Isolation, purity analysis and stability of hyperforin as a standard material from Hypericum perforatum L.

In 1996 131.5 million daily doses of preparations containing extracts of Hy pericum perforatum L. were prescribed in Germany for treating mild to moder ately severe depressive disorders. New pharmacological and clinical results focus on hyperform as the main active ingredient of the drug. Hyperforin (C35H52O4) is one of the main components (2-4%) of the dried her b Hypericum perforatum L. It was isolated after six consecutive steps: extr action of deep-frozen blossoms (-20 degrees C) with n-hexane by means of an Ultra Turrax at room temperature; separation of lipophilic substances on a silica gel column; purification of the relevant fraction by preparative HP LC; evaporation of the mobile phase under reduced pressure; removal of the remaining water by freeze-drying; and storage of hyperforin at -20 degrees C under nitrogen. The identity and purity of the isolated substance were de termined by highperformance thin-layer chromatography (HPTLC), high-perform ance liquid chromatography (HPLC) with diode-array and ultraviolet detectio n (DAD and UV), Fourier-transformed infrared (FTIR) and proton nuclear magn etic resonance (H-1 NMR) spectroscopy, and liquid chromatography coupled wi th positive-ion electrospray-ionization tandem mass spectrometry (LC-ESI(+) -MS-MS). By use of these methods the purity of hyperforin was shown to be > 99.9%. Peroxides present at each step of the isolation were detected by tit ration and by means of Merckoquant analytical peroxide test-strips. Elimina tion of the peroxides and stabilization of hyperforin was achieved by consi stent protection from oxidation-the mobile phases were protected by use of ascorbic acid; evaporation and freeze-drying were performed under nitrogen; and the mobile phase used for preparative HPLC was sparged with helium. St ability testing was performed by HPLC-the samples were stored at -30 degree s C in a normal atmosphere and at -20, 4, and 20 degrees C in a normal atmo sphere or under nitrogen. Results were compared with those obtained after s torage under liquid nitrogen (-196 degrees C). Because of its high sensitivity to oxidation, hyperforin was more stable un der nitrogen under all test conditions. There was no statistically signific ant difference between results obtained after 8 months at -20 degrees C und er nitrogen or at -30 degrees C under a normal atmosphere and those from th e reference sample stored under liquid nitrogen (-196 degrees C), Despite t his, because of the tendency of hyperforin to degrade, long-term storage at -70 degrees C under nitrogen is recommended.