Exploring the Effects of Cyclosporin A to Isocyclosporin A Rearrangement on Ion Mobility Separation

Cyclosporin A (CycA) is a peptide secondary metabolite derived from fungi that plays a crucial role in transplantation surgery. Cyclic traveling wave ion mobility mass spectrometry (IM-MS) revealed an N → O peptidyl shift in singly protonated CycA to isocyclosporin A (isoA), whereas no such isomerization was observed for doubly protonated and sodiated molecules. CycA and isoA were able to be separated by considering doubly protonated precursors using a specific ion fragment. In parallel, sodium ion stabilization facilitated the simultaneous separation and quantitation of singly charged cyclosporin isomers with the limit of detection and coefficient of determination of 1.3% and 0.9908 for CycA in isoA and 1.0% and 0.9830 for isoA in CycA, respectively. Finally, 1H–13C gHSQC NMR experiments permitted parallel recording of up to 11 cyclosporin conformers. The ratios were determined by integrating the volume of cross-peaks of the upfield resonating hydrogen in the diastereotopic methylene group of sarcosine-3.