Naphthenic (NAP) acids have been previously characterized by negative electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), both directly and from acid extracts. Here, we show that collective characterization of NAP acids with negative-ion ESI, both with and without prior extraction, results in a loss of signal from high mass acids because of ion suppression by low mass acids. We have developed an extraction to simultaneously isolate and fractionate NAP acids into distinct molecular weight ranges, thereby grouping acids into fractions with similar ionization efficiency. A NAP acid fraction was isolated (all acids isolated collectively in one fraction) and compared to NAP acid isolation by molecular weight. Characterization of acid molecular weight fractions extended the observed upper mass limit from similar to 850 Da for the collectively isolated acids to similar to 1450 Da for the adds isolated by molecular weight range, thereby approximately doubling the observed mass range for NAP acids. Plots of double bond equivalents (DBE = number of rings plus double bonds to carbon) versus carbon number span both higher carbon number and higher DBE values than are accessed by collective acid characterization. The high mass resolving power and mass accuracy of FT-ICR MS are essential for identification and resolution of acidic species across a wide mass range.