Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's Wort preparations

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's Wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol-water (80:20, v/v) containing 5% HP-beta -cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 degreesC, were injected into a C-18 column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10. v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable. and reproducible. The standard curves were linear over the concentration ranges, 0.5-2.5 mug/ml (hypericin), 0.35-1.6 mug/ml (pseudo hype ricin) and 5-50 mug/ml (hyperforin), The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were less than or equal to 4.4% less than or equal to 5.4%, and less than or equal to 2.8%, respectively; inter-day CVs were less than or equal to 5.8%, less than or equal to 4.9%, and less than or equal to 2.5%, respectively. This method may be applied for the routine standardization of St. John's Wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal. (C) 2001 Elsevier Science B.V. All rights reserved.