Characterization of flavonoid subgroups and hydroxy substitution by HPLC-MS/MS.

HPLC-DAD coupled with mass spectrometry in the positive ionization mode was applied to study the fragmentation of twelve selected flavonoids. Compounds belonging to all the major subgroups found in common plants, i.e. flavonols, flavones, dihydroflavonols, flavanones and flavanols were studied. Compound standards were injected into the spectrometer and produced characteristic mass spectra. The fragmentation of each compound was studied and it was shown that the dehydration and carbon monoxide losses from the [M+H]+ ion by the members of each subgroup produced specific fragments, thus allowing the characterization of the flavonoid subgroups. Moreover, fragments resulting from fission of the C-rings are specific of each subgroup and revealed the substitution pattern of A- and B-rings. In order to verify the identifying efficiency of the positive ionization mode through these characteristic fragmentations, the unknown flavonoids of an Origanum vulgare diethyl ether extract were separated with the HPLC system and the major peaks were successfully identified with the mass spectrometer.