Flavonoids are a class of polyphenolic secondary metabolites found in plants. Due to their ubiquity in our daily dietary intake and their major anti-oxidative, anti-inflammatory and anti-mutagenic activities, they have been a major focus of wide-ranging research for the past two decades. Mass spectrometry combined with liquid chromatography is one of the most popular techniques for the analysis of flavonoids. In this study, high-resolution accurate mass electrospray tandem mass spectrometry was used to study 30 flavonoids in both positive and negative ionisation modes. From the data obtained, common losses were summarised and compiled. Dominating neutral losses were tabulated. The radical loss of CH₃· was observed in flavonoids containing methoxy groups and three key diagnostic product ions were identified. These were m/z 153 (indicative of two OH groups on ring A) m/z 167 (indicative of one OH and one methoxy group on ring A) and m/z 151 (a flavanol, with no ketone oxygen but two OH groups on ring A). These will be useful in structural elucidation of unknown flavonoids and flavonoid metabolites. Energy breakdown graphs were utilised to distinguish between three pairs of structural isomers, and to help rationalise proposed fragmentation pathways. Lastly, a competition of loss of CH₃· and methane was reported for rhamnetin and isorhamnetin in the negative ion mode for the first time. Proposed fragmentation pathways were given to rationalise the differences in peak intensities for this competitive process.

Flavonoids are a class of polyphenolic secondary metabolites found in plants. Due to their ubiquity in our daily dietary intake and their major anti-oxidative, anti-inflammatory and anti-mutagenic activities, they have been a major focus of wide-ranging research for the past two decades. Mass spectrometry combined with liquid chromatography is one of the most popular techniques for the analysis of flavonoids. In this study, high-resolution accurate mass electrospray tandem mass spectrometry was used to study 30 flavonoids in both positive and negative ionisation modes. From the data obtained, common losses are summarised and compiled. Dominating neutral losses are tabulated. The radical loss of CH3. was observed in flavonoids containing methoxy groups and three diagnostic product ions were identified which could be useful in structural elucidation of unknown flavonoids and flavonoid metabolites. Energy breakdown graphs were utilised to dis-tinguish between three pairs of structural isomers, and to help rationalise proposed fragmentation pathways. Lastly, a competition of loss of CH3. and methane were reported for rhamnetin and isorhamnetin in the negative ion mode for the first time. Proposed fragmentation pathways were given to rationalise the differences in peak intensities for this competitive process.

The computational rationalisation of the different MS/MS fragmentation of the isomeric amino acids, l-leucine and l-isoleucine is presented.

Salinomycin is a polyether ionophore natural product widely studied for its anticancer stem cell properties and well established anti-coccidial activity. However, its complex structure and tendency to isomerise in solution complicates its mass spectrometric analysis. In this study, a combination of high-resolution accurate mass electrospray sequential mass spectrometry, ion mobility spectrometry and computational modelling was employed to analyse salinomycin and its isomers for the first time. Product ions generated from salinomycin and its isomer in the MSⁿ analysis are distinguished, and detailed fragmentation mechanisms are proposed. The novel application of ion mobility mass spectrometry to separate isomeric salinomycins provides revolutionary insight into the chelation positions of sodium by salinomycin (‘ionoforms’). The cation position has a fundamental effect on the fragmentation routes observed. These observations were supported by Gaussian modelling and collision cross-section calculations. The relationship between collision energy and peak intensity of all identifiable forms of salinomycin and respective product ions was visualised by a 3D energy breakdown graph. Results from this study provided firm grounding for understanding complex structures such as salinomycin. The methodology demonstrated here could be applied to a wide range of natural products and in other drug development or metabolomic studies.

Natural products such as polyketides are a fertile target for drug discovery. Methodologies relating to discovery, metabolism, synthesis and biosynthesis of polyketides have evolved considerably since they were first studied in the early 20th century. The antibiotic erythromycin, produced by the Streptomyces erythreus bacteria, was the first of the macrolide natural products to be discovered in 1952. The biosynthesis of erythromycin is catalysed by a large multifunctional enzyme, which constructs the polyketide intermediate that is acted upon by tailoring enzymes to produce the final construct. It is during this process that molecular diversity is generated, and commercial samples of erythromycin tend to be mixtures of co-metabolites. To fully identify these compounds, a full fragmentation scheme of the main component (erythromycin A) is required, which is absent from the literature. In this study, accurate-mass sequential mass spectrometry is used to propose a fragmentation scheme which is then used to assign structures to eight co-metabolites including the identification of a previously unpublished form of erythromycin. This clearly demonstrates the successful application of the methodology.