Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC–MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C₁₈ stationary phase, which enabled global analysis of nucleotides and their ¹³C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 min of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD⁺, NADH, NADP⁺, NADPH, cyclic nucleotides, several UDP-hexoses, and all their ¹³C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-¹³C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, ¹³C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their ¹³C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.