Recently, high-resolution mass spectrometry has been largely employed for compound identification, thanks to accurate mass measurements. As additional information, relative isotope abundance (RIA) is often needed to reduce the number of candidates prior to tandem MS(n). Here, we report on the evaluation of the LTQ-Orbitrap, in terms of accurate mass and RIA measurements for building further metabolomics spectral databases. Accurate mass measurements were achieved in the ppm range, using external calibration within 24 h, and remained at <5 ppm over a one-week period. The experimental relative abundances of (M+1) isotopic ions were evaluated in different data sets. First of all, 137 solutions of commercial compounds were analyzed by flow injection analysis in both the positive and negative ion modes. It was found that the ion abundance was the main factor impacting the accuracy of RIA measurements. It was possible to define some intensity thresholds above which errors were systematically <20% of their theoretical values. The same type of results were obtained with analyses from two biological media. Otherwise, no significant effect of ion transmission between the LTQ ion trap and the Orbitrap analyzer on RIA measurement errors was found, whereas the reliability of RIA measurements was dramatically improved by reducing the mass detection window. It was also observed that the signal integration method had a significant impact on RIA measurement errors, with the most-reliable results being obtained with peak height integrations. Finally, automatic integrations using the data preprocessing software XCMS and MZmine gave results similar to those obtained by manual integration, suggesting that it is relevant to use the RIA information in automatic elemental composition determination software from metabolomic peak tables.