Environ. Sci. Technol. 2008, 42, 2060–2065
Reversed-Phase Chromatography Fractionation Tailored to Mass Spectral Characterization of Humic Substances ALEXANDRA C. STENSON Department of Chemistry, University of South Alabama, Mobile, Alabama
Received September 8, 2007. Revised manuscript received December 12, 2007. Accepted December 17, 2007.
Large-scale structural characterization of humic substances via mass spectrometry requires reduction of complexity within nominal mass and separation of isomers, i.e., prefractionation. Humic substances (here loosely defined to encompass all humic, humic-like, and humic-containing material) are notoriously difficult to fractionate. Equally challenging is deriving information on whether and how fractionation has occurred. Here, reversedphase high-performance liquid chromatography was used to induce tailored fractionation of Suwannee River fulvic acid (SRFA) within nominal mass. The fractionation was optimized on synthetic standards that differed in polarity and had elemental formulas similar to SRFA. Fractions were analyzed via electrospray ionization ion-cyclotron resonance mass spectrometry. Kendrick and Van Krevelen comparisons showed that fractionation occurred as predicted based on known molecular formula patterns.
Introduction The importance of humic substances in environmental processes has been nicely reviewed in recent papers (e.g., refs 1 and 2). Lack of detailed structural information remains, however, a huge handicap to most fields of inquiry. The major hurdle to such structural characterization is molecular complexity. The work outlined here therefore focuses on meaningfully reducing sample complexity to facilitate structural characterization. Although great improvements are continually being made (e.g., refs 3–7) fractionation of humic substances remains challenging. In fact, fractionation of humic materials into individual molecules is currently accepted as not feasible, generally reducing the ultimate goal to providing reproducible results that allow for the comparison between humic samples and that might provide clues as to the overall structural/ functional composition of any given sample. The most obvious and immediate stumbling block to fractionation is the immense complexity of humic samples. Continuous, rather featureless, humps are the general result of reversed-phase high-performance liquid chromatography (RPHPLC) separations (3, 5). The same is true for gel permeation chromatography, size-exclusion chromatography (6, 7), and immobilized metal chromatography (4). Whelan et al. (5) is an example of some of the more feature-rich results attainable in RPHPLC with linear gradients (obtained * Author fax: (251) jaguar1.usouthal.edu. 2060
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 6, 2008
through ion pairing). Interestingly, step-gradients generally result in more feature-rich chromatograms (3, 5–7). Notably, however, the number of peak regions thus induced generally corresponds rather tightly to the number of steps in the gradient; Hutta and Góra (3) give a nice account of this effect. While this trends hints to the fact that some of these “features” might be introduced through mechanical means, they have generally proven to be quite reproducible and useful to comparisons between different humic samples (3, 6). A further hurdle is that most standard detectors are neither universal nor particularly information-rich. Frequently, different detectors produce quite different chromatograms for the same humic material (e.g., refs 3 and 6), a phenomenon that can sometimes be used to advantage (e.g., ref 6). Even relatively information-rich detectors such as three-dimensional excitation–emission matrix fluorescence (e.g., ref 7) usually reveal only subtle differences. The inability of most detectors to provide concrete information as to molecular differences between individual humic-fractions is, therefore, a major drawback that needs to be overcome, placing ultrahigh resolution mass spectrometry (i.e., Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) at high magnetic field (9.4 T)) among the most promising tools. FT-ICR MS has been demonstrated to resolve individual humic ions (e.g., refs 8–10) and provide information on the molecular level (e.g., refs 11–14). Kendrick (1, 8, 9, 15–17) and Van Krevelen plots (1, 11, 13, 14, 18) allow for easier visualization of results and have been elegantly employed to derive compositional information on humic/humic-like samples (e.g., refs 11, 13, and 14). Recently, Reemtsma et al. (11) have demonstrated the usefulness of an additional plot relating molecular mass to the number of carbon atoms. Although broadband complexity is not a problem per se in FT-ICR MS, complexity within any given nominal mass and overlap of isomers are. These phenomena impede acquisition of tandem mass spectral (MSn) data to provide structural information. MSn experiments require individual ions (and subsequently their fragments) to be isolated in the mass spectrometer. Humic ions are too closely spaced to allow for such isolation. Previous fragmentation data were therefore limited to one fragmentation (i.e., MS2) on groups of ions (12, 19–21). Full structural characterization will necessitate higher-dimension MSn experiments (n g 3) on individual ions. RPHPLC was chosen for prefractionation based on previous studies (10, 12) indicating that, within nominal mass, humic ions of the same family differ from each other by substitution of CH4 vs O; where families coincide, they differ significantly in degree of saturation. Differing degrees of oxygenation and saturation imply differing polarity, best resolved via RPHPLC. While the ultimate goal is to find the absolute minimum in sample complexity through multidimensional prefractionation, the purpose here was three-fold (1): to establish whether RPHPLC induces fractionation of humic substances (2), whether this fractionation can be detected with FT-ICR MS, and (3) whether it occurred in predictable fashion.
Experimental Section Gradient Development. HPLC separations were run on an X-Bridge (Waters Corporation) phenyl column (3.5 µm, 4.6 × 150 mm). The Shimadzu (Shimadzu Scientific Instruments, Inc.) HPLC system consisted of pump (LC-20AD), autosampler (SIL-20A), oven (CTO-20AC), UV-detector (SPD-20A), fraction collector (FRC-10A), and custom-built 101-slot rack for 13 × 100 mm culture tubes. 10.1021/es7022412 CCC: $40.75
2008 American Chemical Society
Published on Web 02/16/2008
TABLE 1. Retention Times of the Synthetic Standards Used compound
tra
familyb
O/C
H/C
DBEc
DBE - Od
#C ) O
z *e
malonic acid, CH2(COOH)2 gallic acid, (HO)3C6H2CO2H methyl gallate, (HO)3C6H2CO2CH3 hydrocaffeic acid, (HO)2C6H3CH2CH2CO2H chlorogenic acid, C16H18O9f ellagic acid, C14H6O8 trimethylgallic acid, (CH3O)3C6H2CO2H acetylsalicylic acid, 2-(CH3CO2)C6H4CO2H 1-dehydrocortisol, C21H28O5 hydrocortisone, C21H30O5 quercetin, C15H10O7 glycyrrhizic acid, C42H62O16f fluorescein, C20H12O5 tolmetin, C15H14NO3Na 1,3-benzodioxole-5-propanoic acid, C10H10O4 fenoprofen, C30H26O6Ca ibuprofen, C13H18O2
2.0 3.0 5.7 6.2 6.2 7.3 8.3 8.5 9.6 9.7 9.8 10.1 10.8 11.2 11.7 12.5 14.0
-6 -6 -6 -6 -6 1 -6 1 1 1 1 -6 8 1.5 1 1 1
1.33 0.71 0.63 0.44 0.56 0.57 0.50 0.44 0.24 0.24 0.47 0.38 0.25 0.20 0.40 0.20 0.15
1.33 0.86 1.00 1.11 1.13 0.43 1.20 0.89 1.33 1.43 0.67 1.48 0.60 1.00 1.00 0.93 1.38
2 5 5 5 8 12 5 6 8 7 11 12 15 9 6 9 5
-2 0 0 1 -1 4 0 2 3 2 4 -4 10 6 2 6 3
2 1 1 2 1 2 1 2 2 2 1 4 1 2 1 1 1
-8 -12 -12 -14 -10 -6 -12 -2 -4 -2 -6 -4 -4 -9 -2 -10 -4
a tr ) retention time on the RPHPLC column with the gradient shown in Figure 1. b Family ) family score as calculated through eq 1. c DBE ) double bond equivalence (i.e., the number of rings and/or double bonds). d DBE - O ) difference between DBE and the number of O atoms in the compound. e z* ) ((remainder of nominal mass/14) - 14) as defined in ref 15. f Standards previously used in the literature as part of a humic-like test mixture (23).
FIGURE 1. RPHPLC step-gradient optimized on synthetic standards. The structure for each standard and its approximate elution time are indicated (for exact elution times, see Table 1). Gray bars underneath the gradient line indicate where the fractions that were analyzed via FT-ICR MS were collected (fractions 105-152). Given the difficulty of producing feature-rich humic chromatograms, synthetic standards (Table 1) were used to optimize the HPLC method. Mobile phase consisted of 0.1% formic acid in both water and acetonitrile (ACN) and was made fresh each day but left unbuffered to avoid possible signal quenching in ESI- MS (10). Analytes were dissolved in DMSO prior to injection, mainly to avoid solubility problems and dead-volume break-through. Furthermore, Hutta and Góra (3) pointed out that DMSO in the presence of small amounts of acid appears to be the best solvent for disrupting hydrogen bonding in SRFA. DMSO (or DMF) was not, however, used in the mobile phase as in Hutta and Góra (3), because samples needed to be freeze-dried after collection and because inclusion of DMSO in the spray solution can lead to CH3SO2H-adducts (data not shown). A practical upper limit was set to HPLC resolution by the slots available in the fraction collector. Gradient conditions, temperature, and fraction volume were optimized within this constraint. The final gradient was a step-gradient (0–2 min 5% ACN, 2–4 min 15%, 4–7 min 30%, 7–12 min 50%,
12–13 min 70%, 13–15 min 95%, 15–19 min 5%). Oven temperature was 40 °C, and flow rate was 1 mL/min. Figure 1 shows the gradient (after correction for dead volume). Suwannee River Fulvic Acid (SRFA) fractionation. SRFA (International Humic Substances Society, ∼100 mg) was dissolved in 1 mL of DMSO. Injection volume was 20 µL. A total of 167 µL of eluent was collected into each collector vial. Subsequent injections were collected into the same vials, gradually accumulating material. The fractionation was repeated until the collector vials were nearly full. Fractions were then frozen, freeze-dried, and stored frozen or cold until analysis. In all, six fractions were analyzed (vials 105 (2.87 min), 109 (3.60 min), 127 (6.85 min), 134 (8.11 min), 143 (9.74 min), and 152 (11.37 min)). These fractions covered the range where most humic materials seemed to elute and the encompassed fraction that contained relatively large amounts of humic material as well as fractions that contained very little. Vials came from an acid-washed, prelabeled batch starting with vial 100. VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Characterization. The ESI solvent system used was as follows: 70% ACN, 30% H2O, 0.1% ammonium hydroxide. ACN was chosen to avoid self-esterification of fulvic acids (22), although it has been shown (23) that deliberate and complete methylation can sometimes be helpful. Studies in this laboratory with methanol, isopropanol, and acetonitrile on synthetic standards and mixtures thereof showed that, although no solvent system was optimal (i.e., produced good signal for the molecular ion while minimizing dimers, mixeddimers, and solvent adducts), acetonitrile provided the best compromise (data not shown). The data also suggested the use of high concentrations of ACN (g70%). To avoid clogging of the ESI source, samples were centrifuged before introduction into the mass spectrometer at a flow rate of 0.5 µL/min. Dual spray was used to introduce internal standards. Each fraction was analyzed once with and (at least) once without an internal standard (fluorescein plus Electrospray Tuning Mix, Agilent Technologies). FT-ICR MS data were collected in the negative mode on a home-built mass spectrometer (9.4 T magnetic field) housed at the National High Magnetic Field Laboratory in Tallahassee, FL, equipped with dualspray and quadrupole mass-filter; a detailed description of the instrument is given elsewhere (24). Data were processed and converted as described previously (10, 12). Settings were kept identical for all fractions analyzed (capillary heater current: 4 A, tube lens: -320 V, needle: -2000 V, transfer octopoles: 1.5 MHz, accumulation: 20 loops of 0.23 s each, 40 coadds (without internal standard), or 50 coadds (with)). Data Analysis. Kendrick plots were prepared in the usual way (1, 10, 12–14). In order to prepare Van Krevelen plots (1, 11, 13, 14, 18) and carbon-number vs mass plots (11), molecular formulas had to be assigned. Fractions 105, 134, and 152 were chosen as representatives for early, mid, and late eluting material. Formula assignments were performed first by comparison against the originally assigned formulas for SRFA (12) (after adjusting for the difference in ionization mode). As expected, prefractionation allowed for detection of additional ions. Most of these new ions belonged to the same formula families as the previously assigned ones, allowing their formulas to be determined by following the established substitution patterns (i.e., CH4 vs O, addition of H2, etc.). Additionally, some peaks were identified by plugging their measured masses into a molecular formula calculator, (+/-1 ppm, C (1–100), H (2–200), O (1–50)). The latter was also done for a large number of ions identified as described above to confirm the validity of the assignment procedure.
Results and Discussion An early indication that fractionation had occurred was the unequal distribution of color and amounts retrieved after freeze-drying (data not shown). These indications were further supported by subtle differences in solubility in the ESI spray solvent. While these early observations were encouraging, the overall broadband spectrum of each fraction showed relatively little differentiation (Supporting Information, Figure S1), demonstrating the need for high resolution. The only observable difference between fractions in these spectra was that later eluting fractions showed higher signal abundance at higher m/z (above ∼m/z 500). Notably, however, differences in molecular mass distribution (Mn, Mw, and Mn/Mw) were subtle (Supporting Information, Table S1), suggesting that separation did not occur primarily based on mass. From zoomed spectra (e.g., Figure S1b in the Supporting Information and Figure 3), it became apparent, however, that masses within any given m/z unit did increase with retention time. Given previously published SRFA formulas (e.g., ref 12), it is evident that this shift toward larger positive mass-defects coincides with a decrease in the number of O atoms and an increase in the number of H atoms (as O has a negative mass defect, i.e., elemental mass slightly 2062
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FIGURE 2. Data analysis of SRFA fractions’ odd-mass ions: (a) van Krevelen plots, (b) correlation between the number of carbons and molecular mass, and (c) the relationship between DBE and DBE - O. less than 16 Da, and H has a positive mass defect, i.e., elemental mass slightly more than 1 Da). Thus, even this simple comparison of mass segments suggested that fractionation within nominal mass had occurred exactly as predicted. Additionally, Kendrick plots of five of the six fractions (all but 127) were overlaid (Supporting Information, Figure S2) and revealed that significant and gradual fractionation had occurred. Notably, early eluting fractions (105 and 109) were shifted significantly toward higher Kendrick mass defects, suggesting higher oxygen content, possibly in the form of carbonyl/carboxyl groups since the double bond equivalence (DBE) is also increasing in that direction. The convenience of Kendrick plots lies in the fact that molecular formula assignments are not required for their construction. To derive more concrete insight into the molecular basis underlying the observed HPLC fractionation, however, formulas need to be assigned. For this purpose
FIGURE 3. Fractionation within nominal mass: Formulas for peaks 1–7 and a-h are noted in Table 2 (note: since virtually all SRFA ions observed are singly charged, m/z ) mass). three fractions were chosen to represent early, mid, and lateeluting material (105, 134, and 152). Only odd-m/z ions were assigned, since it had previously been discovered (12) that most of the complexity of SRFA is represented in that segment of the spectrum (even-m/z ions can readily be reconstructed from their odd-m/z counterparts by substituting 12C for 13C, or CH2 for NH). Figure 2a shows Van Krevlen plots for the three fractions selected. Based on these plots, it is obvious that (as could be inferred from the Kendrick plots) fractionation occurred based on oxygenation/saturation. As predicted, the more oxygenated/unsaturated analytes evidently are more polar and elute earlier. This excellent agreement between expected and observed results also validates the previously assigned formulas and indicates that increased oxygenation in early fractions may largely be due to additions around the carbon skeleton rather than within (i.e., hydroxyls, carboxyls, and esters vs ether-linkages or heterocycles). It could be hypothesized (considering the type of stationary phase used and the most common structural components of humics) that the observed trend in H/C (Figure 2a) corresponds to an increase in CdO containing functional groups (e.g., carboxylics or esters). Although this hypothesis is plausible, it is by no means proven, as underlined by data for the synthetic standards (Figure 1, Table 1). Table 1 illustrates that no correlation was observed between retention time and the number of double bonds attributable to CdO. Rather, Figure 1 illustrates that the HPLC column was selective for O/C ratio (Table 1, 3rd column) and arrangement of polar groups around the carbon skeleton. The more densely the C-skeleton was covered with hydroxy, carboxy, and/or carbonyl groups, the earlier a compound eluted, regardless of the number of CdO bonds. The relationship between double bond equivalence and the number of oxygen atoms for the various fractions is analyzed in more detail below. Recently, Reemtsma et al. (11) have made an argument for employing plots that correlate molecular mass to the number of carbon atoms. One argument for this inclusion is that, unlike Van Krevelen plots, it takes mass into account. In addition, the authors show that humic data arrange themselves into ‘islands’ (Supporting Information, Figure S3) in which C content decreases and O content increases going down the y-axis. In other words the carbon skeleton
decreases at the same time as the O content increases, leading to a bottom tip of each island where oxygenation is maximized. It should be noted that for such a small C-skeleton, highly oxygenated structures would be the best candidates for initial structural characterization, since the number of possible structures that can be drawn for such structures are mercifully limited. The correlation between molecular mass and number of carbon atoms for the three selected SRFA fractions is shown in Figure 2b. Again, the data clearly show that fractionation on the molecular level has occurred. Furthermore, this plot conveys that later eluting fractions extend to slightly higher overall mass (a parameter that is lost in van Krevelen plots and not readily observable in the broadband spectra (Supporting Information, Figure S1 and Table S1)) and, in general, contain analytes with larger carbon skeletons (higher on the y-axis). Thus, early eluting fractions appear comprised largely of small C-skeleton, highly oxygenated analytes making them the most interesting for structural characterization. To revisit the question of double bond equivalence, a plot of DBE - O versus DBE was constructed (Figure 2c). This plot reveals a preference toward low or negative differences between double bond equivalence and the number of oxygen atoms in the earlier eluting fraction. Later eluting fractions show gradually more preference for ions in which the number of double bonds exceeds the number of O atoms. Not as striking, however, is the difference in overall double bond equivalence between the three fractions (y-axis). This supports the speculation that fractionation is based on the kinds of DBEs that were present (not their sheer number). To investigate how strongly the synthetic standards might undermine this speculation, they were included in Figure 2c. Interestingly, most of the standards fall within or below the DBE regime of the late-eluting SRFA, negating their representativeness for bulk SRFA. One exception to this trend is glycyrrhizic acid (DBE - O ) -4), which falls squarely within the early eluting SRFA signal. Given the skewed nature of the synthetic standard signal in Figure 2c, it appears the standards differ fundamentally from humic substances. A definite conclusion on the nature of the double bond equivalence deviation between early and later eluting fractions can therefore not be drawn. It is, however, evident that it is not DBE alone, but the difference between DBE and the number of O atoms, as well as the O/C ratio, that is indicative of the fractionation incurred. After examining plots representative of the entire spectrum (Figure 2), it is useful to look at individual ions to doublecheck if trends suggested on the large scale ring true there. Figure 3, therefore, compares fractions 105, 134, and 152 based on a single m/z segment. Molecular formulas for each ion identified are given in Table 2. As the table shows, the difference between ions labeled 1-7 is incremental substitution of O for CH4, which decreases both the number of O and the DBE by one each time (the same is true for peaks labeled a-h). It is this previously observed substitution that had led to the initial hypothesis that RPHPLC fractionation should be able to resolve ions of identical nominal mass. This hypothesis was confirmed. The early eluting fraction (vial 105) contains only peaks 1-3, which are the most oxygenated and unsaturated members of the 1–7 peak family. The mideluting material contains peaks 3-7 (with clear preference toward the medium-oxygenated peaks 4–6). The late-eluting fraction exhibits a small contribution from peaks 5–7, but a clear preference for the a-h peak family. The difference between the two peak families (distinguished here by Arabic numerals versus letters) is seven degrees of saturation (compare, for example, formulas for peaks 6 and c (DBE 7 and 14, respectively). These two series of peaks are termed ‘families’ here only for lack of a better term as they VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Formula Assignment for Ions Depicted in Figure 3 peak label 1 a 2 b 3 c 4 d 5 e 6 f 7 g h
in SRFA wholea
frc 105
frc 134
frc 152
455.0107 455.0257 yes yes yes yes yes yes yes yes yes yes
455.0471 455.0835
455.0619 455.0817 455.0982 455.1194 455.1346 455.1558 455.1711 455.1922 455.2075 455.2286
yes
455.0982 455.1347 455.1557 455.1711 455.1923 455.2075 455.2286 455.2439 455.2805
family scoreb
DBE - Oc
DBEd
C
H
O
z *e
accurate mass
-6 1 -6 1 -6 1 -6 1 -6 1 -6 1 -6 1 1
-3 4 -3 4 -3 4 -3 4 -3 4 -3 4 -3 4 4
12 16 11 15 10 14 9 13 8 12 7 11 6 10 9
17 21 18 22 19 23 20 24 21 25 22 26 23 27 28
12 12 16 16 20 20 24 24 28 28 32 32 36 36 40
15 12 14 11 13 10 12 9 11 8 10 7 9 6 5
-6 -6 -6 -6 -6 -6 -6 -6 -6 -6 -6 -6 -6 -6 -6
455.0103 455.0256 455.0467 455.0620 455.0831 455.0984 455.1195 455.1348 455.1559 455.1711 455.1923 455.2075 455.2287 455.2439 455.2803
a Ions previously (12) observed in an unfractionated sample collected in positive mode (1500 coadds) are identified in column 2. b Family score ) value calculated through eq 1. c DBE - O ) difference between DBE and the number of O atoms in the compound. d DBE ) double bond equivalence (i.e., the number of rings and/or double bonds). e z* ) ((remainder of nominal mass/14) - 14) as defined in ref 15.
FIGURE 4. Frequency of occurrence of formula families in each fraction. The number of formulas that fall within each family is plotted on the y-axis. Family score is calculated according to eq 1. merely represent different regions of a continuum of substitution patterns. Despite the separation into families being rather arbitrary, it is useful for tracking differences between fractions. To readily distinguish between these families, they are assigned a family score here, which was calculated following eq 1. familyscore ) 0.5z* + (DBE - O)
(1)
The lower the family score value, the smaller the DBE O. Figure 3, therefore, confirms that the early eluting fraction shows a preference for ions in which the number of O atoms is large compared to the degree of saturation. Figure 4 highlights this trend even further, by demonstrating how many ions within each fraction belong to each ion family. From Figure 4, it is evident that ions with a highly negative family score (-13) do not extend into the late eluting fraction, nor do ions with a large positive family score (+8) extend into the early eluting fraction. Between the two dominant families (family scores -6 and +1), a gradual shift from preference for the -6 family toward preference for the +1 family is evident. Figure 3 shows one example of this shift; peaks marked 1-7 have a family score of -6, and peaks marked a-h have a family score of +1. The late eluting fraction clearly contains more significant contributions from the family with the larger family score. In conclusion, the data strongly support that both goals of the RPHPLC separation were met. Fractionation on the molecular level did occur. Furthermore, the fractionation 2064
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induced changes within each m/z unit in exactly the predicted fashion. The most oxygenated ions within each nominal mass appeared in the early eluting fraction. Ions became gradually less oxygenated as retention time increased. A fortuitous and slightly unexpected result is the significant reduction in nominal mass complexity for the early eluting fractions (Figure 3a). In most cases between two and four ions were observed for any given odd-m/z segment in this fraction (vial 105). In addition, one ion was frequently dominantly abundant. Comparing parts a and b of Figure 3, for instance, it is clear that MSn data in the peak-window shown in Figure 3a would be far easier to interpret than that in the peakwindow of Figure 3b. Based on these results, it is likely that only one additional fractionation step is needed to reduce complexity in the early eluting fractions to only one dominant peak for any given m/z window. That maximum reduction in complexity occurred in the early eluting fractions is additionally fortunate, since the ions present within these fractions represent those of the smallest, most highly substituted carbon-skeletons, i.e., the ones ideal for commencing large-scale structural analysis. One final feature of the prefractionation induced here (which might initially appear as a detractor) is that some ions appear in multiple fractions. Peak 3, in Figure 3, for instance, is present in both the early eluting and the mideluting fractions. Considering the significant difference in retention time between these two fractions (5.24 min), it is likely that peak 3 in vial 105 and peak 3 in vial 134 are structural
isomers (the same is true for peaks 5, 6, and 7, which occur in vials 134 and 152). The fact that ions of identical molecular formula appear in fractions eluting in quite different regions of the chromatogram is taken as an indication that structural isomers are prevalent and that their separation can be achieved via RPHPLC, a feature of extreme relevance since mass spectrometers themselves cannot resolve isomers.
Acknowledgments Financial support was provided by the University of South Alabama (USARC and UCUR). Additional support was derived from the National High-Field FT-ICR MS Facility at the NHMFL (NSF DMR 00-84173) and its students and staff. Dr. Lewis Pannell at the USA College of Medicine Cancer Center is thanked for generous access to the ion-trap instrument (NSF-EPSCoR 0091853-353) for spray-solvent optimization. Gratefully acknowledged are Andrew Harris, Angela Dabbs, and Melissa Stringer who helped with gradient optimization, SRFA fractionation, and lyophilization; as well as Talia America and Vanessa Jarvis for their work on electrospray solvent optimization. Finally, Dr. Christopher Brummel and Eric Block at Vertex Pharmaceuticals Inc. provided the knowledge of DMSO’s ability to prevent/reduce void-volume break-through.
Supporting Information Available Overlays of raw mass spectra (Figure S1), Kendrick plots (Figure S2), zoomed carbon number plots (Figure S3), and molecular mass averages (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.
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