Differentiation of isomeric flavone/isoflavone aglycones by MS2 ion trap mass spectrometry and a double neutral loss of CO

The fragmentation behaviour of seven pairs of isomeric flavone/isoflavone aglycones (solely hydroxylated and/or methoxylated) was studied using ion trap mass spectrometry with atmospheric pressure ionisation (API, both electrospray and APCI) in the positive and negative ion modes. A major difference was found in the neutral loss of 56 u, which was a common feature of all isoflavones in API(+). It was identified as a double loss of CO by accurate mass tandem mass spectrometric (MS/MS) measurements using a hybrid quadrupole time-of-flight (Q-TOF) instrument. Fragmentation of daidzein with (13)C-isotope labelling of the carbon C2 showed that this double loss occurred from the central ring of the molecule. A mechanism for this selective fragmentation is given. Further isoflavone-specific fragmentations were used to develop a guideline for the identification of isoflavone structures. A software-based neutral loss scan of 56 u in the API(+)-MS(2) mode was applied to extracts of leaves of Lupinus albus and to soy flour. The structure elucidation guideline allowed identification of hydroxy and/or methoxy isoflavones. Structures could be confirmed for those available as reference compounds.