RESEARCH ARTICLE Signals of diagnostic ions in the product ion spectra of [M - H] - ions of methoxylated flavonoids Rafał Frański 1 | Błażej Gierczyk 1 | Tomasz Kozik 1 | Łukasz Popenda 2 | Monika Beszterda 3 1 Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89B, 61‐614 Poznań, Poland 2 NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61‐614 Poznań, Poland 3 Department of Food Biochemistry and Analysis, Poznan University of Life Sciences, Mazowiecka 48, 60‐623 Poznań, Poland Correspondence R. Frański, Adam Mickiewicz University, Faculty of Chemistry, Umultowska 89B, 61‐614 Poznań, Poland. Email: franski@amu.edu.pl Rationale: The main feature of the fragmentation of [M - H] - ions of methoxylated flavonoids is the loss of methyl radical (formation of the [M - H - CH 3 ] -• product ion). Subsequent decomposition of [M - H - CH 3 ] -• product ions may be useful for identification of a given compound by HPLC/MS. This paper describes how the selected diagnostic fragment ions can be useful during HPLC/MS(-) analysis of methoxylated flavonoids. Methods: Product ion spectra (ESI‐CID‐MS/MS spectra) of [M - H] - ions of 17 methoxylated flavonoids (flavones, isoflavones and flavonols) were obtained with a Q‐TOF mass spectrometer. Full scan mass spectra (ESI‐MS) were obtained with a single quadrupole type of instrument. Results: A number of product ions were recognized as useful from the point of view of structural elucidation. In most cases they were diagnostic product ions, formed as a result of C ring breaking. Conclusions: The most important conclusions drawn from this study are: the product ion at m/z 132 indicates that the analysed compound is an isoflavone; the product ion at m/z 117 indicates the presence of one hydroxy group at ring B or at the 3‐position; biochanin A and prunetin can be differentiated by their ‘in‐source’ fragmentation, by the relative abundances of product ions at m/z 195, 183 and 167; loss of mass 102 from the [M - H - CH 3 ] -• ion indicates that ring B is not substituted and there is no hydroxy group at the 3‐position; and rhamnetin can be detected using three diagnostic product ions, namely at m/z 121, 165 and 193. 1 | INTRODUCTION The main feature of the fragmentation of [M - H] - ions of methoxylated flavonoids (flavone, flavonol or isoflavone; Scheme 1), observed under collision‐induced dissociation (CID) conditions, is the loss of the methyl radical, thus the formation of the [M - H - CH 3 ] -• product ion. This is an example of violation of the ‘even‐electron rule’ (loss of radical from a deprotonated/protonated molecule 1-7 ) which has been very useful for identification of methoxylated flavonoids. Subsequent decomposition of the [M - H - CH 3 ] -• ion may be quite complicated, as discussed in detail, for example, for biochanin A and formononetin. 8 The mass of a deprotonated one‐methyl‐group‐containing flavone, flavonol or isoflavone can be described as 267 + (16) n (267 is the mass of the deprotonated flavone/flavonol/isoflavone containing one, methoxy group and one hydroxy group, e.g. formononetin). Therefore, the detection of mass 267 + (16) n accompanied by the loss of mass 15 (of course in the negative ion mode) is a very strong suggestion that we are dealing with a one‐methyl‐group‐containing flavone or flavonol or isoflavone. Of course, in order to unequivocally identify a given compound by high‐performance liquid hromatography/mass spectrometry (HPLC/MS), it is necessary to compare the retention time and fragmentation pattern with those of a respective standard. Methoxylated flavones/flavonols/isoflavones are relatively numerous; therefore, for their identification by HPLC/MS, it would be useful if it was possible to limit the number of possible compounds. As mentioned further, the published data concerning the ions assigned to the product ions of [M - H] - ions of methoxylated flavonoids are sometimes disputable. Received: 17 September 2018 Revised: 11 October 2018 Accepted: 16 October 2018 DOI: 10.1002/rcm.8316 Rapid Commun Mass Spectrom. 2019;33:125–132. © 2018 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm 125