Characterisation of extracts of Hypericum perforatum L. using an on-line HPLC system with UV/visible and fluorescence detection prior to and after photochemical conversion of the effluent

An HPLC system designed for the simultaneous determination of the four naphthodianthrone derivatives, protopseudohypericin, pseudohypericin, protohypericin and hypericin, in extracts of Hypericum perforatum L, has been developed. The on-line system permits quantification of the compounds by spectrophotometric detection at 590 nm as well as by fluorescence detection at 470/590 nm, Since protopseudohypericin and protohypericin are converted by exposure to light into pseudohypericin and hypericin, respectively, detection was performed before as well as after photochemical conversion in the on-line system. Verification of the peak identities was made using MS and diode-array detection. The purity of the hypericin reference material was determined by means of quantitative NMR using phenoxyacetic acid as internal standard. Several commercial extracts were characterised regarding the content of the individual naphthodianthrone derivatives using the on-line HPLC system, and the results showed the presence of similar relative amounts of naphthodianthrone derivatives in the investigated extracts. The largest total amount of hypericins in extracts of H. perforatum was obtained using detection at 590 nm after the effluent was illuminated with UV light at 254 nm for 1 min. The apparent content of hypericins was dependent on the mode of detection, and it is therefore of importance to state carefully the mode of detection in any analytical method in order to use the method for reproducible standardisation procedures. Copyright (C) 2000 John Wiley & Sons, Ltd.