Warm ischemia and reperfusion injury (IRI) is a prognostic factor in donation after cardiac death donor transplantation. However, a reliable method to predict IRI before transplantation has not been established. The aim of this study was to identify predictive markers of hepatic IRI by simultaneous measurement of endogenous molecules using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Rats were subjected to hepatic warm ischemia (70%) for 30 or 90 minutes and subsequent reperfusion. The livers were collected at the end of ischemia and 1 hour, 6 hours, and 24 hours after reperfusion. The liver tissue sections were applied to IMS (m/z 200-2000). Candidate molecules were identified by tandem mass spectrometry. Imaging mass spectrometry (IMS) revealed a significant increase in the taurine conjugates of dihydroxycholanoic acid (TDHCA) during ischemia and a tendency to return to the basal level after reperfusion. Notably, high-resolution measurements revealed focal accumulation of TDHCA in the intrahepatic bile duct with ischemic time. In conclusion, IMS is a useful method to detect minute changes provoked by ischemia, which are barely detectable in assays involving homogenization. Accordingly, focal accumulation of TDHCA during ischemia may be a candidate marker for predicting later IRI. • MALDI-IMS is a useful method to detect and identify many molecules that change during ischemia and reperfusion without homogenization and extraction. • MALDI-IMS revealed the accumulation of TDHCA (m/z 498.28) in the intrahepatic bile duct during warm ischemia. • TDHCA, which delayed recovery to the basal level, may become a predictive marker of IRI. • Precise identification of TDHCA remains elusive. Candidate molecules of taurine-conjugated DHCA species (m/z 498.28) are taurine-conjugated CDCA, UDCA, and OCA. [ABSTRACT FROM AUTHOR]