Determination of hyperforin, hypericin, and pseudohypericin in human plasma using high-performance liquid chromatography analysis with fluorescence and ultraviolet detection

Hyperforin, hypericin and pseudohypericin are the main ingredients of St. John’s wort extract, which is available over the counter for treatment of mild to moderate depression. To facilitate clinical studies we developed two sensitive HPLC methods for determination of hypericin/pseudohypericin and hyperforin, respectively, in human plasma samples. The achieved limits of quantitation of 0.25 ng/ml for hypericin and pseudohypericin and 10 ng/ml for hyperforin were low enough to allow determination of pharmacokinetic parameters of the substances. Following liquid–liquid extraction of human plasma the samples were separated by isocratic reversed-phase HLPC and analyzed using fluorimetric detection for hypericin/pseudohypericin and UV detection for hyperforin.