Chromatographic Reduction of Isobaric and Isomeric Complexity of

Sep 2, 2010 - Chemistry Department, University of South Alabama, Mobile, Alabama ... et al.9,18 have demonstrated the usefulness of a plot of molecula...
0 downloads 0 Views 1MB Size
Download PDF
Anal. Chem. 2010, 82, 8194–8202

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 components 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 (MSn). Alternatively, precharacterized fractions can be generated for other types of analysis. As an example, we demonstrate significant reduction of isomeric and isobaric complexity for Suwannee River fulvic acid (SRFA). The combined MS and MSn 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 isolation of individual analytes is generally impossible, limiting spectroscopic techniques such as IR and NMR to providing composite averages. Although spectroscopy provides insight into origin, differentiation, degradation pathways, and overall composition,1-5 the inability to resolve signals from individual analytes precludes * To whom correspondence should be addressed. Phone: +1-251-460-7432. Fax: +1-251-460-7359. E-mail: [email protected]. † 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. Tannins and terpenoids as major precursors of Suwannee River fulvic acid; U.S. Geological Survey Scientific Investigations Report 2004-5276; U.S. Geological Survey, 2004; p 16, http:// pubs.usgs.gov/sir/2004/5276/pdf/Book1Leenheer.pdf. (2) Hertkorn, N.; Benner, R.; Frommberger, M.; Schmitt-Kopplin, P.; Witt, M.; Kaiser, K.; Kettrup, A.; Hedges, J. I. Geochim. Cosmochim. Acta 2006, 70, 2990–3010. (3) Leenheer, J. A.; Nanny, M. A.; McIntyre, C. Environ. Sci. Technol. 2003, 37, 2323–2331.

8194

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

full structural characterization based on spectroscopic techniques 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 Kendrick4,6,7,13-15 and van Krevelen plots4,9,11,12,16,17 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., MSn analysis) is a promising avenue of investigation. Previous MS2 data revealed that fragmentation patterns for different natural organic matter (NOM) samples are highly similar. Reemtsma and co-workers9,19-21 have examined the similarity between (4) Mopper, K.; Stubbins, A.; Ritchie, J. D.; Bialk, H. M.; Hatcher, P. G. Chem. Rev. 2007, 107, 419–442. (5) Perdue, E. M.; Hertkorn, N.; Kettrup, A. Anal. Chem. 2007, 79, 1010– 1021. (6) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. Org. Geochem. 2002, 33, 171–180. (7) Kujawinski, E. B.; Hatcher, P. G.; Freitas, M. A. Anal. Chem. 2002, 74, 413–419. (8) Stenson, A. C.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 4397–4409. (9) Reemtsma, T.; Anja, T.; Springer, A.; Linscheid, M. Environ. Sci. Technol. 2006, 40, 5839–5845. (10) Stenson, A. C.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2003, 75, 1275– 1284. (11) Kujawinski, E. B.; Del Vecchio, R.; Blough, N. V.; Klein, G. C.; Marshall, A. G. Mar. Chem. 2004, 92, 23–37. (12) Tremblay, L. B.; Dittmar, T.; Marshall, A. G.; Cooper, W. J.; Cooper, W. T. Mar. Chem. 2007, 105, 15–29. (13) Hsu, C. S.; Qian, K.; Chen, Y. C. Anal. Chim. Acta 1992, 264, 79–89. (14) Kendrick, E. Anal. Chem. 1963, 35, 2146–2154. (15) Llewelyn, J. M.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 600–606. (16) Kim, S.; Kramer, R. W.; Hatcher, P. G. Anal. Chem. 2003, 75, 5336–5344. (17) Hertkorn, N.; Frommberger, M.; Witt, M.; Koch, B. P.; Schmitt-Kopplin, P.; Perdue, E. M. Anal. Chem. 2008, 80, 8908–8919. (18) Reemtsma, T. J. Mass Spectrom. 2010, 45, 382–390. (19) Reemtsma, T.; These, A.; Linscheid, M.; Leenheer, J.; Spitzy, A. Environ. Sci. Technol. 2008, 42, 1430–1437. (20) Reemtsma, T.; These, A.; Springer, A.; Linscheid, M. Water Res. 2008, 42, 63–72. 10.1021/ac1016216  2010 American Chemical Society Published on Web 09/02/2010

samples of different origin and have hypothesized that their composition is not as source material specific as had been assumed. As the authors point out,19,20 their and others’3,10,22 MS2 results suggest that the prevalence of closely related molecular formulas derives from structural homologies that could enable extrapolation from a small subset of structures to an entire sample. Witt et al.23 reconfirmed the similarity in MS2 fragmentation patterns of different analytes from the same sample. The present work is the first, to our knowledge, that highlights both similarities and differences in fragmentation patterns. In many cases, those differences do not become apparent until extension to MS3 or MS4. Although FTICR MS can resolve 100 or more elemental compositions with the same nominal mass, MSn requires prior isolation of ions of one (or a few) m/z values (and even then cannot distinguish isomers). Moreover, prior attempts at tandem mass spectrometry of NOM have been limited almost exclusively to single-stage fragmentation (i.e., MS2) of groups of ions consisting of isobars and isomers.10,21,22,24 A notable exception is a recent paper by Witt et al.,23 who employed singleshot isobaric isolation with a 9.4 T FTICR mass spectrometer. Here, we introduce a complementary approach that addresses resolution of isomers and isobars through chromatographic prefractionation. Chromatographic reduction of the number of isobars (and, ideally, isomers) to a single dominantly abundant species per nominal mass allows for the characterization of humics via fast MSn analysis with commonly available, low-resolution ion trap mass analyzers. For molecular formula identification (and to determine which nominal mass windows have been sufficiently prefractionated), one high-resolution spectrum must be obtained for each NOM fraction to be characterized. Once a sample has been characterized by MS and MSn, aliquots can be made available for other analysis techniques. Although the present approach reduces isomeric complexity and provides rapid MSn capability, fragment ion elemental compositions cannot be unequivocally deduced from lowresolution MSn data, and isobaric and isomeric isolation are generally not 100% efficient. Nevertheless, the first disadvantage can, to some extent, be overcome by combining MSn analysis with other data (e.g., metal affinity chromatography, hydrogen/deuterium exchange, trends in double-bond equivalents (DBE), and chromatographic retention times). EXPERIMENTAL METHODS Reversed-Phase HPLC Fractionation. Suwannee River fulvic acid (SRFA) was prefractionated as described previously25 and in the Supporting Information. Briefly, the procedure consists of repeated and pooled fractionation of SRFA on a phenyl column (X-Bridge (Waters Corporation) (3.5 µm, 4.6 mm × 150 mm)) via a step gradient. Fractions were collected into vials labeled 1-100. The only modification is the collection of fractions from two (rather (21) These, A.; Winkler, M.; Thomas, C.; Reemtsma, T. Rapid Commun. Mass Spectrom. 2004, 18, 1777–1786. (22) Plancque, G.; Amekraz, B.; Moulin, V.; Toulhoat, P.; Moulin, C. Rapid Commun. Mass Spectrom. 2001, 15, 827–835. (23) Witt, M.; Fuchser, J.; Koch, B. P. Anal. Chem. 2009, 81, 2688–2694. (24) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.; Ferrer, I. Anal. Chem. 2001, 73, 1461–1471. (25) Stenson, A. C. Environ. Sci. Technol. 2008, 42, 2060–2065.

than one) consecutive aliquots of 99 mg of SRFA (International Humic Substances Society, SRFA standard II). Immobilized Metal Affinity Chromatography Post-Treatment. The earliest eluting fractions from the high-performance liquid chromatographic (HPLC) separation are known to exhibit the least isobaric complexity.25 Thus, a fraction that eluted close to the void volume (t0 ) 2.0 mL) was chosen for an additional postcleaning step via immobilized metal affinity chromatography (IMAC), sample 5 (tr ) 2.5-2.7 min). The entire sample was dissolved in 20 µL of mobile phase immediately prior to injection. Two Ni Sepharose columns (HisTrap HP, GE Healthcare, 34 µm, 0.7 cm × 2.5 cm, 0.1% NH4OH mobile phase, pH 10.4) were connected in series. The first was stripped so that it might trap residual cations; the second was left intact to retain SRFA analytes based on affinity for Ni2+. No gradient was run. All visible humic material eluted from the columns in one “totalexclusion” band (tr ∼ 4 min, flow rate ) 0.5 mL/min). Different fractions of the total-exclusion band were collected by drop count and distinguished via different letters (5J-O): no significant compositional differences were detected based on molecular formulas or MSn fragmentation. The different IMACtreated samples (5K, 5M, and 5N) can therefore be considered replicates. Samples were evaporated to dryness and stored frozen. For comparison, two IMAC-untreated samples, one early eluting (6, tr ) 2.9-3.1 min) and one late eluting (53, tr ) 11.3-11.5 min), were analyzed as for the IMAC-treated samples. Sample Preparation. Solvents were Optima grade acetonitrile (ACN) and ammonium hydroxide (Fisher Scientific) as well as Baker Analyzed LC/MS reagent grade isopropyl alcohol (J.T. Baker). Sample 5M was dissolved in 70% H2O (0.1% NH4OH(aq) by volume) and 30% ACN. Samples 5K and 5N, 6, and 53 were dissolved in 70% H2O, 30% isopropyl alcohol, and 1% NH4OH(aq), sonicated, and centrifuged. IMAC postcleaning resulted in improved mass spectral signal-to-noise ratio (S/ N), especially compared with the late eluting sample 53. Switching from ACN to isopropyl alcohol, ISP increasing the percentage of NH4OH (from 0.1% to 1%), sonication, and centrifugation (i.e., 5M vs the other samples) resulted in the removal of sputtering and clogging in the electrospray ionization (ESI) source. The sample containing ACN (5M) resulted in significantly more ions, many of which were in-source reaction products. For example, peaks at 403.12 Da (C19H32O9, not labeled) and 403.23 Da (C20H36O8) in Figure 1 were not observed for any other samples, nor in any of our previously collected unfractionated SRFA samples. Thus, the combination of ACN and NH4OH should be avoided in spray solvents for humics. However, the use of isotopically labeled ACN could potentially provide structural clues based on the in-source derivatizations. For the hydrogen/deuterium exchange, SRFA standard (0.45 mg) was dissolved in 150 µL of deuterated water, 150 µL of deuterated ACN, and 6 µL of NH4OH. Data Collection and Analysis. Details on MS specifications and settings, hydrogen/deuterium (H/D) exchange, metal affinity, and preparation of the analytical standards can be found in the Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

8195

Figure 1. Mass scale expanded (-) ESI FTICR mass spectral segments for IMAC-treated vs untreated early and late eluting components of Suwannee River fulvic acid, showing resolution of isobaric (i.e., same nominal mass) components. Mass labels are for peak identification only. Elemental composition assignments are based on internal calibration (see Supporting Information Figure S-2). Samples 5K, 5M, and 5N are IMAC-treated early eluting samples, 5M is taken under different (offline) solvent treatment that provides slightly different results (see text). Sample 5M is therefore shown first rather than in alphabetical order. Sample 6 is an early eluting IMACuntreated sample. Sample 53 is a late eluting IMAC-untreated sample.

Supporting Information. Each fraction (5K, 5M, 5N, 6, 53) was analyzed with a custom-built 9.4 T FTICR mass spectrometer26 and a modified LTQ FTICR mass spectrometer (Thermo Fisher Corporation, Bremen, Germany) equipped with an actively shielded 14.5 T superconducting magnet (Magnex, Oxford, U.K.).27,28 In all, 74 MS3 and 43 MS4 spectra were collected for six different nominal mass precursor windows (m/z 383, 385, 341, 403, and 431, each with an m/z width of 1.2-1.5) from each of the five different SRFA samples (5K, 5N, 5M, 6, and 53). Most reported ionic species were singly charged, as evident from the unit m/z spacing between 12Cc and 13C12Cc-1 isotopomers. Ten of the MS2 products of the early eluting component at m/z 385 were fragmented further. Although these data significantly reduce the number of possible structures, they are not, in themselves, conclusive, especially lacking independent confirmation of isomeric purity. RESULTS AND DISCUSSION Reduction in Isobaric Complexity by HPLC and IMAC. Although collision-induced dissociation (CID) could be performed in the trapped-ion cell of an FTICR mass spectrometer, the product ions are formed off-axis and it would be necessary to pump away the collision gas in order to regain high mass resolving power. Thus, it is preferable to perform CID external to the FTICR mass (26) Ha˚kansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2003, 75, 3256–3262. (27) Schaub, T. M.; Hendrickson, C. L.; Horning, S.; Quinn, J. P.; Senko, M. W.; Marshall, A. G. Anal. Chem. 2008, 80, 3985–3990. (28) Schwartz, J. C.; Senko, M. W.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2004, 13, 659–669.

8196

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

analyzer, e.g., in a low-resolution quadrupole ion trap. Because low-resolution mass spectrometers cannot resolve humic isobars, it becomes necessary to reduce the number of species to one predominant isobar per nominal mass for low-resolution MSn of individual humic analytes. High resolution then serves to identify the elemental composition of the precursor ion, and the product ions can then be identified by low-resolution CID. Figure 1 and Supporting Information Figures S-1 and S-2 illustrate the reduction in number of isobars afforded by offline HPLC prefractionation followed by IMAC post-treatment for early eluting SRFA. The mass spectra from IMAC fractions 5K and 5N exhibit mostly ions of odd nominal mass with one dominantly abundant species. Although prefractionation is less than 100% efficient, the number of additional isobars is generally small (1-2) and their abundances are sufficiently low that their fragment ions do not significantly contribute to MSn products. High-resolution MS makes it possible to select ions for MSn analysis. Figure 1 also demonstrates that the main reduction in isobaric complexity derives from the HPLC fractionation step. Nevertheless, IMAC postcleaning can improve isobaric purity (e.g., 5K and 5N vs 6 in Figure 1) so long as identical spray-solvent conditions are used. MSn results for the same samples with two different instruments, at two different dilutions, with significantly different number of scans, on two different days were essentially the same (compare Figure 1 and Supporting Information Figure S-1 vs Supporting Information Figure S-2). Sample 5N (tail section near the end of the IMAC totalexclusion band) frequently provided the best results; it also contained the least material. An overlay of plots of Kendrick mass defect versus nominal Kendrick mass (Supporting Information Figure S-3) indicates that no compositional distinction is evident among IMAC-treated fractions. A small portion of the early eluting SRFA material is, however, retained on the IMAC column (Supporting Information Figure S-3, sample 6 vs 5K and 5N). The retained material appears to be the highest molecular weight (MW) subfraction with the highest Kendrick mass defect (KMD). High KMD corresponds to a high oxygen content and/or large double-bond equivalents (DBE equals the number of rings plus double bonds to carbon).10,12,14,29 High carboxyl content results in larger KMD because each carboxyl contributes two oxygens and one DBE. Surprisingly, relatively little material was retained by the IMAC column. Interestingly, Perdue et al.30 observed that for SRFA-Cd2+ complexes, the contribution of N donor atoms to the primary coordination sphere of Cd2+ increased steadily with increasing pH. Thus, nitrogen-containing SRFA (generally underrepresented in ESI MS spectra and therefore not directly observed here) might also be preferentially retained on IMAC columns with intermediate or soft acid31 cations such as Zn2+ or Cd2+sa potential advantage for preconcentration of nitrogenous components. Structural Inferences from Metal Affinity. Because differences in metal affinity can reveal differences in structure, previ(29) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G.; Qian, K. Anal. Chem. 2001, 73, 4676–4681. (30) Perdue, E. M.; Hertkorn, N.; Kettrup, A. Appl. Geochem. 2007, 22, 1612– 1623. (31) Pearson, R. G. J. Chem. Educ. 1968, 45, 581–587.

Table 1. Molecular Characteristics of Precursor Ions Selected for MSn analysisa measured mass

neutral elemental composition

296.9882 323.0045 341.0150

C11H6O10 C13H8O10 C13H10O11

383.0256 385.0413

C15H12O12 C15H14O12

403.0518 425.0362 431.0467

C15H16O13 C17H14O13 C16H16O14

453.0311

C18H14O14

341.1967 385.1656

C18H30O6 C22H26O6

403.1762*b 403.2126*b 431.1711**b 431.2075**b 453.1919

C22H28O7 C23H32O6 C23H28O8 C24H32O7 C26H30O7

DBE

F

max loss

min frag

Early Eluting Analytes (Samples 5K, 5M, 5N, and 6) 9 -6 10 -6 9 -6 5 (44) 97 and 99 10 -6 6 (44) 99 9 -6 6 (44) 93 + (28)c and 95 8 -6 6 (44) 99 11 -6 9 -6 5 (44) 149 +2 (18) 12 -6 7 (44) 109 Late Eluting Analytes (Sample 53) 4 -6 3 (44) 10 1 4 (44) +2 (18)c 9 1 8 1 10 1 9 1 12 1

109 99

O/(C - 6)

DBEnet/O

2.0 1.4 1.6

5/10 6/10 5/11

1.3 1.3

6/12 5/12

1.4 1.2 1.4

4/13 7/13 4/14

1.2

8/14

0.5 0.4

0/6 6/6

0.4 0.4 0.5 0.4 0.4

5/7 4/6 6/8 5/7 8/7

a F stands for family score (ref 32) (i.e., F ) 0.5z* - (DBE - O)), in which z* is the remainder of the nominal mass divided by 14 after subtraction of 14; max loss is the maximum number of successive losses of a given neutral (e.g., -18 Da for H2O, -44 Da for CO2); min frag denotes the lowest-mass fragment; O/(C - 6) is the ratio of number of oxygens to the number of carbons, not counting one assumed benzene ring (see Supporting Information Figure S-4 and related discussion); DBEnet is the number of double-bond equivalents, not counting those from one benzene ring (DBEnet ) DBE - 4). b Asterisks indicate ions from the late eluting IMAC-untreated fraction for which chromatographic isolation of isobars was poor: *, the abundance for ions of 403.2126 Da is at 75% relative to that for 403.1762 Da; **, 431.1711 Da is 61% relative to 431.2075 Da. c Some of the neutral losses of 44 Da for those ions must be C2H4O rather than CO2 because otherwise the combined loss would exceed the total number of oxygens in the molecule.

ously published32 data for SRFA complexed with Be2+, Mn2+, and Cr3+ were searched for complexes between these metals and the SRFA ions listed in Table 1; the results are tabulated in Supporting Information Table S-1. Clear trends in metal complexation are evident. The lowest-mass, early eluting SRFA analytes (m/z 297-341) did not form complexes with any of the metals. For the 2+ metals, there is also a clear preference for the later eluting SRFA analytes. In fact, Be2+ did not complex with any of the early eluting analytes. Mn2+ complexed with some of the higher-MW early eluting analytes, but clearly prefers the later eluting ones. The trivalent metal, Cr3+, forms complexes with both the early and late eluting SRFA analytes. In most cases at least one solvent adduct is required to fulfill the coordination of the metal. However, because a H2O adduct of a given analyte can be isomeric with respect to another analyte, it is impossible to unambiguously assign complexes to any given analyte. Nevertheless, both the IMAC data and the results in Supporting Information Table S-1 indicate that the earliest eluting and lowest-MW SRFA analytes exhibit the lowest affinity for metal cations. Moreover, larger and/or more highly charged cations are better able to complex SRFA. Be2+, Mn2+, and Cr3+ are hard acids31 and therefore expected to complex well with hard bases (e.g., hydroxides, alkoxides, and carboxylates) so that they differ mainly in size: Mn2+, 0.66-0.96 pm, > Cr3+, 0.62 pm (∼ Ni2+, 0.55-0.69 pm) > Be2+, 0.16-0.45 pm.33 The present results combined with our previous findings32 therefore suggest that the lowest-MW and earliest eluting SRFA analytes are too rigid to complex effectively with multivalent metal (32) Stenson, A. C. Rapid Commun. Mass Spectrom. 2009, 23, 465–476. (33) Shannon, R. D. Acta Crystallogr. 1976, A32, 751–767.

cations, especially small cations, such as Be2+. Aromatic rings and/or fused-ring systems substituted with short-chain functional groups are examples of SRFA-likely structures that exhibit such rigidity. Qualitative Differences in MSn Spectra. MSn results confirm the previously noted19,20,23,32 similarity in MS2 fragments formed and their relative abundances but also reveal notable differences. (Figure 2, parts a and b). For example, in spectra II-VII in Figure 2a, the fragmentation pattern for ions of m/z 341 is very similar, irrespective of whether it is a molecular ion or fragment ion. Notably, the fragmentation pattern of m/z 383.0256 (2H removed from the ion of m/z 385.0413, Table 1) is also virtually identical in observed neutral losses and their relative abundances. The 2 Da difference extends up to the fragment at m/z 99 (not shown). The fragment at m/z 99 is likely the virtually bare carbon backbone (see Supporting Information Figure S-4). The differences in fragmentation patterns of the late eluting versus early eluting ions (Figure 2 and Supporting Information Figure S-5) are quite noticeable. For late eluting ions, the higherMW neutral losses are absent or significantly lower in abundance. Although expected based on the difference in molecular formulas (later eluting ions have fewer oxygens: see Table 1 and ref 25), this effect has, to our knowledge, not previously been observed so strikingly. Figure 2b also reveals pronounced differences in fragmentation patterns for MS3 of lower-MW fragments: 253 Da derived from molecular ions at 341 Da (spectra I, III, and IV) versus 385 Da (spectra II and V) or 403 Da (spectrum VI), for both early eluting ions and late eluting ions. Thus, although ions of both 403 and 385 Da produce a fragment at 341 Da that, when refragmented, looks virtually identical to the Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

8197

Figure 2. Mass spectra following one or more stages of collisional dissociation, for each of the various listed values of normalized collision energy. NCE is the normalized collision energy (see text). (a) MS2 and MS3 fragmentation patterns for ions of m/z 341 or 339. I, late eluting IMAC-untreated control sample 53; II-VIII early-eluting IMAC treated samples (5K and 5N). (b) MS3 fragmentation patterns for ions of m/z 253 or 251. I-II, late eluting IMAC-untreated control sample 53; II-VII early-eluting IMAC treated samples.

equivalent molecular ion at 341 Da (Figure 2a), MS3 of lowerMW fragments (Figure 2b) reveals clear structural differences. Because these differences do not become noticeable until most of the labile functional groups have already been lost, the differences likely reside in the carbon backbone rather than in the type of functional groups. Also notable is that the 2 Da difference between 253 Da (from 385 and 403 Da) and 251 Da (from 383 Da) is, once again, maintained for all major peaks (spectrum VII vs spectra V and VI), supporting the hypothesis that the 2H difference is also likely to be in the carbon backbone rather than a carbonyl versus a hydroxyl group. No appreciable difference between fragmentation patterns of the molecular ions at 385 and 403 Da is notable from Figure 2, part a or b. The formulas for the ions in question differ from each other only in number of hydrogens and oxygens, whereas the 341 Da ion differs from those of 385 and 403 Da in number of carbons as well (Table 1). Thus it appears that, for SRFA, differences in the oxygencontaining functional groups are notably harder to detect via MSn analysis than differences in the carbon backbone. 8198

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

The MSn results in Figure 2 are representative for all analytes: late eluting species generally show fewer losses and fewer/less abundant low-m/z fragments. Within each HPLC fraction, MSn differences for isobars are subtle but become more pronounced the smaller the parent ion. Leenheer et al.24 had previously noted that humic-like analytical standards lose H2O and CO2 in MSn experiments until no aliphatic alcohols or carboxylic functional groups remain. Thus, it is interesting to consider the lowest-mass fragment observed for each analyte (Table 1, column 7). Supporting Information Figure S-4 illustrates that, in each case, the lowest-mass fragment is consistent with the presence of an aromatic group. For example, the ion of 107 Da has been noted as the lowest-mass fragment of 6L (Supporting Information Figure S-6) and the ion of 109 Da for 5L.24 Those fragments represent a monosubstituted phenol and a catechol. The aromaticity of Suwannee River dissolved organic matter is furthermore well-established; for example, Perdue et al.5 have recently demonstrated the high complexity of aromatic ring substitution patterns. That both early eluting and late eluting SRFA have aromatic character is further supported by strong UV absorbance at 250, 254, and 280 nm throughout the elution profile (Supporting Information Figure S-7). Throughout the chromatographic run, absorbance decreases with wavelength and is lowest at 365 nm (outside the aromatic absorbance range). The absorbance ratio of 250, 254, and 280 to 365 nm, sometimes used to trace aromaticity,34,35 produces entirely featureless profiles (not shown). We had previously noted that, for analytical standards, retention on the phenyl column did not depend on aromatic character as much as on placement of nonaromatic substituents; e.g., ibuprofen (23, Supporting Information Figure S-6) elutes late, whereas gallic acid (11) elutes early.25 The same appears to hold true for SRFA, suggesting that in the later eluting analytes, aromatic moieties are more accessible for interactions with the stationary phase, whereas in the early eluting fractions they are blocked by more densely crowded polar substituents. Trends in Relative Abundances of MS2 Fragments. Relative abundances of major fragments in MS2 spectra for SRFA ions are tabulated for our data (Supporting Information Table S-2, parts a and b, and Supporting Information Table S-3b, with higher isomeric purity) and that recently presented by Witt et al.23 (Table S-3a, with higher isobaric purity). Witt et al.’s MS2 results23 are uniformly shifted toward lower m/z fragments, probably in part due to use of a heavier collision gas (N2 vs He). In the absence of published MS3 or MS4 data for isobarically resolved SRFA, we restrict the present comparisons to MS2 data. Most of our MS2 data were collected for samples 6 and 53, whereas the more isobarically pure samples (5K and 5N) were reserved for thorough MSn coverage of a few analytes. The late eluting sample was neither isomerically nor isobarically pure, as evidenced by broad peak shape (Supporting Information Figure S-8). In addition, spectra exhibited overlap of doubly and singly charged analytes (Supporting Information Figure S-5). Neutral losses consist of the usual combinations of 18, 28, and 44 (corresponding to H2O, C2H4, or CO, and CO2 or C2H4O, Supporting Information Figure S-5). Not previously observed, to (34) Chin, Y.-P.; Alken, G.; O’Loughlin, E. Environ. Sci. Technol. 1994, 28, 1853– 1858. (35) Peuravuori, J.; Pihlaja, K. Anal. Chim. Acta 1997, 337, 133–149.

our knowledge, are the losses of 28 and 15 (Supporting Information Figure S-5 and Table S-2), discussed in more detail below. The loss of 44 is the most abundant (Supporting Information Tables S-2 and S-3). The loss of 18 is the second most abundant for most analytes although the loss of 28 becomes more prominent for lower-mass species. The prominence of the -18 loss is significant because few analytical standards specifically chosen to represent humic-typical functional groups (Supporting Information Table S-4) exhibit a prominent loss of both 44 and 18 independently (the combined loss of 62 is considerably more common). In the literature,23,24 only three standards ((20L),24 (21L),24 and (25W),23 Supporting Information Table S-4, Figure S-6) exhibited losses of both 18 and 44. In each case, the loss of 18 was more abundant (the reverse of the trend observed for SFRA). Under our instrumental conditions, none of the analytes exhibited both of those losses. For SRFA, the loss of 18 becomes more favorable for higher-mass precursor ions (Supporting Information Table S-2). In the mid-MW range, the abundance of H2O loss is similar for late and early eluting ions. The loss of 36 (2 × 18) is more prominent for late eluting fractions and decreases with increasing precursor mass. The abundance of the -18 loss decreases with number of oxygens for any given DBE (Supporting Information Table S-3a). The same is true for combined losses of 18 and 44 (i.e., -62, -106, -124, -150, and -168). For ions for which both DBE and O increase by the same integer in concert, no clear trends are noted. Loss of H2O has been observed dominantly from aliphatic alicyclic carboxlylic acids, proceeding via formation of five- or six-membered ring anhydrides between adjacent carboxlylic groups in the cis configuration.24 Aliphatic alcohols can also account for a loss of 18, but phenols were found to be stable.24 That is, the loss of 18 from a phenolic group (e.g., 7L,24 Supporting Information Table S-4, Figure S-6) is rare and appears to require a carboxyl group immediately adjacent on the ring (e.g., 7L vs 4-6L, 8, 9, 11, 12, Supporting Information Table S-4, Figure S-6). For a given DBE value, H2O loss becomes more dominant as the number of oxygens increases, suggesting that aliphatic alcohols may (at least in part) be the source. That hypothesis is further strengthened by the observation that no trend exists when both DBE and O increase in concert (as they would if carboxyl groups are added to the same carbon backbone instead of alcohols). Because the loss of 36 is more significant in the late eluting fraction, and the loss of 18 at higher precursor ion mass, the results suggest (a) slightly higher aliphatic alcohol content and/or (b) greater spacing of between OH- groups to allow for stable anhydride ring formation24 for these ions. The trend for the loss of 28 generally runs opposite to that for -18, i.e., less prominent for higher-mass precursors. Because the initial loss of 28 is not observed for carboxyl groups, it most likely derives from a carbonyl functional group (e.g., 3 and 26, Supporting Information Table S-4, Figure S-6). The secondary loss of 28 (after loss of 18 or 44) is, however, common for carboxyl groups. The loss of 28 becomes more prominent at low m/z (and to some extent in the late eluting fraction), suggesting that labile carbonyls (e.g., aldehydes, ketones/quinones, or esters/lactones) might be more common. It is especially notable that the trends

Figure 3. (a) Fragment ion relative abundances, grouped according to common neutral losses, e.g., methyl (-15 Da), water (-18, -36, -62 Da, etc.), carbonyl (-28, -72 Da, etc.), and carboxyl (-44, -88 Da, etc.). (b) Fragment ion relative abundances for each of three different elution stages, grouped according to their ratio of oxygens with exchangeable hydrogens to oxygens without. Because the listed elemental compositions include only CcHhOo species, the maximum number of deuteriums (Dmax) defines the number of oxygens with exchangeable hydrogens. The number of oxygens without exchangeable hydrogens is calculated by difference (i.e., O - Dmax). The error in Dmax can be ∼1-2, given that low-abundance ions with high Dmax may fall below the specified abundance threshold of 3%.

in fragment abundance versus precursor mass are opposite for the losses of 18 and 28 (-18 becomes more abundant as precursor mass increases, -28 less) because microbial degradation of putative source material converts hydroxyls into carbonyls and eventually lactones.3,36 However, the lowest-m/z ions could theoretically also arise from in-source fragmentation of higherMW SRFA, because losses of 28 as a secondary fragment are not uncommon for polycarboxylic acids. The loss of 15, indicative of methyl esters or methyl ethers (e.g., Supporting Information Table S-4), is observed primarily for late eluting ions (Supporting Information Table S-2b, Figure 3a). Thus, CH3O- functional groups may be more common in the late eluting fractionsinteresting, because lignin (one of the putative source materials for SRFA) is known for its appreciable OCH3 content, converted by microbial degradation (demethylation)36 to OH. However, not every compound containing a methyl ether functional group will exhibit a loss of 15, especially if more labile functional groups are present (e.g., 25 and 29, Supporting Information Figure S-6). Carboxylic groups are more prominent in the early eluting fraction, as evidenced by increased abundance for losses of 88, 132, and 176 (combined as “carboxyl” in Figure 3a). Those losses likely correspond to multiple carboxyl groups because they increase in abundance, if both DBE and O increase by the same increment, but decrease when if DBE is constant and the number of oxygens increases (the reverse of the trend for the loss of 18). (36) Leonowicz, A.; Cho, N.-S.; Luterek, J.; Wilkolazka, A.; Wojtas-Wasilewska, M.; Matuszewska, A.; Hofrichter, M.; Wesenberg, D.; Rogalski, J. J. Basic Microbiol. 2001, 41, 185–227.

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

8199

However, not every incremental loss of 44 observed in our data can be due to CO2; e.g., the analyte at 385.0413 Da in the early eluting fraction (Table 1) exhibits up to six incremental losses of 44 plus an additional loss of -18 (and one at -((6 × 44) + 28) as well), but the molecule contains only 12 oxygen atoms. Higher mass resolution following CID could distinguish CO2 from C2H4O. Finally, losses attributable to doubly charged ions (-9, -22, and -31) reside only in the late eluting sample and increase in relative abundance with increasing precursor ion mass. Therefore, high-MW SRFA (MW > 1000 Da) species elute late in the HPLC fractionation and become more available to ESI MS through prefractionation. Notably, however, the doubly charged species are so low in abundance in the FTICR spectra that their 13C12Cc-1 components (from which ion charge is determined) were not observed. The low abundance of doubly charged species for SRFA reveals likely spacing of functional groups based on comparison with analytical standards; MSn data alone does not generally provide such information. Leenheer et al.24 previously noted that, for linear polymers, charges must be 8-10 carbons apart for a stable multiply charged negative ion, consistent with double charging for some of the higher-mass standards (e.g., 25 and 31, Supporting Information Table S-4, Figure S-6)). Leenheer et al.’s data24 and ours additionally suggest that compounds that contain several (g3) carboxyl groups in close proximity to each other around a relatively inert central ring (e.g., 17-19L24 and 21L24) can also produce doubly charged ions. The rarity of doubly charged species from SRFA suggests that for most analytes, carboxyl groups are in close proximity similar to standards 2, 14, 15, 16L, and 29 but that the grouping of three or more carboxyls proximal to each other (as in 18, 19, and 19L) is rare or nonexistent. Isomeric Fractionation. Most SRFA ions contain only C, H, and O atoms, i.e., alcohol, ether, ester, carboxylic acid, ketone, and aldehyde moieties. A mixture of isomers containing these functional groups would therefore be expected to exhibit the most frequently observed losses (-44 and -18) at highest abundance, as is true for SRFA but rare for analytical standards. In addition, the mixture should exhibit significantly more fragments than a pure compound of the same nominal mass. In fact, MS2 of mid/ low-MW (∼200-500 Da) SRFA typically produces >10 fragments (frequently >20)ssignificantly more than most analytical standards in a similar MW range (e.g., Supporting Information Tables S-2 and S-3 vs column 5 in Table S-4). For a mixture of isomers, it would furthermore be expected that low-resolution MSn peaks from higher-mass precursor ions would be broader, as seen from MS2 of SRFA (Supporting Information Figure S-8). However, isobaric complexity also increases with mass, and sample 53 (the only one for which MS2 of high-mass precursor ions was performed) was the least isobarically pure. MSn profiles from different humic materials (i.e., slightly different mixtures of the same or similar isomers) would be expected to exhibit only slight variations, primarily limited to differences in relative abundances of fragments, as noted repeatedly in the literature (e.g., refs 19, 20, and 23). In contrast, we observe pronounced differences in MS2 profiles for early 8200

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

versus late eluting SRFA HPLC fractions (see Supporting Information Tables S-2 and S-3b) as well as consistent differences in fragment abundances for IMAC-treated versus untreated samples (Supporting Information Table S-5). The data (Supporting Information Table S-3b) indicate that ions of both 385.0413 and 341.0150 Da are outliers for which the losses likely to contain H2O are higher than expected; 385.0413 Da also stands out as an outlier in Supporting Information Table S-2a. Few such outliers are noted in the literature (Supporting Information Table S-3a) for unfractionated SRFA. IMAC-treated samples (Supporting Information Table S-5) consistently show higher-mass neutral losses likely to contain H2O (e.g., -62 (-44 and -18), -106 (-88 and -18), and -150 (-132 and -18)) than equivalent ions of the same molecular formula in the IMAC-untreated sample. The loss of 28, meanwhile, is more prevalent in the IMAC-untreated samples. Given that the molecular formulas of the ions are the same and that their isobaric purity is comparable, we hypothesize that the only difference is isomeric content. In general, therefore, MSn data of SRFA are consistent with the idea that they are mixtures of isomers. Our results further demonstrate reduction of isomeric complexity through chromatographic fractionations. Furthermore, because isomers remaining in HPLC and IMAC-treated samples must have identical retention behavior in both the fractionation and posttreatment process, any remaining isomeric complexity is severely constrained. For instance, if the precursor ions giving rise to the MSn profiles in Figure 2 comprised all possible isomers, the 2H differences between molecular ions of 385.0413 and 383.0256 Da could arise from either C-O or C-C unsaturation. If the difference between 385.0413 and 383.0256 Da were based on CdO versus C-OH, however, that difference should disappear as the corresponding functional groups are lost. As noted above, the 2 Da difference between fragments from 385.0413 and 383.0256 Da extends up to the fragment at 99 Da, indicating that the corresponding 2H difference lies in the most inert part of the molecule for all isomers of each mass. Likewise, if the MSn profile for the fragment at m/z 253 (Figure 2b) represents a group of random isomers, there should not be such notable differences between spectra deriving from molecular ions at 385 and 403 Da versus those deriving from the molecular ion at m/z 341. It appears, therefore, that isomers still present in HPLC and IMAC-treated samples consist of similar or identical functional groups in slightly different spatial arrangements around a relatively invariant core. H/D Exchange Data. Another intrinsic problem with interpreting MSn data of true unknowns is that rearrangement can occur during fragmentation. In low-resolution MSn, it is also impossible to distinguish isobaric losses (e.g., C2H4O vs CO2, as noted above). Quercetin (26, Supporting Information Figure S-6, Table S-4), for example, shows a loss of 44, even though it does not contain a carboxylic functional group. We therefore applied solution-phase H/D exchange to distinguish hydroxyl from ether/carbonyl oxygens. Unfortunately, not enough sample remained from the 5, 6, or 53 fractions, so the experiment was limited to unfractionated SRFA (Figure 3b and Supporting Information Figure S-9).

In general, the abundance of each isobar decreased with the number of exchanged D atoms, presumably because H/D exchange is not 100% complete for each potentially exchangeable hydrogen. Late eluting analytes generally contain fewer oxygens, but more of them are exchangeable (Supporting Information Figure S-9, top). The ratio of oxygens with exchangeable H to oxygens without would be 1, if all oxygens were carboxylic. The greater the reduction in ratio from 1.0, the higher is the ether, ester, ketone, and aldehdye content; the greater the increase in ratio above 1.0, the higher is the aliphatic alcohol and phenol content (see Supporting Information Table S-4 for the analytical standards). Figure 3b illustrates that early eluting SRFA exhibits ratios of 1 or less, whereas late eluting material are more diverse and trend toward the noncarboxylic values of 1. In the late eluting fraction, more of the double bonds are associated with carbon-carbon rather than carbonyl linkages. For instance, species from the late eluting fraction generally exhibit DBE - O ) 1-3, requiring several double bonds between C atoms. The reverse is true for the early eluting fraction (DBE O ) -1.5 to -4.0). Also, the absolute minimum number of unsaturations that must be associated with carbon (DBE - (O exchangeable H)) generally shifts more toward higher values (maximum ≈ 7-8) compared with the early eluting fraction (maxima ≈ 0, 3-4, and 7). Finally, for any given DBE, the late eluting fraction always has a significantly higher ratio of exchangeable to nonexchangeable O. Whereas the late eluting fraction appears to have more carbon-carbon double bonds and rings, the early eluting fraction appears to have approximately the correct DBE to allow for one benzene ring (DBE ) 4) and virtually all of its oxygens as carboxylic acids. For early eluting SRFA (Table 1, column 9) DBEnet/O is about one-half, as it should be if there were one benzene ring and all oxygens were from carboxyls. For late eluting SRFA, DBEnet/O is closer to one. Furthermore, the ratio of the number of oxygens divided by the number of carbons left after one benzene ring has been formed (i.e., C - 6, (Table 1, column 8)) is always >1 for early eluting fractions, suggesting that each carbon not in the aromatic ring has a high likelihood of being bound to at least one oxygen, and several are bound to two. For the late eluting fraction the ratio is less than one, once again indicating fewer O-C and more C-C bonds. Together these trends (Figure 3) suggest that early eluting material might be enriched in highly oxidized (carboxyl, ester, and/or ketone/aldehyde) functional groups, i.e., functional groups for which (exchanged oxygens)/(nonexchanged oxygens) e 1, a significant fraction of double bonds is associated with oxygen, and most carbons are bound to oxygen. Late eluting material exhibits fewer carboxyls but more alcohol/phenol ((exchanged oxygens)/(nonexchanged oxygens) > 1) or ether ((exchanged oxygens)/(nonexchanged oxygens) < 1) functional groups in which fewer double bonds are associated with oxygen (leaving more as carbon-carbon bonds). Note that (exchanged oxygens)/ (nonexchanged oxygens) ) 1 means only that the number of oxygens with exchangeable H equals the number without, but does not require each oxygen to derive from a carboxyl group (e.g., 31, Supporting Information Table S-4, Figure S-6).

CONCLUSIONS Taken together, the present results suggest that our HPLC fractionation separates SRFA into younger, less degraded (i.e., oxidized) material in the late eluting fractions and older, more oxidized material in the early fractions. The late eluting fraction exhibits higher aliphatic alcohol, phenol, and ether content (based on H/D exchange, DBE, and MS2 neutral losses of 15, 18, and 36). That fraction also contains the highest molecular weight material (based on losses of 9, 22, and 31, indicating doubly charged species). Furthermore, chargeable groups separated by a sufficient number of carbons to allow for multiple charging are more common. Finally, late eluting SRFA appears more flexible (i.e., better able to complex divalent metal ions). If degraded tannins, terpenoids, and lignin1,7 are the main sources of SRFA, that late eluting fraction most resembles its biological precursors (e.g., methyl ether and aliphatic alcohol content from lignin, additional aliphatic alcohol content from double-bond oxidation of terpenes, high phenolic content from tannins, and some polymeric/higher-MW species from each). Furthermore, van Krevelen plots of the HPLC fractions25 show overlap between the late eluting fraction and the region of the van Krevelen plot associated with biomolecular precursors and condensation products thereof,17 whereas the early eluting fractions occur in the most highly oxidized, least saturated region that does not overlap with known biomaterial and eventually terminates in CO2. The early eluting fractions appear to be older/more highly oxidized with a high content of carboxyl groups (based on H/D exchange, DBE, and MS2 losses). Relative increase in oxygens and decrease in precursor character and aromatic content (fewer C-C unsaturations) have previously been observed for artificially aged11,37 fulvic acids, in the difference between “young” freshwater and “old” deep ocean water,19 as well as during outswelling to coastal estuaries.12,38 Through microbial degradation, formation of carboxylic acids and esters or lactones is also common for lignin and terpenoids, for example.36,39 Demethylation36 decreases the overall methyl ether and methyl ester content, possibly explaining why the -15 Da neutral loss is significantly less abundant for this fraction (Figure 3a). Depolymerization may explain the more prevalent loss of 28 Da for lower-mass/more degraded components. Because degradation produces analytes that are lower in mass, more highly substituted, and more polar, the older/more degraded material should be less retained on the HPLC column, as observed. Multiple very short-chain (although highly oxidized) functional groups bound to a rigid, inert, aromatic, and/or fused-ring core could further explain the relatively low metal affinity for this fraction. Future work will focus on ever more selective isomeric isolation, as well as on combining various analytical and synthetic approaches. The combination of in-cell derivatization (as in refs 40 and 41) with MSn analysis, for instance, is powerful in distinguishing functional groups and retaining isomeric resolution of the prefractionation step(s). Extensive MSn characterization of a wide range of analytes (37) These, A.; Reemtsma, T. Environ. Sci. Technol. 2005, 39, 8382–8387. (38) Gonsior, M.; Peake, B. M.; Cooper, W. T.; Podgorski, D.; D’Andrilli, J.; Cooper, W. J. Environ. Sci. Technol. 2009, 43, 698–703. (39) Leenheer, J. A.; Nanny, M. A.; McIntyre, C. Environ. Sci. Technol. 2009, 34, 2323–2331. (40) Alomary, A.; Solouki, T.; Patterson, H. H.; Cronan, C. S. Environ. Sci. Technol. 2000, 34, 2830–2838. (41) Solouki, T.; Freitas, M. A.; Alomary, A. Anal. Chem. 1999, 71, 4719–4726.

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

8201

should enable semiquantitative comparisons, particularly by use of multivariate statistics (as in ref 42). Tetraalkylammonium hydroxide pyrolysis-GC/MS (as in refs 43 and 44) could reveal the most common placement of functional groups relative to each other. Spectroscopic experiments can provide additional detail on differences in carbon backbone and functional group content. The improvement in interpretability of NMR data for SRFA subjected to extensive prefractionation has already been demonstrated1 as has the great potential of combining NMR with FTICR MS.2,4,45 Particularly promising are difference NMR spectra (as in ref 2), spectral editing techniques (as reviewed in ref 4), and computational peakmatching algorithms (as in ref 5). (42) Kujawinski, E. B.; Longnecker, K.; Blough, N. V.; Vecchio, R. D.; Finlay, L.; Kitner, J. B.; Giovannoni, S. J. Geochim. Cosmochim. Acta 2009, 73, 4384–4399. (43) Lehtonen, T.; Peuravuori, J.; Pihlaja, K. J. Anal. Appl. Pyrolysis 2003, 6869, 315–329. (44) Xiaoli, C.; Shimaoka, T.; Qiang, G.; Youcai, Z. Waste Manage. 2008, 28, 896–903. (45) Hockaday, W. C.; Grannas, A. M.; Kim, S.; Hatcher, P. G. Geochim. Cosmochim. Acta 2007, 71, 3432–3445.

8202

Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

ACKNOWLEDGMENT The authors thank Nicole Novotny, Priscillia Ajaegbu, and Dr. Amy M. McKenna for assisting in data collection and also Dr. June E. Ayling for access to the ion trap instrument at the USA College of Medicine, supported through funding from NSFEPSCoR 0091853-353. We also thank Jerry Koncar (ThermoFisher) for providing the conversion between NCE and collision energy in volts. Financial support from NSF EAR-0848635, NSF DMR-0654118, the State of Florida, and the University of South Alabama is also gratefully acknowledged. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 18, 2010. Accepted August 6, 2010. AC1016216