Separation of hypericins and hyperforins in extracts of Hypericum perforatum L. using non-aqueous capillary electrophoresis with reversed electro-osmotic flow.

The separation of the lipophilic compounds in extracts of Hypericum perforatum L. is demonstrated in a non-aqueous capillary electrophoresis system with reversed electro-osmotic flow. Solvent mixtures of methanol, dimethylsulfoxide and N-methylformamide were used for the electrophoresis media, with addition of ammonium acetate and sodium acetate as electrolytes. The flow was reversed by the addition of the polycation hexadimethrine bromide, and thus negative voltage was applied. The method shows baseline separation between the four hypericins-protopseudohypericin, pseudohypericin, protohypericin and hypericin-whereas total baseline separation between the two hyperforins-hyperforin and adhyperforin-was not achieved. Using a fused-silica capillary (30 cm x 25 microm ID) and a voltage of -25 kV the analysis time of the hypericins and hyperforins was obtainable within 3 min. Application of the method with a fused-silica capillary of a larger internal diameter (48.5 cm x 50 microm ID) and a voltage of -20 kV resulted in analysis times of 8 min, but also lower limits of detection. The maximal attainable voltage was applied in both cases. Simultaneous separation of the flavonoids-although less efficient-may also be achieved. The technique of non-aqueous capillary electrophoresis with reversed electro-osmotic flow provides a very fast technique to evaluate the composition of hypericins and hyperforins in extracts of Hypericum perforatum L.