Background: Polymyxin E (PME), a complex of cationic cyclic lipodecapeptides, is used to treat multidrug-resistant gram-neg. bacterial infections. Besides the main components PME1 and PME2, polymyxin containing unsaturated fatty acyl (FA) group with lower contents can hardly determine the structure without chromatog. preparations and NMR. Introduction: The peptide sequences of PME components have been carried out based on highperformance liquid chromatog.-quadrupole / time-of-flight mass spectrometry (HPLCQ/ TOF-MS). However, the components with double bonds on the FA, such as 2′, 3′-dehydro PME1, were difficult to be determined or easily misjudged by MS/MS. The transformation of such unsaturated components to be epoxidized or di-hydroxylated components can promote the acquisition of more fragment ions in the MS/MS to assist in judging the position of double bonds on FA. Methods: In this paper, the PME mixtures were dissolved in an equal proportion of 20% ACN aqueous solution and 2-acetylpyridine. The above PME solution was transferred to a quartz cuvette and irradiated with the UV lamp at 254 nm for 8h. The dehydro PME components were converted to epoxy PMEs and dihydroxy PMEs. A fragmentation pathway of epoxidized or di-hydroxylated components based on Q/TOF-MS/MS was proposed for the first time. Results: According to the characteristic ions of epoxidized components and di-hydroxylated components, 2′, 3′-epoxy PME1/E2 and 2′, 3′-dihydroxy PME1/E2 were confirmed. It can be inferred that the double bond is located at the 2′, 3′-position of FA. Conclusion: The structure of unsaturated PME components with double bonds on the FA is elucidated by HPLC-Q/TOF-MS combined with photochem. reaction. This strategy applies to other lipopeptides containing unsaturated FA chains.