Analytical and Bioanalytical Chemistry

, Volume 385, Issue 4, pp 716–723 | Cite as

Optimization of the assay of naphthodianthrones in dry St John’s wort extract by reversed-phase liquid chromatography

  • Guilhem Pages
  • Michael Mazarin
  • Michelle Sergent
  • Roger Phan-Tan-Luu
  • Corinne DelaurentEmail author
Original Paper


An optimization procedure for the reversed-phase liquid chromatography (RPLC) assay of naphthodianthrones in St John’s wort extract from the European Pharmacopoeia project is described. The results obtained from two screening designs showed that light exposure, recommended in the monograph as sample pretreatment, does not permit one to obtain a reproducible quantification of the main ingredients. Improvement of the method robustness implies the need to overcome the problem of light exposure, to subsequently quantify protonaphthodianthrones and to perform the separation on octadecyl (ODS)-bonded phase at optimized flow rate. The method robustness was checked by using a bifurcation sequential approach investigating the influence of 13 factors. The eluent recommended in the monograph is a ternary mixture of methanol, phosphate buffer and ethyl acetate. For the sake of simplicity, the phosphate buffer was substituted by an acetate buffer. The best composition of the ternary mixture was determined by a combined design including three mixture variables and the temperature as an independent variable. Chromatographic parameters were modelled in terms of analysis time, resolution and asymmetry. Desirability functions permit one to cope with these parameters and to determine the best compromise. The naphthodianthrones were separated on a conventional endcapped octadecyl silica gel column eluted by a ternary mobile phase at 40 °C in 10 min.


HPLC St John’s wort Hypericum perforatum L. Naphthodianthrones Chemometric methodology 



This work was supported by the French Conseil Régional Provence-Alpes-Côte-d’Azur. The authors are also grateful for financial support received from the Pharmaceutical Laboratory DISTRI B3 (Marseille, France). We thank Patrick Fournier for his revision of the English version of this paper.

Supplementary material


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Guilhem Pages
    • 1
  • Michael Mazarin
    • 1
  • Michelle Sergent
    • 2
  • Roger Phan-Tan-Luu
    • 2
  • Corinne Delaurent
    • 1
    Email author
  1. 1.JE 2421 TRACESUniversité Paul CézanneMarseilleFrance
  2. 2.LMRE, Case D52Université Paul CézanneMarseilleFrance

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