission, 590 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%, respectively, for 3 sample mixtures containing concentrations ranging from approximately 0.2 to 1.5% combined hyperforins per gram dry weight. Average relative standard deviation (RSD) values for hyperforin and adhyperforin for all 3 mixtures were 18.9 and 18.0%, respectively. Average recoveries of hypericin and pseudohypericin were 88.6 and 93.3% respectively, from 3 sample 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.