In the present work the distribution of oxygen compounds in the total organic acid content of ten crude oils was assessed by means of negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry ((−) ESI FT-ICR-MS). As a first attempt, the relative abundance of the O2 class was related to the total acid number (TAN) for samples following the state of the art, and no positive correlation was achieved. Therefore, we performed the selective isolation of acidic compounds via solid phase extraction using amino-propyl silica (APS), finding an acceptable correlation (R2 = 0.98) between acidic fraction percentage and TAN. Both the reliability and performance of the APS method were confirmed using a chosen sample as control. FT-IR spectroscopy was employed to validate the acidic nature of the isolated fraction. In the IR spectrum of the acidic fractions, characteristic signals of carboxylic acids, such as the sharp band around 1700 cm–1 and the wide band around 2300–3500 cm–1, were identified. Additionally in such fractions, oxygenated classes such as O2, NO2, O3, SO2, and O3S were detected through (−) ESI FT-ICR-MS. Nevertheless, it can be said that none of these classes exclusively belong to the acidic fraction since for instance, O2 and NO2 compounds were found in both nonacid and acid fractions. In this sense, some O2 compounds may be considered to be bifunctionalized alcohols, phenols, ketones, or ethers. Finally, by comparing the contour plots DBE vs carbon number of chosen samples, it was possible to infer that the contribution of the O2 class over the TAN is structure dependent for samples with TAN lower than 0.5 mg KOH/g. Thus, the DBE distribution within the acidic and nonacidic fractions must be carefully considered in order to estimate their relevance over the total acid content.