Optimized and Validated HPLC Analysis of St. John's Wort Extract and Final Products by Simultaneous Determination of Major Ingredients

Aim of this work was to develop a validated high performance liquid chromatography method for the analysis of extracts and final products of St. John's wort, according to international guidelines for bioanalytical method validation. Chromatographic separation was performed on a C18 column with a combination of gradient and isocratic steps; the mobile phase composed of ammonium acetate solution (pH 4.5; 10 mM), acetonitrile and methanol. Quantification and method validation was performed using extract spiked with external reference standards of chlorogenic acid, rutin, hyperoside, isoquercitrin, quercetin and hypericin. Validation study revealed that trans-chlorogenic acid is partially transformed into its cis-isomer during analysis. The method showed good linearity, precision and accuracy. Hyperforin was completely unstable. All other ingredients were stable at -18°C and after three freeze-thaw cycles, while stability of most ingredients was limited at room temperature and 4 - 8°C; quercetin was the most unstable one. The major ingredients of methanolic extracts, infusions and final products of Hypericum perforatum were completely resolved and quantified. Beyond its potential usefulness in the analysis of St. John's wort products, this study addresses the issue of validation from the perspective of the field of bioanalysis and reveals the wealth of critical information which can be derived.