Lipidomics data require consideration of ions with near-identical masses, which comprises amongst others the Type-II isotopic overlap. This overlap occurs in series of lipid species differing only by number of double bonds (DB) mainly due to the natural abundance of 13C-atoms. High-resolution mass spectrometry, such as Fourier-Transform mass spectrometry (FTMS), is capable of resolving Type-II overlap depending on mass resolving power. In this work, we evaluated FTMS quantification accuracy of lipid species affected by Type-II overlap. Spike experiments with lipid species pairs of various lipid classes were analyzed by flow-injection-analysis (FIA)-FTMS. Accuracy of quantification was evaluated without and with Type-II correction (using relative isotope abundance) as well as utilizing the first isotopic peak (M+1). Isobaric peaks, which were sufficiently resolved, were most accurate without Type-II correction. In cases of partially resolved peaks, we observed peak interference causing distortions in mass and intensity, which is a well described phenomenon in FTMS. Concentrations of respective species were more accurate when calculated from M+1. Moreover, some minor species, affected by considerable Type-II overlap, could only be quantified by M+1. Unexpectedly, even completely unresolved peaks were substantially overcorrected by Type-II correction due to peak interference. The described method was validated including intra and inter-day precisions for human serum and fibroblast samples. Taken together, our results show that accurate quantification of lipid species by FTMS requires resolution-depended data analysis
