Use of an on-line, precolumn photochemical reactor in high-performance liquid chromatography of naphthodianthrones in Hypericum perforatum preparations

A method has been developed for the determination of naphthodianthrones in Hypericum perforatum L. extracts and phytopharmaceutical preparations by high-performance liquid chromatography combined with on-line, precolumn photochemical conversion followed by photodiode-array detection. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions. An on-line, precolumn photochemical reactor equipped with a knitted PTFE reaction coil around a visible light source was used in order to transform the light sensitive naphthodianthrones, protohypericin and protopseudohypericin, very easily into the non-protoforms, hypericin and pseudohypericin, respectively. Two UV chromatograms (photochemical reactor “on” and “off”) were compared and were quite useful in characterizing the sample. Validation studies demonstrated that this HPLC method is simple, rapid, reliable and reproducible. The time-consumptive manual irradiation of the samples is omitted by this automated on-line irradiation step. The developed method was successfully applied to the quality control of Hypericum perforatum L. extracts and its phytopharmaceutical preparations.