Influence of Ionization Source Conditions on the Gas-Phase Protomer Distribution of Anilinium and Related Cations
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The gas-phase-ion generation technique and specific ion-source settings of a mass spectrometer influence heavily the protonation processes of molecules and the abundance ratio of the generated protomers. Hitherto that has been attributed primarily to the nature of the solvent and the pH. By utilizing electrospray ionization and ion-mobility mass spectrometry (IM-MS), we demonstrate, even in the seemingly trivial case of protonated aniline, that the protomer ratio strongly depends on the source conditions. Under low in-source ion activation, nearly 100% of the N-protomer of aniline is produced, and it can be subsequently converted to the C-protomer by collisional activation effected by increasing the electrical potential difference between the entrance and exit orifices of the first vacuum region. This activation and transformation process takes place even before the ion is mass-selected and subjected to IM separation. Despite the apparent simplicity of the problem, the preferred protonation site of aniline in the gas phase—the amino group or the aromatic ring—has been a topic of controversy. Our results not only provide unambiguous evidence that ring- and nitrogen-protonated aniline can coexist and be interconverted in the gas phase, but also that the ratio of the protomers depends on the internal energy of the original ion. There are many dynamic ion-transformation and fragmentation processes that take place in the different physical compartments of a Synapt G2 HDMS instrument. Such processes can dramatically change the very identity even of small ions, and therefore should be taken into account when interpreting product-ion mass spectra.
KeywordsProtomers Anilinium ion Protonated aniline Ion mobility Tautomerization
The question of protonation sites of gas-phase aromatic compounds has been the subject of extensive studies [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12]. For many mass spectrometric (MS) investigations, protonated gas-phase molecules are generated under ambient conditions by electrospray (ESI), atmospheric-pressure, or in vacuo chemical and plasma ionization processes . In the process of ionization, the charge-imparting proton can sometimes attach to different sites, thus forming a mixture of several isomeric tautomers [9, 14]. We prefer to designate such prototropic isomers as protomers, with the caveat that the term has been used previously in the entirely different context of structural biology to name distinct protein chains that form a larger hetero-oligomer .
Many previous investigations have addressed whether the protonation regio-selectivity of a given gaseous molecule formed from sprayed droplets directly reflects the situation in the original bulk liquid; however, the subject remains highly debatable [16, 17, 18, 19, 20, 21]. Generally, the preferred protonation site of a polyfunctional molecule can be predicted by computational methods . Nevertheless, even for a simple molecule such as aniline, theoretical studies can often be inconclusive in predicting an unequivocal protonation site [22, 23, 24, 25, 26, 27]. In computational studies, the final predictions are strongly dependent on the levels of theory and the basis sets of functions employed [25, 28]. It has been noted that some protonated species formed under certain MS ionization conditions are not always the thermodynamically most favored species . This is not unexpected because usually species investigated under MS conditions are not in their lowest energy states. Sometimes, kinetically controlled protonation could take place at a less preferred site due to steric, thermal, or other factors [18, 19, 20, 21, 29, 30]. Whatever the case may be, different protomers of the same molecule can undoubtedly coexist in the gas phase .
Although not much attention has been paid to post-ionization isomerization processes that could take place upon collisional activation of mass-selected ions, as early as 1993 Bakhtiar et al reported that the Si(C2H5)H2 + ion could isomerize to Si(CH3)2H+ upon collisional activation .
Extensive experimental and theoretical studies have been carried out to determine the factors influencing the abundance ratio of protomers formed during positive-ion-generating ESI . The general consensus is that the protic or aprotic nature of the solvent, the ionic strength and pH of the solution, and the solution-based chemical equilibrium that exists between the base and its conjugate acid determine the manifested protomer ratio . On the other hand, the existence of protomers has been noted even under atmospheric-pressure chemical ionization (APCI) conditions . Because analytes are often sprayed as solutions, the nature of the solvent has been regarded as the major factor that controls the ratio of protomers, even under APCI conditions.
For tandem MS, ions generated in an ion source are often isolated by a quadrupole filter and transferred to a collision cell, where they are subjected to collision-induced dissociation (CID). In a Synapt G2 HDMS instrument, an ion mass-selected by the quadrupole analyzer is transferred to a Triwave cell consisting of three traveling-wave assemblies. Ions are briefly accumulated in the Trap region, and released to a traveling-wave ion-mobility (TWIM) cell via a chamber filled with helium (Supplementary Figure S1) . Ions separated by IM then pass a Transfer cell and undergo acceleration by the pusher before they are mass-separated by the time-of-flight (ToF) analyzer. A product-ion mass spectrum can be generated by subjecting an ion to CID either in the Trap or the Transfer region. Generally, when MS fragmentation experiments are conducted, the experimental conditions that are primarily tuned are the collision-cell parameters. Customarily, when ion-fragmentation pathways are deliberated, one specific structure is selected to represent the precursor ion. The selected structure is generally the lowest-energy configuration predicted by computational methods. Although it is well known that ions undergo activation and fragmentation while being transferred from the atmospheric-pressure source area to the high-vacuum analyzer region , the possibility that ions could also experience conformational or structural isomerization during this transfer process has not been sufficiently appreciated. Such transformations occur particularly in the first low-vacuum region of a mass spectrometer (Supplementary Figure S1). If isomerizations occur prior to the ion isolation and CID, the strong possibility exists that the precursor ion can be represented by several distinct structures. In other words, a CID spectrum recorded in this way may well be that of a composite from several isomeric precursor ions.
There have been many attempts to design artificial intelligence (AI) programs to automate the interpretation of CID spectra in order to identify compounds [37, 38]. However, if the definitive structure of the precursor ion is uncertain, many spectra currently compiled in databases could well be regarded as composites representing mixtures of protomers (or deprotomers). In this respect, AI programs currently available for the interpretation of CID spectra are clearly inadequate for the unambiguous identification of compounds.
Révész et al. have noted that diastereomeric interconversion of some Tröger bases can be caused by activation in the in the ESI ion source . We recently reported that negative-ion-generating ESI source conditions exert a dramatic effect on the tautomer distribution of deprotonated p-hydroxybenzoic acid in the gas phase . Herein, we report a similar effect on the abundance ratio of the protomers derived from aniline and related compounds under positive-ion ESI-MS.
All chemicals, including aniline, p-toluidine, p-aminophenol, and p-fluoroaniline were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and used without further purification. Ultrapure water was obtained from a Milli-Q purification system (Millipore Corporation, Bedford, MA, USA). All test solutions (50 ppm) were prepared in 50:50 v/v acetonitrile:water containing 1% formic acid.
Spectra were acquired on a Synapt G2 HDMS instrument (Waters, Milford, MA, USA) equipped with an ESI, an ASAP, or a HePI source. For ion-mobility (IM) experiments, the ions of interest were mass-selected and transferred to the Triwave cell, where they were briefly accumulated in the Trap region, and released to the TWIM cell . After the IM separation, the ions passed through the Transfer cell and underwent acceleration by the pusher before they were mass-separated by the time-of-flight analyzer. Unless otherwise specified, all IM separation experiments were carried out under the following typical instrumental conditions: Trap collision energy 4 eV, Transfer collision energy 2 eV, IM wave velocity 1500 m/s, IM wave height 40.0 V, scroll-pump pressure 3.13 mbar, source pressure 1.36 × 10−3 mbar, helium-cell pressure >14.1 mbar, IM-cell pressure 4.45 mbar (N2), TOF analyzer pressure 9.68 × 10−7 mbar, Trap pressure 4.49 × 10−2 mbar (Ar), and Transfer pressure 4.87 × 10−2 mbar (Ar).
The general source conditions for electrospray-ionization (ESI) experiments were as follows: capillary voltage 2.55 kV, extraction cone 3.0 V, desolvation-gas flow rate 477 L/h, sample infusion flow rate 15 μL/min, and Vernier-probe-adjuster position 5.92 mm. The source- and desolvation-gas temperatures were held at 80 and 100 °C, respectively. To investigate individual effects, the magnitude of the sampling-cone voltage was varied between 10 and 70 V while other parameters were kept constant.
For the ASAP-APCI experiment, a glass tube smeared with aniline was inserted to the probe. Spectra were recorded under the following conditions: corona voltage 5.0 kV, extraction cone 3.0 V, desolvation-gas flow rate 477 L/h, Vernier-probe-adjuster position 5.92 mm, probe temperature 100 °C, source 80 °C, and desolvation-gas temperature 100 °C.
For helium-plasma ionization (HePI) , a stream of high-purity helium at a flow rate of about 30 mL/min was passed through a stainless steel capillary needle (100 μm i.d.) held at an electrical potential of +3.67 kV. Other instrument parameters for the HePI experiment were as follows: extraction cone 3.0 V, desolvation-gas flow rate 477 L/h, and Vernier-probe-adjuster position 5.92 mm. The source- and desolvation-gas temperatures were held at 80 and 100 °C, respectively. To investigate sampling-cone activation effects, the voltage was varied between 10 and 120 V while other parameters were kept constant. For atmospheric-pressure ion generation, a few milligrams of pure sample in a 2 mL vial was placed near the cone entrance and the capillary tip within the ion-source chamber.
All calculations were done using Gaussian 09 . Geometries for all species proposed for the fragmentation pathways were fully optimized by the B3LYP  method (Becke’s three-parameter nonlocal-exchange functional with the nonlocal correlation functional of Lee et al. ), using a large 6-311++G(2d,2p) basis set. Frequency calculations were performed at the same level to verify the nature of each stationary state on the potential-energy surface. Reactants/products were associated with all positive vibrational frequencies, and transition states (TS) were associated with only one imaginary frequency, for which the normal vibrational mode corresponded to the expected bond formation/breaking movements in a specific reaction pathway. Gibbs free energies were calculated for room temperature and ambient-pressure conditions.
Results and Discussion
For ESI-MS analysis, gaseous ions generated under atmospheric-pressure ESI conditions are transferred through several modular units of independently pumped physical compartments maintained under different vacuum conditions, after which the ions are subjected to mass analysis in the high-vacuum region. The first low-vacuum compartment, immediately after the atmospheric-pressure section, has an entrance nozzle (sampling cone entrance) and an exit orifice (extraction cone) (Supplementary Figure S1). An electrical potential difference is maintained between these two orifices to increase the ion transmission efficiency (often called the sampling-cone voltage) . It is known that in addition to increased transfer efficiency, the ions gain acceleration and endure vibrational activation due to collisions between themselves and the residual gaseous molecules in this region [36, 45, 46, 47].
However, we noticed that the ratio of the two species is highly dependent on the ion-source parameters. In particular, the sampling-cone voltage exerts a profound influence on the protomer ratio. For example, the ATD recorded from the m/z 94 ion generated at a sampling-cone voltage setting of 10 V showed essentially one peak – for the second-to-arrive N-protomer (Figure 1a). Upon gradual increase of the sampling-cone voltage, a dramatic increase of the abundance of the first-to-arrive ion was observed. In fact, our cone-voltage-varying experiments showed that the N-protomer is the dominant species when the cone-voltage difference is relatively low (<30 V) (Figure 1d). However, as the cone voltage was gradually raised, the formation of the C-protomer became more favored. At a setting of 70 V, we observed a ratio of 1:1.1. At about 100 V, the only peak in the arrival-time profile was that at 5.55 ms (Figure 1c). Since the abundance of the C-protomer observed at lower voltages is much lower than that in the ratio reported by Lalli et al. , it is evident that the 1:5 ratio should not be considered as either intrinsic or constant for the aniline protomers. We propose that the N-protomer undergoes a conversion to the C-protomer in the supersonic expansion region due to post-ionization collisional isomerization.
Previously, Kaufmann et al.  reported that the IM arrival-time profiles of protomers of norfloxacin change when the cone-voltage settings were varied from low to high values. However, such changes had been attributed to differences in the fragmentation rates of the individual protomers.
Although not as dramatic as that instigated by the sampling-cone voltage, a similar change in the protomer ratio was also noted when the extraction-cone voltage was increased (Supplementary Figure S2). This less significant effect was understandable because far fewer neutral molecules, needed for collisional activation, make their way through the extraction-cone (skimmer) orifice.
From our observations, it is evident that the dissimilarities in the spectra are primarily attributable to the variations in the protomer abundances brought about by changes in the source conditions, rather than by the changes ensuing from differences in fragmentation rates. For example, the 5.55 ms arrival-time peak in Figure 1b, which was recorded at a sampling-cone setting of 70 V, represents a composite mixture of both the precursor (m/z 94) and its product ions generated by fragmentation (primarily, the m/z 93 ion). However, the fragmentation of the precursor, mediated by the sampling-cone voltage, is not the fundamental reason for the changes of the intensity ratios of the IM peaks at 5.55 ms and 6.38 ms. To support this proposition, we extracted absolute signal intensities of the m/z 94 ion from the IM profile data recorded from protonated aniline at different sampling-cone voltages (Supplementary Figure S5). Clearly, the intensity of the m/z 94 peak at 5.55 ms (Supplementary Figure S5, B1-B10) increases as the sampling-cone voltage is raised, demonstrating the increasing formation of C-protonated aniline as the cone voltage is raised. The peak-intensity increase is evident even if we disregard the contributions due to fragmentation (Supplementary Figure S5, C1–C10). Intensity profiles normalized to a scale of 100 showed that the intensity of the m/z 93 peak can be used as a diagnostic marker for the C-protomer (Supplementary Figure S6). Although the fragmentation rates of the two protomers are different (Supplementary Figure S4), it is the tautomerization effected by ion activation in the source that is primarily responsible for the dissimilarities in the overall fragmentation spectra. The m/z 93 ion, which originates from the C-protomer, can be formed in the Trap or the Transfer region of the Triwave cell by activation of the m/z 94 ion (Supplementary Figure S4). An arrival-time profile recorded from the IM-separated mass-selected m/z 93 ion (C6H7N+●, the molecular ion of aniline) generated by HePI at a sampling-cone voltage of 70 V from a sample of aniline placed in the source, showed that C6H7N+● ion bears the same mobility as that of the C-protomer (Supplementary Figure S7); in fact, the 20 eV Transfer-cell fragmentation spectrum was comparable to the electron-ionization library spectrum of aniline. Thus, if the m/z 93 ion is generated in the trap region from the C-protomer, it will co-elute with the m/z 94 ion of the undissociated C-protomer, thereby generating a composite mobility peak (Figure 1b).
The induced in-source formation of C-protonated aniline can also be indirectly demonstrated even without the need of an IM separation: with increasing sampling-cone voltage, the abundance of the C-protomer-specific fragment-ion at m/z 93 shows an upward trend even when the empty Triwave cell is operated simply as a transfer line (Supplementary Figure S3). The m/z 94 ions generated at high sampling-cone voltages and isolated by the quadrupole analyzer arrive sufficiently activated, and undergo unimolecular fragmentations, even when there is no argon in the Trap and Transfer collision cells (Supplementary Figure S3, Panel B7).
Ab initio calculations suggested that the preferred protonation site of p-toluidine is the nitrogen atom (Supplementary Figure S9). This result is congruent with our experimental observation that at low sampling-cone voltages, only one peak appeared in the arrival-time profile (Figure 5a). The structure of the slower-moving protomer that corresponded to the 7.28 ms peak was confirmed by the post-IM mass spectrum recorded at a Transfer collision energy of 20 eV. The product-ion spectrum obtained showed an intense fragment-ion peak at m/z 91 attributable to an ammonia loss from the precursor ion (Supplementary Figure S8 B1). In contrast, the product-ion spectrum recorded from the faster-moving protomer, for which a mobility peak appeared only when the sampling-cone voltage was raised above 35 V, showed intense peaks at m/z 107 and 106 for the respective H● and H2 losses from the precursor ion (Supplementary Figure S8 A1). Presumably, the formation of 4-methylenecyclohexa-2,5-dien-1-iminium cation is the driving force for this H2 elimination (Supplementary Scheme S1). This result confirmed that the peak with an arrival time of 6.24 ms represents the C-protonated p-toluidine. According to our computations, the protonation on the ortho-ring carbon is only slightly less favored than the formation of the N-protomer (Supplementary Figure S9). The in-source ion activation provides the energy required to surmount the barrier for the isomerization of the N-protomer to the C-protomer via a proton migration.
Experiments conducted with protonated p-fluoroaniline also showed that its N-protomer predominates at low cone voltages. As the cone voltage is raised (Figure 6e), the population of the C-protomer increases. The Transfer-CID spectra recorded at 20 eV from the N-protomer demonstrated that it loses NH3 and HF (Supplementary Figure S11 B1), whereas the C-protomer loses a H● (Supplementary Figure S11 A1). The presence of a peak at m/z 95 in the spectrum of the C-protomer indicated that the C-protomer re-tautomerizes to a small extent to the N-protomer upon activation (Supplementary Figure S11 A1).
Flammang et al.  noted that ESI mass spectra of aniline recorded at three different cone voltages were different, and recognized the significance of the co-occurrence of its N- and C-protomers. Owing to the different proportions of protomers that could be present at different cone-voltage settings, such dissimilarities are expected. The solvent composition and the pH of the solution have been considered to be the most important factors determining protomer abundance ratios for aromatic compounds under ESI conditions . However, our results demonstrate that ESI instrumental parameters also exert dramatic effects on the abundances of different protomers generated from the same compound. We have shown that the protomer ratio is affected by the cone voltage regardless of whether the ions are generated by ESI or by plasma ionization of analytes after direct thermal desorption from neat liquids or solid residues without the intervention of a solvent.
For identification and characterization of drugs and their metabolites by mass spectrometry, it is important to develop reliable interpretation rules. Although enormous research efforts have been focused in this direction, nearly all attempts to interpret fragmentation spectra begin by preselecting and assigning one specific structure to represent the precursor ion. The results reported herein indicate that most of the interpretation mechanisms proposed on the basis of composite spectra must be improved by considering the fact that an activated protomer or, respectively, a deprotomer in the negative ionization mode, can exist in multiple tautomeric forms. The exact structural nature of a precursor ion must be correctly recognized before a rational fragmentation mechanism is proposed. However, establishing an unambiguous precursor structure for activated ions is not an easy task, particularly when the energy differences between the putative structures are small . As we gather more information, it is becoming clearer that results based on solution chemistry and parameters such as gas-phase basicity cannot be used as sufficiently reliable criteria to predict fragmentation mechanisms of activated gaseous ions. Evidently, even for a simple ion such as protonated aniline, high-level theoretical methods are unable to provide unambiguous conclusions because structures proposed by computational energy-optimization methods are not very reliable for activated ions. Moreover, an extensive study carried out by Russo et al. to determine the preferred protonation site of aniline in the gas phase by computing the energetic parameters at many different levels of theory and the DFT-based reactivity descriptors demonstrated that energy values predicted by various methods can sometimes differ by up to 10 kcal/mol . Some library mass spectra are listed from data collected without the appreciation of the fact that the initial precursor-ion structures may be composites. Thus, CID libraries should be used with caution for computer-based identification of compounds.
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