High-Resolution Electrospray and Ion Mobility Sequential Mass Spectrometry for Structural Characterisation of Anticancer Stem Cell Agent Salinomycin and Its Isomers

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.