Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G 4, G5 and G6 according to their R-F values in normal-phase thin-layer chrom atography, is reported. The pure flavones were purified after maceration wi th methanol by normal-phase solid-phase extraction, normal-phase medium-pre ssure Liquid chromatography, normal-phase preparative thin-layer chromatogr aphy and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the subs tances G was accomplished by UV detection at 213-216 nm, diode array UV det ection, or fluorescence detection (lambda(cx)=330 nm; lambda(em)=440 nm). T he molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrom etry (EI-HRMS). Substance G4 was identified as 3',4',5'-trimethoxyflavone. The substances G1-G6 were shown to be mono-, di- tri- and pentamethoxyflavo nes. HPLC-electrospray ionization tandem mass spectrometry (ESI-MS-MS) of t he flavones was carried out employing a 150X2 mm I.D. column packed with a 3 mu m/100 Angstrom octadecylsilica stationary phase and a mobile phase com prising 1.0% acetic acid in water-acetonitrile (50:50). Comparative RP-HPLC -ESI-MS of the raw methanol extract and the isolated substances G1-G6 prove d that the isolated compounds were pure and were not artifacts. Finally, RP -HPLC-ESI-MS-MS was used to identify substances G1-G6 in phytopharmaceutica l drugs. (C) 2000 Elsevier Science B.V. All rights reserved.