Compound-specific isotope analysis (CSIA) by liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) has until now been based on ion-exchange separation. In this work, high-temperature reversed-phase liquid chromatography was coupled to, and for the first time carefully evaluated for, isotope ratio mass spectrometry (HT-LC/IRMS) with four different stationary phases. Under isothermal and temperature gradient conditions, the column bleed of XBridge C18 (up to 180 °C), Acquity C18 (up to 200 °C), Triart C18 (up to 150 °C), and Zirchrom PBD (up to 150 °C) had no influence on the precision and accuracy of δ13C measurements, demonstrating the suitability of these columns for HT-LC/IRMS analysis. Increasing the temperature during the LC/IRMS analysis of caffeine on two C18 columns was observed to result in shortened analysis time. The detection limit of HT-RPLC/IRMS obtained for caffeine was 30 mg L–1 (corresponding to 12.4 nmol carbon on-column). Temperature-programmed LC/IRMS (i) accomplished complete separation of a mixture of caffeine derivatives and a mixture of phenols and (ii) did not affect the precision and accuracy of δ13C measurements compared with flow injection analysis without a column. With temperature-programmed LC/IRMS, some compounds that coelute at room temperature could be baseline resolved and analyzed for their individual δ13C values, leading to an important extension of the application range of CSIA. Copyright © 2011 John Wiley & Sons, Ltd.