Chromatographic Reduction of Isobaric and Isomeric Complexity of Fulvic Acids To Enable Multistage Tandem Mass Spectral Characterization Erin N. Capley, † Jeremiah D. Tipton, ‡ Alan G. Marshall, ‡,§ and Alexandra C. Stenson* ,† Chemistry Department, University of South Alabama, Mobile, Alabama 36688, Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310-4005, and Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, Florida 32306 Humic substances and related material commonly grouped under the designation of natural organic matter (NOM) are of interest in fields ranging from marine chemistry and geochemistry to industry, agriculture, and pharmacology. High-field Fourier transform ion cyclotron resonance mass spectrometry enables resolution and identification of elemental compositions of up to thousands of compo- nents from a single mass spectrum. Here, we introduce an offline prefractionation to reduce the number of species of the same nominal (nearest-integer) mass, allowing for isolation of ions of one or a few m/z values, from which structural information can be obtained by low-resolution multistage tandem mass spectrometry (MS n ). Alterna- tively, precharacterized fractions can be generated for other types of analysis. As an example, we demonstrate significant reduction of isomeric and isobaric complex- ity for Suwannee River fulvic acid (SRFA). The com- bined MS and MS n analyses support the hypothesis that early eluting material comprises older, highly oxidized SRFA, whereas later eluting material is younger, retaining some similarity with precursor material. The major analytical obstacle to structural characterization of humic substances is their heterogeneity. Chromatographic isola- tion of individual analytes is generally impossible, limiting spectro- scopic techniques such as IR and NMR to providing composite averages. Although spectroscopy provides insight into origin, dif- ferentiation, degradation pathways, and overall composition, 1-5 the inability to resolve signals from individual analytes precludes full structural characterization based on spectroscopic tech- niques alone. Ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) resolves individual humic isobars (e.g., refs 6-8) and provides elemental compositions (e.g., refs 9-12). Visualization tools such as Kendrick 4,6,7,13-15 and van Krevelen plots 4,9,11,12,16,17 allow for easier interpretation of results and reveal compositional distributions and differences. Reemtsma et al. 9,18 have demonstrated the usefulness of a plot of molecular mass versus number of carbon atoms. These techniques, although powerful for comparison of broadband spectra, unfortunately offer limited direct structural information. Given that mass spectrometry affords the best resolution of humic analytes, multistage tandem mass spectrometry (i.e., MS n analysis) is a promising avenue of investigation. Previous MS 2 data revealed that fragmentation patterns for different natural organic matter (NOM) samples are highly similar. Reemtsma and co-workers 9,19-21 have examined the similarity between * To whom correspondence should be addressed. Phone: +1-251-460-7432. Fax: +1-251-460-7359. E-mail: astenson@jaguar1.usouthal.edu. † University of South Alabama. ‡ National High Magnetic Field Laboratory, Florida State University. § Department of Chemistry and Biochemistry, Florida State University. (1) Leenheer, J. A.; Rostad, C. E. 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Technol. 2008, 42, 1430–1437. (20) Reemtsma, T.; These, A.; Springer, A.; Linscheid, M. Water Res. 2008, 42, 63–72. Anal. Chem. 2010, 82, 8194–8202 10.1021/ac1016216  2010 American Chemical Society 8194 Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 Published on Web 09/02/2010