Temperature fluctuations occurring during the cold chain logistics of salmon contribute to lipid oxidation. This study aimed to simulate cold chain interruption through freeze-thaw operations and evaluate the lipidomics data from salmon samples subjected to different numbers of freeze-thaw cycles by using rapid evaporative ionization mass spectrometry (REIMS) combined with an intelligent surgical knife (iKnife). The results indicated significant differences in the relative abundance of characteristic ions among the samples (p < 0.05). A total of 34 ions with variable importance for the projection values ≥1 were identified as potential biomarkers, including m/z 719.4233 ([PC_C36:5-NH(CH₃)₃]^-), m/z 337.3134 ([FA_C22:1]^-), m/z 720.4666 ([PE_C35:6-H]^-), m/z 309.2780 ([FA_C20:1]^-), m/z 777.4985 ([PC_C40:4-NH(CH₃)₃]^-), m/z 745.4421 ([PC_C38:6-NH(CH₃)₃]^-/[PE_C38:6-NH₃]^-), m/z 747.4665 ([PC_C38:5-NH(CH₃)₃]^-/[PE_C38:5-NH₃]^-), etc. The degree of lipid oxidation was found to be associated with the number of freeze-thaw cycles, exhibiting the most significant alterations in the relative abundance of lipid ions in the 8T samples. Additionally, sensory evaluation by the CIE-L*a*b* method and volatile analysis by headspace solid-phase microextraction gas chromatography-mass spectrometry demonstrated significant differences (p < 0.05) in color and odor among the salmon samples, with a correlation to the number of freeze-thaw cycles. The primary compounds responsible for alterations in salmon odor were aldehydes with lower odor thresholds. In summary, the iKnife-REIMS method accurately differentiated salmon muscle tissues based on varying levels of lipid oxidation, thus expanding the application of REIMS.

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