Simultaneous determination of hypericin and hyperforin in human plasma and serum using liquid–liquid extraction, high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry

A method for the simultaneous extraction of hypericin and hyperforin from a St. John’s Wort extract, which is used in case of moderate depressions and skin injuries, from human plasma and serum by liquid–liquid extraction (LLE) with n-hexane–ethylacetate (70:30, w/w) was developed. A reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV, fluorescence (FLD) and mass spectrometric (MS) detection using electrospray ionization (ESI) was used to identify and quantify hypericin and hyperforin in the extracts from blood samples. Linearity was obtained in the ranges 8.4–28.7 ng/ml (hypericin) and 21.6–242.6 ng/ml (hyperforin). Recoveries were between 32.2 and 35.6% for hypericin and 100.1 and 89.9% for hyperforin. Intra-day accuracy and precision for this method ranged between 3.2 and 4.3% and 2.6 and 2.8%, respectively. After validation of the LLE, the method was tested on real plasma samples which were obtained by ingestion of St. John’s Wort extract capsules. Blood samples were taken 2, 4, and 6 h after ingestion. Finally, this method proved to be highly suitable for clinical and pharmacologically relevant studies.