Identification and Counting of Oxygen Functionalities in Aromatic

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Identification and Counting of Oxygen Functionalities in Aromatic Analytes Related to Lignin by Using Negative-Mode Electrospray Ionization and Multiple Collision-Activated Dissociation Steps Lucas M. Amundson,† Ryan J. Eismin,† Jennifer N. Reece,† Mingkun Fu,‡ Steven C. Habicht,§ Allan B. Mossman,|| Ryan C. Shea,|| and Hilkka I. Kentt€amaa*,† †

Department of Chemistry, Purdue University, Brown Building, 560 Oval Drive, West Lafayette, Indiana 47907, United States Millennium, 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States § CNA, 4825 Mark Center Drive, Alexandria, Virginia 22311, United States British Petroleum, 150 West Warrenville Road, Naperville, Illinois 60563, United States

)

‡

ABSTRACT: Identification and counting of different oxygen-containing functional groups in 40 small aromatic analytes, including a lignin monomer, was explored using a linear quadrupole ion trap (LQIT) mass spectrometer. The analytes were evaporated and ionized by negative-mode electrospray ionization (ESI). In an effort to cleave off all of the functionalities, one at a time, the deprotonated analytes were then subjected to multiple consecutive collision-activated dissociation (CAD) events until no more fragmentation was observed (up to MS5). In most cases, the number and types of functionalities could be determined. This approach was demonstrated to be feasible on the high-performance liquid chromatographic (HPLC) time scale. Hence, valuable structural information can be obtained for previously unknown aromatic analytes directly in complex mixtures, such as lignin degradation products.

’ INTRODUCTION Development of biofuels has become increasingly important in the search for alternatives to the use of fossil fuels. In fact, the U.S. Department of Energy is planning to replace 30% of liquid petroleum transportation fuel with renewable biofuels by the year 2025.1 Currently, biomass supplies over 3% of the total energy consumption in the U.S., and it recently surpassed hydropower as the largest domestic source of renewable energy.2 The ability to characterize the molecular composition of biomass degradation products and biofuels is crucial for the rational development of biofuels. Tandem mass spectrometry (MS/MS) is a uniquely powerful method for complex mixture analyses because it allows for the direct molecular-level characterization of mixture components without isolation. This capability, coupled with the many additional beneficial characteristics of MS/MS, such as high specificity, sensitivity, and speed, has resulted in its wide use in applications in pharmaceutical, environmental, biological, and petroleum fields.3 The coupling of gas chromatography (GC) or high-performance liquid chromatography (HPLC) with MS/MS provides an even more powerful tool for mixture analysis.4 Despite all of these advances, the unambiguous identification of previously unknown compounds directly in mixtures is often problematic when using only mass spectrometry. The interest in the development of biofuels from lignin degradation products has created a need to be able to structurally characterize previously unknown aromatic compounds with several oxygen-containing functionalities directly in complex mixtures. Unfortunately, oxygen-containing compounds often do not produce stable protonated molecules, which hinders their analysis using traditional experiments, such as positive-mode electrospray ionization (ESI).5 Derivatization reactions have been employed to modify oxygenr 2011 American Chemical Society

containing functional groups, so that stable protonated molecules can be formed in ESI.6 These derivatization reactions rely on the modification of a targeted functional group to enable its ionization without dissociation in mass spectrometry, which is not always feasible for analysis of unknown mixtures.7 Further, the derivatization step is time-consuming, and the reaction itself may be incomplete, yield unwanted products, and not derivatize all analytes equally efficiently. In contrast to positive-ion ESI, many oxygen-containing analytes can be efficiently ionized via negative-mode ESI because of their acidity.8 This eliminates the problems associated with derivatization. The studies reported thus far include using negative-mode ESI for the detection of aromatic and aliphatic carboxylic acids in drug metabolites and environmental toxins.7c,8b,9 In these experiments, the analytes were first separated by HPLC and then ionized by negative-mode ESI. Upon collision-activated dissociation (CAD), the negatively charged analyte molecules fragmented by losing CO2 and, in some cases, H2O as well. In 2007, an extensive study was published on the fragmentation of various types of deprotonated (and protonated) analytes upon CAD in MS/MS experiments (mostly MS2).10 This study covered 121 small organic compounds (mainly aromatic). From these compounds, 28 had only oxygen-containing functionalities, including up to two carboxylic acid, hydroxyl, or keto functionalities. While several of these functionalities could be identified in deprotonated monofunctional compounds, only one functionality, COOH, could be conclusively identified in polyfunctional analytes.10 Furthermore, several oxygen compounds, Received: January 25, 2011 Revised: April 9, 2011 Published: April 15, 2011 3212

dx.doi.org/10.1021/ef200141w | Energy Fuels 2011, 25, 3212–3222

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Table 1. ESI/CAD Tandem Mass Spectraa Obtained for Analytes with at Least One Carboxylic Acid Functionality

a

Product ions with abundances of at least 5% of the total product ions’ abundance are listed; the most useful products are in bold. Consecutive CAD and fragment ion isolation steps were repeated until no more fragment ions were observed.

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dx.doi.org/10.1021/ef200141w |Energy Fuels 2011, 25, 3212–3222

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Table 2. ESI/CAD Tandem Mass Spectraa Obtained for Carboxylic Acids with Benzoyl Functionalities

a

Product ions with abundances of at least 5% of the total product ions’ abundance are listed; the most useful products are in bold. Consecutive CAD and fragment ion isolation steps were repeated until no more fragment ions were observed. b Proposed product composition for ion/molecule reaction products involving water.

including alcohols, phenols, and aldehydes, could not be ionized in the negative-ion mode. A later study found HPLC/negative-mode ESI/MS3 mass spectrometry to yield useful information for the major bonding structures that are encountered in lignins, including dimers (dilignols), trimers, and higher order oligomers (oligolignols).11 However, only limited structural information was obtained for the aromatic subunits themselves, likely because only two consecutive CAD events were performed.11

To address the limitations of the above studies and to expand the general understanding of fragmentation pathways of oxygencontaining aromatic anions, we have probed the utility of HPLC/ negative-mode ESI coupled with multi-stage MS/MS (continued until no more fragmentation was observed) for the identification and counting of many different types of oxygen-containing functionalities in small aromatic analytes. A total of 40 such analytes were examined using a commercial linear quadrupole ion trap (LQIT). 3214


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Scheme 1

’ EXPERIMENTAL SECTION Sample Preparation. Analytes readily formed stable deprotonated molecules in solutions of 50:50 (v/v) and 20:80 (v/v) methanol and HPLC-grade water, with the exception of analytes containing hydroxy groups. These compounds only formed stable deprotonated molecules in a 20:80 (v/v) solution of methanol and HPLC-grade water but not in as high abundance as the analytes containing other functionalities. Test compounds were prepared in a 50:50 (v/v) solution of methanol and HPLC-grade water. While analytes in a 50:50 (v/v) solution were ionized by negative-mode ESI, a 20:80 (v/v) solution of methanol and HPLC-grade water was used to optimize the ionization efficiencies of some of the analytes. Mass Spectrometry. The experiments were carried out using a Thermofischer Scientific LTQ linear quadrupole ion trap (LQIT) mass spectrometer equipped with an ESI source. The LQIT was operated using the LTQ Tune Plus interface and Xcalibur 2.0 software. A pressure from 0.52 105 to 0.58 105 Torr was maintained within the LQIT, and helium was used as the buffer gas. Sample solutions were prepared with analyte concentrations from 0.01 to 1 mg/mL (105103 M) in either a 50:50 (v/v) solution of H2O and CH3OH or a 20:80 (v/v) solution of H2O and CH3OH. The solutions were introduced into the instrument via the LQIT syringe drive (10 μL/min), followed by ionization with negativemode ESI. Typical ESI conditions were a spray voltage of 4.55 kV, a sheath gas (N2) flow of 10 (arbitrary units), and a capillary temperature of 275 °C. Ion optic voltages were optimized for each analyte using the tune function of the LTQ Tune Plus interface. Collision-Activated Dissociation. Using the advanced scan feature of the LTQ Tune Plus interface, all analyte ions formed were isolated with a m/z window of 23 Th and a q value of 0.25 and then subjected to CAD using helium. The activation time was 30 ms. Collision energies were varied from 20 to 30% of the “normalized collision energy”. All of the formed fragment ions were subjected to further isolation/CAD events, until no further fragmentation was observed. For mixture analysis, high performance liquid chromatography was used for separation prior to online mass spectrometric analysis. Xcalibur 2.0 software was used for both data acquisition and processing. All mass spectra shown are an average of 10 spectra. Fragmentation products with abundances of above 5% of the total ion abundance are reported. Liquid Chromatography. All HPLC experiments were performed using a Surveyor HPLC system, consisting of an autosampler, thermostatted column compartment, quaternary pump, and photodiode

Figure 1. (a) Negative-ion mass spectrum of 4-hydroxymethylbenzoic acid obtained after ESI, isolation of the deprotonated analyte, and subjection to CAD shows one CO2 loss. (b) Subsequent isolation of the fragment ion and CAD resulted in one CH2O loss. array (PDA) detector. Analytes were separated on a Waters Xbridge C18 column (150 2.1 mm inner diameter, 3.5 μm particle size). The mobile phases used in the gradient separation were a mixture of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A linear gradient mixture of 5% B and 95% A to 70% B and 30% A over 30 min was used as the mobile phase. The flow rate was 0.2 mL/min, with an injection volume of 10 μL. The PDA detector was set to acquire a single channel at a wavelength of 254 nm. Chemicals. Chemicals were either purchased from Sigma-Aldrich or provided by British Petroleum, and they were used without further purification. HPLC-grade water was purchased from Burdick and Jackson, and HPLC-grade methanol and acetonitrile were purchased from Mallinckrodt.

’ RESULTS AND DISCUSSION Ionization Reactions. A total of 40 aromatic (and a few aliphatic) analytes containing various oxygen functionalities were ionized via negative-mode ESI and subjected to consecutive CAD events in a LQIT mass spectrometer. As opposed to the earlier report on negative-mode ESI,10 wherein hydroxyl- and most aldehyde-functionality-containing analytes could not be ionized, all such compounds studied here were ionized by ESI to give deprotonated molecules with high enough abundances to isolate them and examine their CAD reactions. Furthermore, as opposed to this earlier study,10 structurally informative fragment ions were obtained for all of the oxygen-containing aromatic 3215


Energy & Fuels analytes examined (with the exception of phenol). This may be partially due to the fact that the earlier study was mostly limited to MS2, while in the current study, the ion isolation and CAD experiments were continued until no fragment ions were observed. Fragmentation Reactions. All of the deprotonated monoand bifunctional carboxylic acids were found to fragment exclusively by loss of a carboxylic acid functionality as CO2, as expected10 (Table 1). When the fragment ions of the bifunctional carboxylic acids were subjected to a second CAD event, the second carboxylic acid functionality was eliminated as CO2 (Table 1). The same was observed for trifunctional carboxylic acids: all of the three carboxylic acid functionalities were removed as CO2 molecules upon three consecutive CAD events (Table 2).

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Interestingly, the relative location of the carboxylic acid groups did not matter. Hence, the most obvious mechanism involving spatially proximate carboxylic acid moieties, shown in Scheme 1a, cannot rationalize all of these losses. Scheme 1b shows a more likely mechanism involving charge-remote fragmentation.12 This mode of fragmentation has been reported for many negative ions; it appears to be more prevalent for negative than positive ions.13 The charge-remote loss of the first CO2 molecule, as opposed to the second, is shown in Scheme 1b because this loss would yield a more stable ionic product (an oxygen anion instead of a phenide ion). Calculations at the B3LYP/aug-cc-pVDZ// B3LYP/aug-cc-pVDZ level of theory revealed that the final product, the biphenyl anion (Scheme 1b), is not likely to

Table 3. ESI/CAD Tandem Mass Spectraa Obtained for Analytes with at Least One Keto, Hydroxy, or Aldehyde Functionality

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Table 3. Continued

a

Product ions with abundances of at least 5% of the total product ions’ abundance are listed; the most useful products are in bold. Consecutive CAD and fragment ion isolation steps were repeated until no more fragment ions were observed. b Proposed product composition for ion/molecule reaction products involving air.

undergo ring closure to form a delocalized carbanion because the cyclization is endothermic by 21.7 kcal/mol. The loss of the carboxylic acid moieties as CO2 also occurred upon CAD of deprotonated carboxylic acids with other oxygen functionalities (Table 2). Hence, the carboxylic acid moiety appears to be easy to identify, as reported in the literature.10 However, information on the other functionalities was obtained after all of the carboxylic acid moieties had been cleaved off. For example, the hydroxymethyl group of 4-hydroxymethylbenzoic acid was lost as CH2CO (Figure 1 and Table 3) after the loss of the carboxylic acid moiety as CO2. Again, the relative locations of the substituents seem not to matter. Hence, charge-remote fragmentations are probably involved. Observations made for functionalities other than the carboxylic acid are discussed in detail below. Deprotonated benzophenones (with carboxylic acid functionalities) lose their carbonyl functionalities as CO after the cleavage of all carboxylic acid functionalities as CO2 (Table 2). In most cases, the number of the carbonyl groups is revealed by the number of CO losses. CO loss was also observed for an unsaturated lactone (Table 2) and for one aldehyde (Table 3). In contrast, the earlier study reported the loss of CO also for deprotonated carboxylic acids.10 Acetyl groups can be lost as CH2CO (Figure 2 and Table 3). Loss of a CH3 or CH3O group is indicative of methoxy functionalities (Tables 3 and 5). Hydroxymethyl groups are lost as CH2O. The number of these groups is revealed by the number of CH2O losses (Table 3). The observation that the loss of the first CH2O molecule from deprotonated 2,20 -biphenylmethanol yields an ion that fragments almost identically to the 2-biphenylmethanol

Figure 2. Negative-ion mass spectrum of 4-hydroxyacetophenone obtained after ESI, isolation of the deprotonated analyte, and subjection to CAD shows CH2CO loss and CH3 loss.

(both lose CH2O and C6H6 with branching ratios of 78 and 22%, and 64 and 36%, respectively; Table 3) indicates that the first CH2O group was probably lost via a charge-remote fragmentation mechanism, as shown in Scheme 2. No fragmentation patterns characteristic to the phenol functionality were observed. In addition to only oxygen-containing analytes, also a few nitrogen-containing analytes were examined (Table 4). Aniline did not fragment under these conditions. The nitro substituent in the two nitro compounds studied cleaved off as NO. 3217


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Table 4. ESI/CAD Tandem Mass Spectraa Obtained for Analytes with Nitrogen-Containing Functionalities

a

Product ions with abundances of at least 5% of the total product ions’ abundance are listed. Consecutive CAD and fragment ion isolation steps were repeated until no more fragment ions were observed.

Table 5. ESI/CAD Tandem Mass Spectraa Obtained for Analytes with Methyl Groups

a

Product ions with abundances of at least 5% of the total product ions’ abundance are listed; the most useful products are in bold. No fragment ions were observed after the first CAD event.

Deprotonated fluorenones with carboxylic acid functionalities behaved similarly as the other aromatic compounds discussed above in that they first lose all of the carboxylic acid functionalities as CO2 molecules (Table 6). However, the identification of their carbonyl groups was not possible because the ions formed upon the loss of all of the carboxylic acid functionalities do not

fragment further. The reasons for this behavior are not clear at this time. Finally, several aliphatic carboxylic acids were investigated (Table 7). With the exception of 2-methylvaleric acid, a loss of H2O was observed for all of the aliphatic carboxylic acids upon the first CAD event, as expected.10 The methyl group in 3218


Energy & Fuels 2-methylvaleric acid may sterically hinder proton transfer from the alkyl chain to the hydroxyl group, which is necessary for H2O loss. All of this behavior differs drastically from that of the aromatic carboxylic acids that only exhibit CO2 loss upon CAD (Table 1). It is obvious that this analytical approach is much more valuable for aromatic than aliphatic analytes. Examination of a Lignin Monomer, Coniferyl Alcohol. Coniferyl alcohol was ionized as described above, and the deprotonated analyte was subjected to multiple ion isolation and CAD experiments (Scheme 3). The most facile fragmentation involved the loss of water (for a likely11 mechanism involving an intermediate ion/molecule complex,13 see Scheme 4), which is indicative of the presence of an aliphatic hydroxyl functionality.11 The loss of a methyl group (for a likely mechanism,11 see Scheme 4) revealed the presence of a methoxy group. After these reactions, the loss of CO occurred. On the basis of the results presented here, this reaction erroneously indicates the presence of a carbonyl group. Hence, characterization of even more model compounds is indicated. The lack of losses of

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CH2O, CH2CO, CO2, and NO reveal the lack of hydroxymethyl, acetyl, carboxylic acid, and nitro functionalities. Mixture Analysis. A mixture of four compounds (Figure 3) containing oxygen functionalities was examined to test the feasibility of this experimental approach on the chromatographic time scale. All four compounds were separated using HPLC prior to mass spectrometric analysis. As the components eluted from HPLC, they were successfully ionized by negative-mode ESI, isolated, and subjected to multiple CAD and ion isolation events. Scheme 3

Scheme 2

Table 6. ESI/CAD Tandem Mass Spectraa Obtained for Analytes with a Fluorenone Functionality

a

Product ions with abundances of at least 5% of the total product ions’ abundance are listed; the most useful products are in bold. The consecutive CAD and fragment ion isolation steps were continued until no more fragment ions were observed. 3219


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Table 7. ESI/CAD Tandem Mass Spectraa Obtained for Analytes with Aliphatic Alcohol Functionalities

a

Products corresponding to at least 5% of the total product ions’ abundance are listed; the most useful products are in bold. No fragment ions were observed after the first CAD event.

Scheme 4

The expected characteristic fragmentations were observed for all compounds (for an example, see Figure 4).

’ CONCLUSION The CAD fragmentation patterns obtained in MSn experiments for 40 deprotonated aromatic oxygen-containing analytes (evaporated and ionized via negative-mode ESI) are shown to facilitate the identification and counting of different oxygencontaining functionalities, including carboxylic acid, hydroxymethyl, acetyl, keto, aldehyde, methoxy, aliphatic hydroxy, and nitro groups. The loss of all of the carboxylic acid functionalities exclusively as CO2 molecules almost always occurs before the loss of other functionalities. Hence, multiple CAD events are usually needed to identify all functionalities in multiply substituted analytes. Most carbonyl groups are lost as CO. Most hydroxymethyl functionalities are lost as CH2O. Most nitro 3220


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The above experiments involved analytes with as many as four functionalities and, hence, MS/MS up to MS5. For more complex analytes, further MS stages are probably needed. Despite the need for multiple consecutive CAD and ion isolation events, these experiments were demonstrated to be fast enough to be applicable to HPLC/ion trap mass spectrometry. The analytical methodology discussed here will facilitate the structural characterization of previously unknown aromatic analytes directly in complex mixtures, such as lignin degradation products. Application of this methodology to the characterization of the products obtained upon catalytic hydrogenation and fast pyrolysis of lignin is underway.

’ AUTHOR INFORMATION Corresponding Author Figure 3. Analytes selected for mixture analysis: (a) 9-fluorenone-1carboxylic acid, (b) 2-(hydroxymethyl)anthraquinone, (c) 2,40 ,5-tricarboxybenzophenone, and (d) diphenyl-4,40 -dicarboxylic acid.

*Telephone: (765) 494-0882. Fax: (765) 494-0239. E-mail: [email protected].

’ ACKNOWLEDGMENT Dr. John Nash is acknowledged for the computational data. The authors gratefully acknowledge British Petroleum for partial financial support of this work. Jennifer N. Reece was supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center, funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award DE-SC0000997. ’ REFERENCES

Figure 4. Negative-ion mass spectra measured for one of the mixture components, 2,40 ,5-tricarboxybenzophenone, after HPLC separation, ESI, isolation of the deprotonated analyte (m/z 313), and four consecutive CAD and fragment ion isolation events. (a) First CAD after the isolation of the deprotonated 2,40 ,5-tricarboxybenzophenone results in CO2 loss (m/z 269) and a minor loss of two CO2 molecules (m/z 225). (b) Second and (c) third CAD events for the isolated primary and secondary fragment ions of m/z 269 and 225, respectively, show a second (m/z 225) and third (m/z 181) CO2 loss, respectively, and the (d) final CAD event of the fragment ion of m/z 181 shows the final CO loss (m/z 153). The ion of m/z 197 is a CO2 adduct of the ion of m/z 153.

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