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Ion-Molecule Reactions for the Characterization of Polyols and Polyol Mixtures by ESI/FT-ICR Mass Spectrometry Michael A. Watkins,† Brian E. Winger,‡ Ryan C. Shea,† and Hilkka I. Kentta 1 maa*,†
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, and Eli Lilly and Company, Indianapolis, Indiana 46285
A mass spectrometric method is described for the identification and counting of hydroxyl groups in an analyte. Analytes introduced into a FT-ICR mass spectrometer and ionized by positive mode ESI were allowed to react with the neutral reagent diethylmethoxyborane. This results in derivatization of the hydroxyl groups of the analytes by replacement of a proton with a diethylborenium ion. Protonated polyols react by consecutive derivatization reactions, wherein all, or nearly all, of the hydroxyls are derivatized. The polyol derivatization products are separated by 68 mass units in the mass spectrum. This 68 Da mass shift, along with 30 Da mass shifts arising from intramolecular derivatization of the primary derivatization products, makes it easy to count the number of functional groups present in the analyte. The utility of this method for the analysis of polyols as single-component solutions, as mixtures, or in HPLC effluent (LC-MS analysis) is demonstrated. The identification of drug metabolites, degradation products, and impurities is critical in the course of drug discovery and development. Structure elucidation of these compounds sheds light on the drug’s metabolic and degradation pathways and aids the identification of the source of process impurities. This information can assist in formulation efforts, aid in selecting drug candidates, and provide information on the stability and bioavailability of a drug substance and on the potential toxicity of degradation products and metabolites.1-5 Rapid structure identification methods are important for the acceleration of the drug development process.1,2,4 The advent of electrospray ionization (ESI) has significantly increased the speed of mass spectrometric analysis of complex mixtures by providing * To whom correspondence should be addressed. E-mail:
[email protected]. † Purdue University. ‡ Eli Lilly and Co. (1) Lee, M. S.; Kerns, E. H.; Hail, M. E.; Liu, J.; Volk, K. J. LC-GC 1997, 15, 542-558. (2) Wu, Y. Biomed. Chromatogr. 2000, 14, 384-396. (3) Haskins, N. J.; Eckers, C.; Organ, A. J.; Dunk, M. F.; Winger, B. E. Rapid Commun. Mass Spectrom. 1995, 9, 1027-1030. (4) Hopfgartner, G.; Husser, C.; Zell, M. J. Mass Spectrom. 2003, 38, 138150. (5) Wolff, J.-C.; Monte´, S.; Haskins, N.; Bell, D. Rapid Commun. Mass Spectrom. 1999, 13, 1797-1802. 10.1021/ac049031t CCC: $30.25 Published on Web 01/26/2005
© 2005 American Chemical Society
a convenient method for extracting ions from solution and volatilizing them for gas-phase analysis.6,7 Exact mass measurement is commonly used to obtain the molecular formula of the protonated analytes, while tandem mass spectrometric (MSn) collision-activated dissociation (CAD) analysis is often employed to provide some insight into the elemental connectivity. However, these experiments do not allow the direct determination of the elemental connectivity and, therefore, can only provide inferences into structures of analytes. Since the functional groups present in the analyte may still be unknown, accurate assignment of the analyte’s structure may be impossible based on these data alone. Spectroscopic methods, such as NMR and X-ray crystallography, are commonly used to obtain information regarding the elemental connectivity of the analyte. However, these techniques require time-consuming isolation of mixture components prior to analysis. Further, their relatively low sensitivities have traditionally necessitated access to rather large quantities of analytes. We have shown previously that the functional groups present in simple protonated monofunctional oxygen-containing compounds can be identified via gas-phase ion-molecule reactions in a mass spectrometer.8,9 These studies revealed that protonated oxygen-containing molecules react with selected methoxyborane reagents by proton transfer, followed by a nucleophilic substitution reaction at the boron atom wherein methanol is displaced by the analyte. This reaction results in the derivatization of the functional group. The net reaction is the replacement of a proton with a borenium ion. This derivatization allows for easy identification of molecules with oxygen-containing functionalities due to the unique boron isotope ratio (24.8% 10B relative to 11B) of the derivatization (6) Cole, R. B. Electrospray Ionization Mass Spectrometry; John Wiley & Sons: New York, 1997. (7) Fenn, J. B. Presentation at the 2002 Nobel Prize Ceremony, Stockholm, Sweden, October 9, 2002. (8) Watkins, M. A.; Price, J. M.; Winger, B. E.; Kentta¨maa, H. I. Anal. Chem. 2004, 76, 964-976. (9) For additional work discussing gas-phase borane reactions, see: (a) Leeck, D. T.; Stirk, K. M.; Zeller, L. C.; Kiminkinen, L. K. M.; Castro, L. M.; Vainiotalo, P.; Kentta¨maa, H. I. J. Am. Chem. Soc. 1994, 116, 3028-3038. (b) Tho ¨lmann, D.; Gru ¨ tzmacher, H.-F. J. Am. Chem. Soc. 1991, 113, 32813287. (c) Gronert, S.; O’Hair, R. A. J. Am. Soc. Mass Spectrom. 2002, 13, 1088-1098. (d) Gao, H.; Petzold, C. J.; Leavell, M. D.; Leary, J. A. J. Am. Soc. Mass Spectrom. 2003, 14, 916-924. (e) Ren, J.; Workman, D. B.; Squires, R. R. J. Am. Chem. Soc. 1998, 120, 10511-10522. (f) Murphy, M. K.; Beauchamp, J. L. J. Am. Chem. Soc. 1976, 98, 1433-1440. (g) Gronert, S.; Huang, R. J. Am. Chem. Soc. 2001, 123, 8606-8607. (h) Suming, H.; Yaozu, C.; Longfei, J.; Shuman, X. Org. Mass Spectrom. 1985, 20, 719723.
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product. Further analysis of the derivatized molecules by H/D exchange, CAD, or both provides the identity of the oxygencontaining functional group. This report describes the extension of the above method to polyols and to LC-MS. Herein we describe the ion-molecule reaction-assisted structure elucidation of polyols in singlecomponent solutions, in a multicomponent mixture, and in HPLC effluent (LC-MS analysis). EXPERIMENTAL SECTION All experiments were performed in a Finnigan MAT (Bremen, Germany) TQ-80 dual-cell FT-ICR mass spectrometer which was equipped with an 8-T superconducting magnet (Intermagnetics General Corp., Latham, NY), an Odyssey data station, an ESI source (Analytica of Branford, New Haven, CT), an rf hexapole for ion accumulation, a pulsed valve assembly for introduction of reagents, and a Waters 2690 separations module for LC-MS experiments. A detailed description of this instrument has been presented previously.10 The ESI source allows for ions to be generated external to the magnetic field while the rf hexapole serves as a linear ion trap, allowing for ion accumulation and collisional cooling (hexapole region pressures are ∼10 mTorr). Once a sufficient ion population had been accumulated (accumulation time of 0.3-1 s), the ion packet was axially ejected from the hexapole and guided into the ICR cell by a series of high-voltage focusing optics. Once trapped in the ICR cell, the ions were allowed to react with a selected neutral reagent that was pulsed into the cell via the pulsed valve assembly (∼(1-5) × 10-7 Torr peak nominal pressure in the cell). Multiple reagent pulses (up to 5) were required to produce the highest order reaction products. A mass spectrum (consisting of 20 averaged transients) was collected for the reaction of each protonated polyol by using 0, 1, 3, and 5 reagent pulses. These spectra were then summed to generate a single mass spectrum that displays the protonated analyte and all of its derivatization products. Each reaction mass spectrum was background subtracted by a spectrum collected using identical conditions except that the polyol ion was ejected from the cell prior to the introduction of the neutral reagent. Since the polyol ions were generated in an external ion source, only one cell of this dual-cell instrument was utilized for ion-molecule reactions and detection. Stock solutions of each polyol were prepared at 1 mg/mL concentrations in 50:50 (% v/v) acetonitrile/water. Prior to analysis, each stock solution was diluted 100 fold with 50:50 (% v/v) acetonitrile/0.2% (vol) HCl to prepare 10 µg/mL solutions for single-component and mixture analysis. These samples were infused directly into the ESI source by a syringe pump at flow rates of 5-10 µL/min. For LC-MS analysis, a 1 mg/mL solution of the sample was prepared in 85:15 (% v/v) acetonitrile/water with 0.2% (vol) formic acid. An Agilent Zorbax Stable Bond (5 µm, 4.6 × 250 mm) CN column and a Waters 2690 separations module were utilized for the LC-MS analysis. A 25-µL aliquot of sample was injected into the column with a mobile phase (85:15 (% v/v) acetonitrile/water with 0.2% (vol) formic acid) flow rate of 1 mL/min and was split 1/20 postcolumn prior to the ESI source, resulting in a flow rate of ∼50 µL/min. Three polyols commonly used as drug excipients were chosen for this study. The polyols selected are meso-erythritol (Aldrich (10) Winger, B. E.; Kemp, C. A. J. Am. Pharm. Rev. 2001, 4, 55-63.
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Chemical Co., Milwaukee, WI), xylitol (Aldrich Chemical Co.), and D-mannitol (ICN Biomedicals, Aurora, OH). These compounds were ionized by positive ion ESI, which produced singly protonated ([M + H]+) polyols. These ions were allowed to react with the neutral reagent diethylmethoxyborane. The number of exchangeable hydrogen atoms in the protonated analyte and in its derivatization products was determined by preparing the analytes in fully deuterated solvents (with the exception of acetonitrile due to its lack of exchangeable protons) and subsequently allowing the [dn-(M + D)]+ (where n represents the number of exchangeable hydrogens) to react with diethylmethoxyborane in the mass spectrometer. All theoretical energies reported in this work were calculated with the Gaussian 98 suite of programs.11 The proton affinity (PA) of diethylmethoxyborane was calculated at the BLYP/6-311G(d,p) level of theory by using protonated methanol as the Brønsted acid in an isodesmic reaction scheme. All polyol calculations were performed at the BLYP/3-21G(d) level of theory and also utilized protonated methanol as the Brønsted acid in isodesmic reaction schemes. All stationary points were verified by vibrational frequency analysis to possess zero imaginary frequencies. All theoretical energies are presented at 0 K and include zero point vibrational energy corrections. RESULTS AND DISCUSSION Single-Component Studies. Erythritol. Protonated erythritol reacts with diethylmethoxyborane in the FT-ICR mass spectrometer by a proton transfer/nucleophilic substitution reaction resulting in a 68 Da mass increase. The proposed mechanism (Scheme 1; note: only one possible site of protonation and derivatization is shown) involves the transfer of a proton from erythritol to the basic methoxy group of diethylmethoxyborane. Prior to dissociation of the gas-phase collision complex, one of the hydroxyl group oxygens in erythritol acts as a nucleophile and adds into the vacant p-orbital of the boron of diethylmethoxyborane. This newly formed adduct dissociates into methanol and a derivatized erythritol ion. The net reaction observed is the replacement of a proton of erythritol with a diethylborenium ion. This reactivity is analogous to that previously observed for monofunctional oxygen-containing compounds.8 The presence of multiple hydroxyl functionalities allows for multiple derivatization reactions to take place (each reaction product is separated by 68 Da from the others). In fact, if sufficient reaction time is provided (reagent at static pressure) or after multiple reagent pulses (1-5 pulses at ∼(1-5) × 10-7 Torr peak nominal pressure), all of the hydroxyl groups in protonated erythritol are derivatized (Figure 1). While this reaction proceeds, the abundances of the lower order reaction products decrease as those of the higher order reaction products increase. This is (11) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Buran, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, Revision A.9; Pittsburgh, PA, 1998.
Figure 1. Reaction of protonated erythritol with diethylmethoxyborane. All functional groups are derivatized. Each reaction product is separated by 68 Da and incorporates the unique boron isotope pattern (24.8% 10B relative to 11B). Isotope ratio analysis confirms the number of boron atoms incorporated and therefore the number of functional groups.
Scheme 1
consistent with a consecutive reaction mechanism. The secondary, and higher order, reaction products are generated by transfer of the acidic proton of the derivatized analyte to another diethylmethoxyborane reagent, which, while still in the collision complex, reacts with another hydroxyl functional group (Scheme 1; note: the exact order in which the different sites are derivatized is not known). This consecutive reaction continues until all of the functional groups are derivatized. Counting the number of de-
rivatization products generated by this reaction reveals the number of hydroxyl groups present in the analyte polyol. The incorporation of a boron atom in each reaction product facilitates their identification in the mass spectrum and provides confirmation of the number of derivatized functional groups (via isotope ratio analysis). For example, since the first derivatization product contains one boron atom, the 10B isotope abundance is ∼25% of the 11B isotope abundance, while the second derivatization product Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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Scheme 2
contains two boron atoms, which results in a 10B isotope abundance of ∼50% of the 11B isotope abundance. The third derivatization product contains three boron atoms, which leads to a 10B isotope abundance that is ∼75% of the 11B isotope abundance, while the fourth product has four boron atoms and has a 10B isotope abundance that is nearly equal to that of the 11B isotope abundance (see Figure 1). In addition to the observation of derivatization product formation, it was also found that some of the derivatization products are capable of one or more unimolecular dissociation reactions resulting in the loss of one or more ethane molecules, respectively (see Figure 1). Similar observations have been made for the reaction of protonated 2-hydroxymethyltetrahydropyran with trimethylborate.12 This reaction was found to proceed by proton transfer/nucleophilic substitution wherein methanol is lost. The derivatization product of this reaction was found to decompose by loss of another methanol molecule, resulting in the formation of what is believed to be a bicyclic product. The unimolecular dissociation of the protonated polyol derivatives may follow a similar mechanism (Scheme 2). Similar dissociations have been reported for the reaction of hydroxide with cyclic boronate esters where the adduct formed dissociates by the loss of methane or ethane.13 Determining the number of labile hydrogen atoms that can be exchanged for deuterium atoms in a molecule provides information about the functional groups present. For example, ketones, ethers, and esters do not have readily exchangeable hydrogen atoms; however, alcohols and carboxylic acids do. Therefore, H/D exchange experiments serve to limit the list of possible candidates for an unknown chemical functionality by eliminating functional groups that do not match with experimental H/D exchange results. Similarly, determining the number of exchangeable hydrogen atoms in derivatization products can give insight into the structure of the product by providing the identity of the derivatized functional group (and hence of the analyte).8 In accordance with the proposed derivatization mechanism (Scheme 1), one exchangeable hydrogen will be lost to the methanol byproduct when each hydroxyl functionality is derivatized. Therefore, if the [M + D]+ of the perdeuterated analyte is allowed to react with diethylmethoxyborane until all functionalities are derivatized, and one deuterium is lost during each derivatization reaction, the identity of all derivatized functional groups will be known to be hydroxyl. Dissolving erythritol in completely deuterated solvents (D2O and DCl; nondeuterated acetonitrile was used due to its lack of (12) Somuramasami, J.; Duan, P.; Watkins, M. A.; Winger, B. E.; Kentta¨maa, H. I. In preparation. (13) Kiplinger, J. P.; Crowder, C. A.; Sorensen, D. N.; Bartmess, J. E. J. Am. Soc. Mass Spectrom. 1994, 5, 169-176.
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exchangeable hydrogens) results in the exchange of all hydroxyl hydrogen atoms for deuterium atoms. ESI forms the [d4-(M + D)]+ ion in the mass spectrometer. The number of deuterium atoms lost during the formation of each reaction product can be determined by comparing the mass spectra obtained for the reaction of the perdeuterated (Figure 2) and the nondeuterated (Figure 1) analyte with diethylmethoxyborane. For example, Figure 1 shows the m/z value of the first reaction product to be 191, while in Figure 2, the first derivatization product has an m/z value of 195. The four Da mass shift indicates that four deuterium atoms are incorporated in the first derivatization product in Figure 2. Furthermore, the observation of the loss of one exchangeable deuterium for each derivatization reaction positively identifies each functional group as a hydroxyl based on the proposed reaction mechanism (Scheme 1). This finding is consistent with previous experiments carried out on monofunctional analytes.8 It should be noted here that the lower mass isotope peaks observed for each reaction product in the mass spectrum are due to the exchange of deuterium atoms back to hydrogen atoms from ubiquitous proton sources at various locations in the mass spectrometer, e.g., in the atmospheric pressure interface, etc. (most likely due to residual solvent molecules arising from the use of ESI). Xylitol and Mannitol. Protonated xylitol and mannitol were found to react with diethylmethoxyborane much like protonated erythritol. The similarities include a consecutive reaction sequence resulting in the derivatization of multiple hydroxyl functionalities and dissociation of the derivatized ions via ethane losses. However, unlike erythritol, it was found that the reactions of xylitol and mannitol with diethylmethoxyborane result in incomplete derivatization; all but one of the functional groups were derivatized in each (Figure 3). The incomplete derivatization phenomenon was examined via density functional theory calculations (BLYP/3-21G(d) +ZPVE). Theory predicts that the derivatization products of xylitol and mannitol can fold up, providing solvation for the acidic hydrogen that must be transferred to a diethylmethoxyborane molecule in order to promote the derivatization of the last hydroxyl. This intramolecular proton solvation was found to reduce the acidity of the ion to the point that the proton transfer/nucleophilic substitution reaction is no longer favorable. For example, the acidity of xylitol’s third derivatization product is calculated to be 203.3 kcal/mol while that of the fourth derivatization product is calculated to be 236.6 kcal/mol. Previous studies have shown that diethylmethoxyborane is only capable of reacting by proton transfer/nucleophilic substitution with analytes that have PAs at or below ∼212 kcal/mol.8 Therefore, the third derivatization product of xylitol can react with a diethylmethoxyborane molecule
Figure 2. Reaction of perdeuterated erythritol with diethylmethoxyborane. Each reaction product has one less exchangeable deuteron than its lower order precursor, just as predicted by the reaction mechanism proposed for polyols. This finding confirms that all functional groups are hydroxyl groups.
to produce the fourth derivatization product, but the acidity of the fourth derivatization product is too low (236.6 kcal/mol) for the proton transfer/nucleophilic substitution reaction to occur. Hence, the fifth derivatization product is not observed. Although not all of the functional groups are derivatized by diethylmethoxyborane, the number of functional groups present in the analyte can still be determined by counting the consecutive ethane losses arising from intramolecular derivatization of derivatization products (see Figure 3). For example, the third derivatization product of xylitol undergoes two unimolecular dissociation reactions (forming the ions of m/z 327 and 297), which reveals the presence of two additional functional groups (one can be determined to be a hydroxyl, the other can be inferred to be a hydroxyl; vide infra). The third and fourth derivatization products of mannitol undergo three (m/z 357, 327, and 297) and two (m/z 425 and 395) unimolecular dissociation reactions revealing the presence of three and two additional functional groups, respectively. These results indicate that there are six functional groups present in the analyte (five can be determined to be a hydroxyl, one can be inferred to be a hydroxyl; vide infra). Furthermore, analysis of the abundance of the 10B isotope peak relative to 11B can be used to determine the number of boron atoms incorporated into each dissociation product (vide supra). Therefore, the number of functional groups in these analytes can be accurately assigned based on the number of inter- and intramolecular derivatization products. As for erythritol, reactions of the perdeuterated xylitol and mannitol can be used to confirm the identity of the functional groups to be hydroxyl. Due to incomplete derivatization, the identity of all but the last derivatized functional group can be
determined to be hydroxyl. Although the mechanism proposed for the intramolecular derivatization reactions requires the last functional group to be a hydroxyl, the identity of the functional group cannot be directly determined and therefore can only be inferred to be hydroxyl. Multicomponent Mixture Studies. A recent trend in highresolution mass spectrometry is to carry out direct mass spectrometric analysis of mixtures without prior chromatographic separation.14 This method has proven to be effective, for example, in identifying thousands of different species in extremely complex petroleum samples. Similarly, this work seeks to bypass chromatographic separation in order to increase the speed and ease of analysis of mixtures while providing more information about molecular structure (i.e., the identity and number of the functional groups) than is available from traditional ESI-MS analysis. The functional group enumerating and hydroxyl group elucidating ion-molecule reactions discussed above can also be applied to mixtures. The information obtained from the analysis of multiple analytes isolated from a mixture can be acquired by the direct (14) For some examples, see: (a) Marshall, A. G.; Hendrickson, C., L.; Shi, S. D.-H. Anal. Chem. 2002, 74, 252A-259A. (b) Hughey, C. A.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2002, 74, 4145-4149. (c) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G.; Qian, K. Anal. Chem. 2001, 73, 4676-4681. (d) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G. Energy Fuels 2001, 15, 1186-1193. (e) Petzold, C. J.; Leavell, M. D.; Leary, J. A. Anal. Chem. 2004, 76, 203-210. (f) Miyabayashi, K.; Naito, Y.; Miyake, M.; Tsujimoto, K. Eur. J. Mass Spectrom. 2000, 6, 251-258. (g) Alomary, A.; Solouki, T.; Patterson, H. H.; Cronan, C. S. Environ. Sci. Technol. 2000, 34, 2830-2838. (h) Brown, T. L.; Rice, J. A. Anal. Chem. 2000, 72, 384-390. (i) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. Org. Geochem. 2002, 33, 171-180.
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Figure 3. Reaction of (a) xylitol and (b) mannitol with diethylmethoxyborane. Although not all of the functional groups are derivatized by the diethylmethoxyborane reagent, the number of functional groups can be determined by counting the consecutive ethane losses from derivatization products.
analysis of the mixture. To demonstrate this, a mixture containing erythritol, xylitol, and mannitol was prepared and directly infused into the ESI source for subsequent ionization for mass spectrometric analysis. The [M + H]+ of each mixture component was observed and was allowed to react with the neutral diethylmethoxyborane reagent. Derivatization reaction products were observed for each of the mixture components. The resulting mass spectrum (Figure 4a) provides the same information as the spectra measured for the individual components. Functional group identification can be achieved in the same way as for single-component analysis by dissolving the polyol mixture in deuterated solvents 1390
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(vide supra) and subsequently allowing the perdeuterated polyols to react with diethylmethoxyborane. The number of exchangeable hydrogens in each reaction product (Figure 4b) was determined as for the single-component analysis. LC-MS/Ion-Molecule Reaction Experiment. Although the ion-molecule reaction-assisted structure elucidation method described here is designed to be used without prechromatographic separation, it may be advantageous in some situations to apply this method to LC-MS analysis. To ascertain the feasibility of coupling LC-MS and ion-molecule reaction analysis, a sample containing a single polyol, erythritol, was analyzed. The width of
Figure 4. Direct infusion ESI of a solution of erythritol (E), xylitol (X), and mannitol (M). Spectrum a shows that all derivatization products observed in single-component solution analysis are observable in the three-component mixture without prechromatographic separation. Peak labels indicate the analyte and how many functional groups have been derivatized. For example, E-4 indicates that the derivatized analyte is erythritol and four functional groups have been derivatized. Spectrum b shows the reaction of deuterated analytes with diethylmethoxyborane. The number of incorporated deuterons in each derivatization product reveals the identity of the functional groups. Peak labels indicate the derivatized analyte and how many deuterons are incorporated. For example, (E)-2D indicates that the derivatized analyte is erythritol and that the derivatization product has two deuterons incorporated.
the erythritol chromatographic peak was determined by conventional LC-MS (total ion current data (not shown) collected at a rate of ∼1 Hz; 1 scan/s) to be ∼21 s (FWHM). The [M + H]+ generated by ESI of the effluent was injected into one cell of the dual-cell FT-ICR and was subsequently probed by ion-molecule reactions. For each scan, two consecutive pulses of diethylmethoxyborane (1 × 10-7 Torr peak nominal pressure) were
introduced into the ICR cell while collecting data at ∼0.14 Hz (1 scan was acquired every 7 s), which provides enough time for the ion-molecule reactions to take place and for the borane reagent to be evacuated prior to detection. A maximum of two MS scans could be taken during the elution of erythritol. The LC-ESI/ion-molecule reaction mass spectrum (Figure 5) acquired during the erythritol elution is analogous to that Analytical Chemistry, Vol. 77, No. 5, March 1, 2005
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Figure 5. LC-ESI/ion-molecule reaction analysis. The feasibility of coupling LC-MS and ion-molecule reaction analysis was established by the successful derivatization of all of the functional groups of erythritol ionized by ESI as it eluted from an HPLC column. This mass spectrum is a single LC-ESI/ion-molecule reaction MS scan acquired during the elution of erythritol and is analogous to that collected from direct infusion ESI/ion-molecule reaction MS analysis.
collected without HPLC (direct infusion; see Figure 1). Hence, the feasibility of coupling LC-MS and ion-molecule reaction analysis was demonstrated by the successful derivatization of all of the functional groups in protonated erythritol as it eluted from the HPLC column. Substantially faster data acquisition than described here can be obtained by optimizing the experiment, e.g., by using a higher boron reagent pressure and both cells of the dual-cell FT-ICR for the experiment.
groups present in an unknown analyte. This ion-molecule reaction approach for functional group identification, along with exact mass measurement and tandem CAD, holds promise for rapid and complete structure elucidation of unknown molecules directly in mixtures by using mass spectrometry. Future work will focus on applying this methodology to other, more commonly available (i.e., triple quadrupole and quadrupole time-of-flight), mass spectrometers and to expand this method for the identification of a wider variety of functional groups.
CONCLUSIONS Ion-molecule chemistry in a mass spectrometer is a powerful tool that can be used to provide detailed information about the structures of unknown molecules. The method presented here for polyol analysis provides more information about the functional groups in the molecule than exact mass measurements and tandem mass spectrometric CAD methods. Unlike exact mass and tandem CAD methods, ion-molecule reaction-based methods provide direct evidence for the type and number of functional
ACKNOWLEDGMENT The authors gratefully acknowledge Eli Lilly and Co. for financial support of this work. The authors also thank Dr. Steven W. Baertschi of Eli Lilly for useful discussions and guidance during this project.
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Received for review July 1, 2004. Accepted November 19, 2004. AC049031T
