Different chemometric approaches to optimize the assay of St. John's Wort active ingredients

In this paper, we describe the optimization procedure for the quantitative assay of naphthodianthrones in St. John's Wort dry extract by reversed phase liquid chromatography. Lately, a project for European Pharmacopoeia monograph including an HPLC assay for the active moieties in St. John's Wort dry extract was published with a view to provoking reactions from other researchers. We therefore decided to use different chemometric approaches to evaluate the influence of both analytical and preparative factors to demonstrate the robustness of the optimized method. An asymmetric screening design was built in order to evaluate the weight of each level for each factor–the sonication duration, the light exposure duration, the flow rate and the type of column–on the response: the total hypericin content. Considering the results so obtained, we were compelled to modify some parameters. Thus we built a screening design to apprehend the reliability of the new sample pre-treatment process, the interpretation and identification of active factors were performed according to various methods. We used a third chemometric approach: a sequential bifurcation to check out the method's robustness. In a second step, an eluent compatible with Mass Spectrometry detection was determined by a combined design. To cope with both separation and analysis time, desirability functions were used. Optimal conditions are finally given by ternary system at an optimized temperature (40 °C) and all the naphthodianthrones are separated in 10 min on conventional endcapped octadecyl silica gel column.