Identification of the Carboxylic Acid Functionality by Using

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Anal. Chem. 2008, 80, 3416-3421

Identification of the Carboxylic Acid Functionality by Using Electrospray Ionization and Ion-Molecule Reactions in a Modified Linear Quadrupole Ion Trap Mass Spectrometer Steven C. Habicht, Nelson R. Vinueza, Enada F. Archibold, Penggao Duan, and Hilkka I. Kentta1 maa*

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907

A mass spectrometric method has been developed for the identification of the carboxylic acid functional group in analytes evaporated and ionized by electrospray ionization (ESI). This method is based on gas-phase ion-molecule reactions of ammoniated ([M + NH4]+) and sodiated ([M + Na]+) analyte molecules with trimethyl borate (TMB) in a modified linear quadrupole ion trap mass spectrometer. The diagnostic reaction involves addition of the deprotonated analyte to TMB followed by the elimination of methanol. A variety of analytes with different functionalities were examined, and this reaction was only observed for molecules containing the carboxylic acid functionality. The selectivity of the reaction is attributed to the acidic hydrogen present in the carboxylic acid group, which provides the proton necessary for the elimination of methanol. The diagnostic products are easily identified based on the m/z value of the product ion, which is 72 Th (thomson) greater than the m/z value of the charged analyte, and also by the characteristic isotope pattern of boron. The applicability of this method for pharmaceutical analysis was demonstrated for three nonsteroidal anti-inflammatory drugs: ibuprofen, naproxen, and ketoprofen. The ability to rapidly identify unknown degradation products and impurities in drugs is of great importance to the pharmaceutical industry.1-7 Since such impurities may arise from a variety of sources (e.g., change in manufacturing conditions, physical contamination, drug instability), analytical techniques that can rapidly provide information-rich data are integral to these structural elucidation processes.2 Mass spectrometry (MS) has proven to be a powerful tool in mixture analysis, due to its high sensitivity, * Corresponding author. Phone: 765-494-0882. Fax: 765-494-0239. E-mail: [email protected]. (1) Ahuja, S. Impurities Evaluation of Pharmaceuticals; Marcel Dekker, Inc.: New York, 1998. (2) Van Rompay, J. J. Pharm. Biomed. Anal. 1986, 4, 725-732. (3) Kassel, D. B. Chem. Rev. 2001, 101, 256-268. (4) Lee, M. S. LC/MS Applications in Drug Development; John Wiley and Sons, Ltd.: New York, 2002. (5) Krstulovic, A. M.; Lee, C. R.; Firmin, S.; Jacquet, G.; Van Dau, C. N.; Tessier, D. LC‚GC Eur. 2002, 15, 31-41. (6) Deng, G.; Sanyal, G. J. Pharm. Biomed. Anal. 2006, 40, 528-538. (7) Prakash, C.; Shaffer, C. L.; Nedderman, A. Mass Spectrom. Rev. 2007, 26, 340-369.

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specificity, and speed. With the onset of atmospheric pressure ionization methods, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), high-performance liquid chromatography (HPLC) coupled with MS has become a valuable tool for pharmaceutical analyses.4-8 However, this technique only allows for the determination of the molecular weights and elemental compositions of unknown analytes, thus leaving questions about the structural details of the analytes unanswered. Tandem mass spectrometry has provided a partial solution to the problem discussed above. With the use of tandem mass spectrometry, analytes ionized, for example, by ESI, can be separated in the first mass analysis stage and then subjected to collision-activated dissociation (CAD) in order to obtain structural information.4,7,9 However, these experiments usually cannot provide unambiguous information on the elemental connectivity of unknown analytes, and thus only inferences can be drawn about the structure of the analytes.7 Gas-phase ion-molecule reactions of neutral boron compounds with protonated analytes were recently demonstrated to facilitate the identification and counting of various oxygen functional groups in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer.10-12 The general mechanism proposed for the diagnostic reaction of trimethyl borate (TMB) with a protonated oxygen-containing analyte is shown in Scheme 1. The products formed in these reactions are easy to identify due to the unique isotope ratio of boron (25% 10B relative to 11B). Although useful, this method is limited to analytes that can be ionized to form stable protonated molecules. Many common oxygen-containing compounds do not produce stable protonated molecules when ionized by positive mode ESI, including carboxylic acids. Hence, these compounds are often analyzed by using negative mode ESI, which is successful due to their acidic nature.13-15 Unfortunately, analysis of complex mix(8) Thomson, B. A. J. Am. Soc. Mass Spectrom. 1998, 9, 187-193. (9) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry; VCH Publishers: New York, 1988. (10) Watkins, M. A.; Price, J. M.; Winger, B. E.; Kentta¨maa, H. I. Anal. Chem. 2004, 76, 964-976. (11) Watkins, M. A.; Winger, B. E.; Shea, R. C.; Kentta¨maa, H. I. Anal. Chem. 2005, 77, 1385-1392. (12) Somuramasami, J.; Duan, P.; Watkins, M. A.; Winger, B. E.; Kentta¨maa, H. I. Int. J. Mass Spectrom. 2007, 265, 359-371. 10.1021/ac800002h CCC: $40.75

© 2008 American Chemical Society Published on Web 03/26/2008

Scheme 1

tures by using negative mode ESI is complicated by the fact that reversed-phase HPLC separations often rely on the use of acidic mobile phase additives to improve chromatographic resolution, which tends to suppress negative ion formation.16 Therefore, derivatization reactions have been employed to modify carboxylic acid groups so that they will form stable protonated molecules via positive mode ESI.17,18 However, these reactions can be timeconsuming and may result in the formation of unwanted side products. Although carboxylic acids do not generally form stable protonated molecules upon ESI, they often form abundant adducts with ammonium, sodium, and other cations. This inspired us to examine the utility of gas-phase reactions of TMB with the ammonium and sodium adducts of oxygen-containing analytes generated by ESI. The experiments were carried out using a commercially available linear quadrupole ion trap mass spectrometer (LTQ) that was modified to allow the introduction of neutral reagents into the ion trap via an external mixing manifold. EXPERIMENTAL SECTION Mass Spectrometry. The experiments were performed in a Finnigan LTQ linear quadrupole ion trap (LTQ) mass spectrometer equipped with an ESI source. Sample solutions were prepared at analyte concentrations of 0.01 to 1 mg/mL (10-5 to 10-3 M) in either a 50/50 (v/v) solution of H2O and CH3OH or a 50/50 (v/v) solution of 10 mM aqueous ammonium acetate and CH3OH. The solutions were directly infused into the ESI source at 3-5 µL/min by using an integrated syringe drive. Typical ESI conditions were as follows: spray voltage, 4.5-5 kV; sheath gas (N2) flow, 10 (arbitrary units); capillary temperature, 275 °C. Voltages for the ion optics were optimized for each analyte by using the tune feature of the LTQ Tune Plus interface. Ion-Molecule Reactions. To allow introduction of neutral reagents into the helium buffer gas line, an external reagent mixing manifold was designed based on the apparatus described by Gronert19,20 for use with the Finnigan LCQ quadrupole ion trap (13) Schug, K.; McNair, H. M. J. Sep. Sci. 2002, 25, 760-766. (14) Schug, K.; McNair, H. M. J. Chromatogr., A 2003, 985, 531-539. (15) Bandu, M. L.; Watkins, K. R.; Bretthauer, M. L.; Moore, C. A.; Desaire, H. Anal. Chem. 2004, 76, 1746-1753. (16) Jemal, M.; Ouyang, Z.; Teitz, D. S. Rapid Commun. Mass Spectrom. 1998, 12, 429-434. (17) Cartwright, A. J.; Jones, P.; Wolff, J. C.; Evans, E. H. Rapid Commun. Mass Spectrom. 2005, 19, 1058-1062. (18) Lamos, S. M.; Shortreed, M. R.; Frey, B. L.; Belshaw, P. J.; Smith, L. M. Anal. Chem. 2007, 79, 5143-5149.

Figure 1. Schematic of the external mixing manifold used to examine ion-molecule reactions in the linear ion trap mass spectrometer.

mass spectrometer. A diagram of the manifold is shown in Figure 1. The neutral reagent (TMB) was introduced into the manifold via a syringe drive at 30-60 µL/h where it was diluted with a known flow of helium (100-500 mL/min). The syringe port and surrounding area of the manifold was heated to ∼70 °C to ensure rapid evaporation of TMB into the flow of helium. In the standard operating mode, the appropriate flow of helium into the trap is maintained by use of a flow splitter. For the experiments discussed here, this flow splitter was bypassed to allow control over the concentration of the reagent introduced into the trap. The He/reagent mixture was split before entering the trap by the use of two Granville-Phillips leak valves; one was set to allow ∼2 mL/min of the mixture to enter the trap to establish a helium pressure of ∼3 mTorr,21 while the other was used to control the flow of that part of the mixture that was diverted to waste. On the basis of the relative flow rates of the TMB and helium, and accounting for differential effusion from the trap, the typical pressure of TMB present during the experiments is estimated as 10-7 to 10-6 Torr.22 After experiments were completed each day, the manifold was isolated from the LTQ and placed under vacuum to minimize contamination of the helium line. The experiments were performed using the advanced scan features of the LTQ Tune Plus interface. The analyte ion was isolated using an m/z window of 2-3 Th and a Q value of 0.25, then allowed to react with TMB for 10 ms to 10 s before being ejected from the trap and detected. CAD experiments involved isolation of the reaction product by using an m/z window of 4-6 m/z Th and the application of an appropriate activation voltage (generally 10-30% “normalized collision energy”, as defined by the LTQ Tune Plus interface) for 30 ms. Xcalibur 2.0 software was used for both data acquisition and processing. All mass spectra shown are an average of at least 10 scans. (19) Gronert, S. J. Am. Soc. Mass Spectrom. 1998, 9, 845-848. (20) Gronert, S. Mass Spectrom. Rev. 2005, 24, 100-120. (21) Schwartz, J. C.; Senko, M. W.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2002, 13, 659-669. (22) Adapted from ref 20: pressure(TMB) ) P(He)[flow(TMB)/flow(He)] xMW(TMB)/MW(He) where P(He) is the nominal helium buffer gas pressure in the ion trap, flow(TMB) is the molar flow rate of TMB, flow(He) is the molar flow rate of helium, MW(TMB) is the molecular weight of TMB, and MW(He) is the molecular weight of helium.

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Figure 2. Structures of the simple analytes used in this study.

Figure 4. Positive ion mass spectra of (a) 6-hydroxy-2-hexanone, (b) ethyl butanoate, and (c) hexanoic acid obtained after ESI, isolation of the sodiated analyte, and exposure to TMB. No reactions were observed for sodiated 6-hydroxy-2-hexanone and ethyl butanoate. However, incorporation of TMB in the reaction product observed for sodiated hexanoic acid (c) is confirmed by the unique boron isotope ratio (inset).

Figure 3. Positive ion ESI mass spectra of ibuprofen dissolved in a 50/50 (v/v) solution of methanol and (a) HPLC grade water or (b) 10 mM aqueous ammonium acetate. Peak intensities of [M + NH4]+ and [M + Na]+ are given in arbitrary units.

Chemicals. HPLC grade water was purchased from Burdick & Jackson, and HPLC grade methanol was purchased from Mallinckrodt. All other chemicals used were purchased from Sigma-Aldrich and used without further purification. RESULTS AND DISCUSSION Method Development. Several simple analytes containing various oxygen functionalities (Figure 2) were ionized via positive mode ESI in the LTQ to examine what sorts of charged species are formed. Test solutions of each compound were prepared in a 50/50 (v/v) solution composed of methanol and either HPLC grade water or 10 mM aqueous ammonium acetate. Ammonium acetate was used to facilitate production of adduct ions. Analytes containing basic functionalities, such as ketones and amides, were 3418 Analytical Chemistry, Vol. 80, No. 9, May 1, 2008

found to readily form protonated molecules in either solvent system. However, analytes with less basic functionalities, such as carboxylic acids and alcohols, predominantly formed adduct ions, including ammoniated ([M + NH4]+) and sodiated ([M + Na]+) analyte molecules, even when the H2O/CH3OH solution was used. When the buffered solution was used, these analytes formed very abundant adduct ions. An example is shown in Figure 3. Addition of ammonium acetate into the solvent system resulted in a substantial increase in the intensity of the peaks corresponding to adduct ions (over 20 times increase for [M + NH4]+ and almost 5 times increase for [M + Na]+) for ibuprofen. Thus, the remainder of the experiments dealing with adduct ions were carried out by using this solvent system. Following optimization of the ESI conditions, the protonated, ammoniated, and sodiated analytes were isolated and allowed to react with TMB. The expected diagnostic product ion (formed by addition followed by elimination of methanol) was observed for all protonated analytes, in agreement with previous results

Scheme 2

Scheme 3

obtained using an FT-ICR mass spectrometer.12,23 In sharp contrast, almost all of the sodiated and ammoniated analytes were found to be unreactive toward TMB (within the maximum tested reaction time of 10 s; this is the maximum allowed by the software). However, analytes containing a carboxylic acid functional group formed the diagnostic product ion within a reaction time of 500 ms. The diagnostic product ions have an m/z value 72 Th higher than that of the positively charged analytes, consistent with the product being an adduct of the analyte and the dimethoxyborenium ion. As an example, Figure 4 shows the positive ion mass spectra obtained after the reactions of TMB with three isomeric sodiated analytes: 6-hydroxy-2-hexanone, ethyl butanoate, and hexanoic acid. No reaction products were observed for the first two sodiated analytes, whereas the last one, sodiated hexanoic acid, yielded a product ion of m/z 206, corresponding to addition to TMB followed by loss of methanol. This product ion was easily identified in the mass spectrum due to the unique boron isotope ratio (25% 10B relative to 11B; see inset of Figure 4c). The product ion was observed for both the ammoniated and the sodiated analytes containing a carboxylic acid group. Diagnostic Reactions of TMB. The diagnostic reaction of TMB with protonated oxygen-containing analytes is likely initiated by deprotonation of the analyte by a methoxy group in TMB (Scheme 1).12 This is followed by nucleophilic addition of the analyte to the boron in TMB and elimination of a methanol molecule. This mechanism is supported by the fact that the reaction does not occur for the ammoniated and sodiated analytes other than carboxylic acids since only sodiated/ammoniated (23) Somuramasami, J. Ph.D. Thesis, Purdue University, West Lafayette, IN, 2007.

Figure 5. (a) Positive ion mass spectrum obtained for the monomethyl ester of succinic acid after ESI, isolation of the ammonium adduct, and exposure to TMB. (b) CAD mass spectrum obtained for the isolated ion-molecule reaction product (m/z 222). Ammonia loss dominates to yield a fragment ion of 205. The minor fragment ion observed at m/z 133 corresponds to the protonated monomethyl ester of succinic acid and was not useful in determining the site of derivatization. (c) CAD mass spectrum obtained for the isolated fragment ion formed by loss of ammonia (m/z 205). The loss of HOB(OCH3)2 from the fragment ion confirms that the diagnostic reaction occurs at the carboxylic acid functionality.

carboxylic acids have an acidic hydrogen that they can donate to TMB (Scheme 2). It should be noted here that ammoniated and sodiated analytes with an ester functionality also formed the diagnostic product ion, albeit only very slowly (observable after very long (>5 s) reaction times). This is partially explained by the slightly acidic hydrogens present at acyl R-carbons (Scheme 3) that have been exploited in reactions used to synthesize esters, such as Claisen condensation.24 These hydrogens are much less acidic than the OH group of the carboxylic acid, which accounts for the substantially slower reaction. The absence of this reaction for benzoate esters with no hydrogens at the acyl R-carbon provides support for the (24) Carey, F. A. Organic Chemistry, 4th ed.; McGraw-Hill: New York, 2000.

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Figure 7. CAD mass spectra obtained for (a) the isolated fragment ion of ammoniated naproxen (m/z 231) and (b) the isolated fragment ion of the ion-molecule reaction product of ammoniated naproxen (m/z 303). The only fragment ion in both spectra has the same m/z ratio.

Figure 6. Positive ion mass spectra obtained for (a) ibuprofen, (b) naproxen, and (c) ketoprofen after ESI, isolation of the ammonium adduct of the analyte, and reaction with TMB. The region of each mass spectrum indicating the formation of a product ion has been magnified for clarity.

proposed mechanism (Scheme 3). Further, although many aldehydes and ketones contain hydrogens at the acyl R-carbon position that are more acidic than those of esters,24 the reaction was not observed for ammoniated or sodiated aldehydes and ketones. Indeed, on the basis of the proposed mechanism (Scheme 3), these ions should not undergo the reaction since they lack a nucleophilic site that is required for the second step of the reaction, addition of the deprotonated analyte to the boron atom in TMB. To probe whether the above methodology allows the differentiation of carboxylic acids from esters, two compounds were examined that contain both the carboxylic acid and ester functionalities. The two compounds are the monomethyl ester of succinic acid and the monomethyl ester of phthalic acid. Both analytes formed abundant ammonium and sodium adducts when ionized by positive ion ESI. The ammoniated analytes were isolated for further examination. The mass spectrum obtained after the reaction of the isolated ammonium adduct of the monomethyl ester of succinic acid with TMB is shown in Figure 5. The analyte formed the anticipated product ion consistent with the addition of the ammoniated deprotonated analyte to the TMB molecule 3420 Analytical Chemistry, Vol. 80, No. 9, May 1, 2008

followed by the loss of methanol. Also shown in Figure 5 are the MS2 and MS3 CAD mass spectra measured for this diagnostic product ion. The MS2 experiment demonstrated that the ionmolecule reaction product ion fragments predominately by the loss of NH3. In the MS3 experiment, this fragment ion was isolated and subjected to CAD. The biggest peak in the MS3 spectrum corresponds to loss of the HOB(OCH3)2 molecule. Assuming that CAD did not cause isomerization of the fragmenting ion, this reaction can only occur if the boron moiety was initially attached to the carboxylic acid group in the analyte (a loss of ROB(OCH3)2, where R ) the O-alkyl chain of the ester, would be expected if the boron moiety was attached at the ester group). The TMB reaction product of the ammonium adduct of the monomethyl ester of phthalic acid behaved in a similar manner and also lost the HOB(OCH3)2 molecule when subjected to CAD. It is concluded that the reaction of TMB with the carboxylic acid functionality in these bifunctional analyte adducts occurs so much faster than with the ester functionality that carboxylic acids can be differentiated from esters by using this method. Nonsteroidal Anti-Inflammatory Drugs. Three nonsteroidal anti-inflammatory drugs (ibuprofen, naproxen, and ketoprofen) were examined to test the validity of this method for the identification of the carboxylic acid functionality in more complex analytes. All three analytes formed intense ammonium and sodium adducts upon positive mode ESI. For all three compounds, the expected diagnostic product ion was observed upon reaction of TMB with both the ammoniated and sodiated analytes. Mass spectra obtained after the reaction of the ammoniated analytes with TMB are shown in Figure 6. A peak corresponding to the diagnostic product ion is present in each spectrum at an m/z value 72 Th higher than that of the ammoniated analyte. Since ibuprofen only contains one oxygen functionality, the boron moiety must be attached to this functionality in the diagnostic product ion. However, both naproxen and ketoprofen

contain an additional oxygen functional group (ether and keto, respectively) and hence could form two isomeric borenium ion adducts. In order to probe the structures of the borenium ion adducts of the ammoniated analytes, CAD experiments were performed. For both drugs, the expected ammonia loss dominated, analogous to the results shown in Figure 5b. For naproxen, isolation of this fragment ion followed by CAD (i.e., MS3) yielded a fragment ion of m/z 185. The fragment ion of m/z 185 does not display the characteristic boron isotope pattern. Further, this fragment ion has the same m/z value as that obtained by loss of HCOOH when ammoniated naproxen is subjected to CAD (see Figure 7). Therefore, formation of the fragment ion of m/z 185 upon CAD of the ammonia-loss fragment ion of the diagnostic product of naproxen involves the loss of dimethoxyborenium ion with the carboxylate group. Similar results were obtained when ketoprofen was subjected to the same experimental sequence, confirming the carboxylic acid as the site of attachment of the boron moiety.

cation adducts (an example mass spectrum for the reaction of a potassium adduct, [M + K]+, with TMB has been included in the Supporting Information). The method was shown to be specific for the carboxylic acid functionality even in the presence of additional oxygen-containing functional groups. Successful detection of the carboxylic acid functionality in ibuprofen, naproxen, and ketoprofen, demonstrates the applicability of this method to pharmaceuticals. The short time needed for the diagnostic reaction should make this methodology suitable for LC-MS applications.

CONCLUSIONS A specific gas-phase ion-molecule reaction of neutral TMB with ammoniated and sodiated analytes (generated via positive mode ESI) has been shown to be diagnostic for the carboxylic acid functionality. This is most likely due to the requirement of an acidic hydrogen in the analyte ion to initiate the diagnostic reaction. Thus, although this methodology was only demonstrated for ammoniated and sodiated adducts, it may be applied to other

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT The authors gratefully acknowledge the National Science Foundation for financial support of this work. The authors thank Dr. David G. Harman and Dr. Stephen J. Blanksby for helpful discussions regarding the design of the reagent mixing manifold. S.C.H. acknowledges fellowship support from the Department of Education through the Graduate Assistantships in Areas of National Need (GAANN) program.

Received for review January 1, 2008. Accepted February 18, 2008. AC800002H

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