Determination of hypericin and pseudohypericin in pharmaceutical preparations by liquid chromatography with fluorescence detection.

The quantification of the naphthodianthrones, hypericin and pseudohypericin, to which most St. John's wort pharmaceutical preparations are standardized, can be accomplished with a high degree of specificity and sensitivity with LC-fluorescence. Extraction into ethanol is rapid and recovers more than 95% of both pseudohypericin and hypericin following 1 h of sonication. This LC-fluorescence method can separate and detect both pseudohypericin and hypericin in the presence of other constituents in Hypericum perforatum with a greater degree of specificity than LC-UV at either 236 nm or 592 nm and may even be more specific than LC-MS if only the molecular ion is monitored. LC-fluorescence is also more sensitive than LC-UV at either 236 nm or 592 nm. The LC-fluorescence method can quantify on-column amounts of hypericin and pseudohypericin as low as 0.18 ng with less than 5% deviation from known and method replication error (reproducibility) less than 5.5%. Hypericin and pseudohypericin can be accurately and reproducibly quantified from capsules, tablets and tinctures of St. John's wort.