Characterization of monohydroxylated derivatives of the anticancer agent flavone-8-acetic acid by liquid chromatography with on-line UV and mass spectrometry.

The experimental anticancer agent flavone-8-acetic acid (FAA) is metabolized into several monohydroxylated derivatives using mouse microsomes. Because these metabolites could be involved in the biological effects of FAA, the aim of this study was to characterize all its possible monohydroxylated derivatives. To do so, we have developed a methodology using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) to analyze and identify FAA derivatives hydroxylated at the 2', 3', 4', 3, 5, 6, or 7 position. In RP-HPLC, 4'-, 3'-, 2'-, 6-, and 7-OH-FAA eluted before FAA, whereas 3- and 5-OH-FAA eluted after FAA. UV spectra showed a bathochromic shift of band I for all derivatives and of band II for 5- and 6-OH-FAA. In addition, the position of the OH group could be determined by the presence of certain product ions in MS. Ions at m/z 133 and 151 were specific for 2'-, 3'-, 4'-, and 3-OH-FAA, whereas the ion at m/z 177 was specific for 3-OH-FAA only. The ions m/z 133, 151 and 167 were specific for 2'-OH-FAA. Ions at m/z 149 were specific for the presence of the OH group on cycle A only (i.e., 5-, 6- or 7-OH-FAA). The presence of both product ions m/z 149 and 179 were specific for 7-OH-FAA. Finally, ions at m/z 149 and several product ions of even m/z values were specific for 5-OH-FAA. In conclusion, the methodology described can be used to identify all possible monohydroxylated FAA derivatives.