Oxidative Ionization Under Certain Negative-Ion Mass Spectrometric Conditions
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1,4-Hydroquinone and several other phenolic compounds generate (M – 2) –• radical-anions, rather than deprotonated molecules, under certain negative-ion mass spectrometric conditions. In fact, spectra generated under helium-plasma ionization (HePI) conditions from 1,4-hydroquinone and 1,4-benzoquinone (by electron capture) were practically indistinguishable. Because this process involves a net loss of H• and H+, it can be termed oxidative ionization. The superoxide radical-anion (O2 –•), known to be present in many atmospheric-pressure plasma ion sources operated in the negative mode, plays a critical role in the oxidative ionization process. The presence of a small peak at m/z 142 in the spectrum of 1,4-hydroquinone, but not in that of 1,4-benzoquinone, indicated that the initial step in the oxidative ionization process is the formation of an O2 –• adduct. On the other hand, under bona fide electrospray ionization (ESI) conditions, 1,4-hydroquinone generates predominantly an (M – 1) – ion. It is known that at sufficiently high capillary voltages, corona discharges begin to occur even in an ESI source. At lower ESI capillary voltages, deprotonation predominates; as the capillary voltage is raised, the abundance of O2 –• present in the plasma increases, and the source in turn increasingly behaves as a composite ESI/APCI source. While maintaining post-ionization ion activation to a minimum (to prevent fragmentation), and monitoring the relative intensities of the m/z 109 (due to deprotonation) and 108 (oxidative ionization) peaks recorded from 1,4-hydroquinone, a semiquantitative estimation of the APCI contribution to the overall ion-generation process can be obtained.
KeywordsOxidative ionization HePI APCI ESI Hydroquinone Thermometer ions Ambient ionization
Gaseous negative ions for mass spectrometry are usually generated from neutral molecules by four specific mechanisms: deprotonation, electron capture, charge exchange, or anion attachment . Typically, acidic compounds form their conjugate bases by deprotonation in the presence of a stronger base. In the electron-capture process, an external thermal electron is incorporated into an available orbital of a molecule [2, 3, 4]. For charge exchange, an ion-neutral reaction takes place in which the charge from an anion is transferred to the neutral molecule [2, 3]. For anion attachment, both species travel together through the mass spectrometer and are detected as a single entity [5, 6, 7].
It is known that analytes can sometimes undergo redox transformations in the ion source of a mass spectrometer [8, 9, 10]. However, these transformations are typically known to occur in non-plasma-based ionization sources, such as those for electrospray ionization (ESI), by an electrochemical mechanism. For example, the cysteine content in tryptic peptides was determined using ESI-MS by tagging them with a substituted quinone; the quinone was generated in the ion source by the electrochemical oxidation of a 1,4-hydroquinone to 1,4-benzoquinone [8, 9, 10]. 1,4-Hydroquinone has also been used as a redox buffer to prevent the oxidation of compounds such as morphine, amodiaquine, and imipramine during the ESI process .
Some oxidative modifications are also known to occur in ambient ion sources such as desorption electrospray ionization (DESI) or direct analysis in real time (DART); however, hydroquinones have been considered to be resistant to redox changes [12, 13]. Under positive-ion DESI mass spectrometric conditions, certain pharmaceutical compounds (e.g., morphine, reserpine, levodopa) are prone to producing oxidation products .
The helium-plasma ionization (HePI) technique [1, 14] is an ambient-ionization method to generate gaseous ions for mass spectrometric analysis, by direct insertion of a solid, liquid, or gaseous sample into the ion source, or by performing in situ reactions, which yield volatile products [15, 16, 17, 18, 19, 20, 21, 22, 23]. Analytes may be both organic [14, 15, 18, 19, 20, 21, 22, 23] and inorganic [16, 17]. Hitherto reported HePI-generated gaseous ions include radical-cations , radical-anions , protonated [14, 18, 19, 20] and deprotonated [22, 23] species, ion adducts [15, 17, 22], as well as simple inorganic anions . Herein we report that under HePI, atmospheric-pressure chemical ionization (APCI), and certain ESI mass spectrometric conditions, phenolic compounds undergo a redox transformation to either quinones or semiquinones by an oxidative process, and discuss the mechanisms of this ionization pathway, which involves the superoxide radical-anion. Previously, the formation of (M – 2H)- ions has been observed when atomic oxygen radical-anion was allowed to react with gaseous molecules such as cyclopropanecarbonitrile [24, 25].
All chemical were either synthesized or obtained from commercial sources. See Supplementary Materials for details.
Gas Chromatography-Mass Spectrometry (GC-MS)
To check purity, samples (1.0 mg/mL in dichloromethane) were introduced by split injection to a Hewlett Packard 5890 Series II gas chromatograph fitted with a 30 m × 0.25 mm capillary column coated with a 0.25-μm film of XTI-5 (5% diphenyl/95% dimethyl polysiloxane; Restek, Bellefonte, PA, USA), connected to an HP 5970 quadrupole mass spectrometer. The oven temperature was held at 40 °C for 4.0 min and increased (8 °C/min) to a final temperature of 250 °C. The temperature was then held at 250 °C for 10 min. Electron ionization (EI) mass spectra (70 eV) were recorded from all synthetic samples and compared with the EI spectra in the NIST/EPA/NIH mass spectral database ; 1,4-hydroquinone: EI-MS m/z (%), 110(M+•, 57), 111(4), 81(17), 53(15), 40(100); 1,4-benzoquinone: EI-MS m/z (%), 108(M+•, 100), 82(46), 80(33), 54(27), 50(15).
HePI Mass Spectrometry
For the generation of the HePI plasma, a stream of high purity helium (99.999%, Airgas, Radnor, PA, USA) was passed through a metal capillary held at a voltage of 3.2 kV . The metal capillary tip was set about 10 mm from the mass analyzer entrance-cone orifice of a Waters Micromass Quattro Ultima mass spectrometer (Waters Corp., Manchester, UK). Typically, the helium flow was maintained at less than 10 mL/min.
The source temperature was held at 150 °C. The cone voltage was typically set at 10 V, and hexapole 1 transfer lens was held at 20 V. For MS/MS experiments, the pressure of the argon in the collision cell was maintained at 2.5 × 10–5 mbar. A stream of heated desolvation gas (N2) was used to facilitate the thermal desorption of analytes. Typically, the “desolvation” temperature was set between 250 and 400 °C. Samples were deposited on glass slides and placed in the open source about 1 cm from the capillary tip (Supplementary Figure S1a).
Electrospray Ionization Mass Spectrometry
A methanolic solution of 1,4-hydroquinone (1 mg/mL) was sprayed at a flow rate of 20 μL/min and spectra were recorded on a Waters Micromass Quattro Ultima mass spectrometer under the following instrumental conditions: capillary voltage 3.2 kV, cone voltage 10 V, hexapole 1 transfer lens voltage 50 V, desolvation temperature 150 °C, source temperature 100 °C, and a desolvation gas flow rate of 450 L/h.
Selective Ion Recording (SIR) Experiment
Two-channel selective ion recording (SIR) was performed for the m/z 108 and 109 ions generated under enclosed-source ESI conditions (Supplementary Figure S1b) from a methanolic solution of 1,4-hydroquinone (1 mg/mL) sprayed at 20 μL/min, at the following settings: capillary voltage 3.20 kV, cone voltage 10 V, hexapole 1 transfer lens voltage 10 V, dwell time 0.3 s, interchannel delay 0.10 s. After 2 min of signal acquisition, the source was engulfed with sulfur hexafluoride. At 7.5 min, the flow of SF6 was switched off and the system was allowed to reach its initial conditions.
Electrospray Ionization (ESI) Using a Nanospray Ion Source
A methanolic solution of 1,4-hydroquinone (1 mg/mL) was infused into the nanospray source of a SYNAPT G2 HDMS instrument (Waters, Manchester, UK) at a rate of 2 μL/min. The sample solutions were sprayed through a fused-silica tube (outer diameter 375 μm, orifice diameter 25 μm). The instrument was operated in the sensitivity mode; the signal was acquired over m/z range 100–120; the capillary voltage was varied between 1.7 and 4.0 kV; the source temperature was kept at 100 °C; the sampling cone was set at 43.0 V; the extraction cone was set at 2.5 V. The desolvation temperature was set at 400 °C; the cone gas flow was at 50 L/h; the desolvation gas flow was at 200 L/h.
Atmospheric-Pressure Chemical Ionization
A methanolic solution of 1,4-hydroquinone (1 mg/mL) was sprayed through a fused-silica capillary with an inner diameter of 75 μm at 20 μL/min and spectra were recorded on a Waters Micromass Quattro Ultima mass spectrometer under the following instrumental conditions: corona needle 5 μA, cone voltage 10 V, Hexapole 1 transfer lens voltage 50 V, desolvation gas temperature 150 °C, source temperature 100 °C, and desolvation gas flow rate of 450 L/h.
Capillary Voltage Sweep Experiment
A methanolic solution of 1,4-hydroquinone (1 mg/mL) was sprayed at 20 μL/min and spectra (m/z 107.5 to 110.5) were recorded on a Waters SYNAPT G2 HDMS mass spectrometer under the following instrumental conditions: negative-ion resolution mode, extraction-cone voltage 1.5 V, sampling-cone voltage 20 V, Vernier probe adjustment screw 5.92 mm, source temperature 100 °C, desolvation gas flow rate 450 L/h. The capillary voltage was varied between 1.4 and 4.0 kV, trap cell collision energy 2.0 eV, transfer cell collision energy 2.0 eV
Synthesis of 1,4-Hydroquinone-d4
1,4-Benzoquinone-d 4 (20 mg, 0.179 mmol) was suspended in ether (1 mL) and shaken with a solution of sodium dithionite (1 mL, 10% w/w Na2S2O4 in 0.5 M NaOH) until the yellow color of the quinone disappeared . The aqueous layer was separated, cooled on ice, and acidified with concentrated HCl. The product was extracted with diethyl ether and recrystallized from absolute ethanol; 1,4-benzoquinone-d 4 : EI-MS m/z (%), 112(M+•, 65), 84(50), 56(100); 1,4-hydroquinone-d 4 : EI-MS m/z (%), 114(M+•, 100), 85(20), 57(28), 40(97).
The Gaussian 09 program , utilizing the unrestricted HF  method and 6-31G + (d,p) basis set for all atoms, was used to perform quantum mechanics (QM) calculations. All structures were fully optimized without any constraints. Frequency analysis was done at the same level to confirm that the optimized structures correspond to the minimum-energy states on the respective potential energy surfaces. Annihilation of the first-spin contaminant was achieved for all molecules.
Calculation of Electronic Energies
Electronic energy values were calculated at a temperature of 298.15 K and a pressure of 1.0 atm and obtained from the output file. Zero-point corrections were applied.
Results and Discussion
The HePI mass spectrum from 1,4-hydroquinone (Fig. 2b) was practically indistinguishable from that recorded from 1,4-benzoquinone (Fig. 2a). For neutral 1,4-hydroquinone to generate an anion of m/z 108, it must formally lose H• and H+. In other words, under HePI conditions 1,4-hydroquinone undergoes ionization by an oxidative process. Although redox transformations under electrospray [8, 9, 10] and laser ionization  conditions have been reported, the phenomenon we describe here is uncommon. For simplicity, the observed ionization process will be referred to as “oxidative ionization.” Although less intense, the ESI mass spectrum of 1,4-hydroquinone also showed an analogous m/z 108 peak for the (M – 2)–• radical-anion (Fig. 1a). However, the ESI mass spectrum showed the base peak at m/z 109 for the deprotonated species (Fig. 1a), while the base peaks in both the HePI (Fig. 2b) and APCI (Fig. 1b) spectra corresponded to the oxidatively ionized species (m/z 108). In contrast, in the HePI spectra of catechol (Fig. 2c) and resorcinol (Fig. 2d), practically no (M – 2)–• ion was observed. It stands to reason this is because the corresponding oxidized species are of very low energetic stability.
The next question to be addressed was the identity of the oxidizing agent. We hypothesized that the ion plasma present in the HePI and APCI ion sources may play an important role in the oxidative ionization process [14, 31, 32]. Dioxygen is a molecule with significant electron affinity that is present in the ambient atmosphere [33, 34]. Consequently, one of the common gaseous anions found in the plasma of discharge ion sources operated under negative-ion-generating conditions is the superoxide radical-anion, O2 –•, which is primarily responsible for ionizing many analytes in negative-ion-generating HePI and DART [22, 31]. However, spectra recorded on many commercial mass spectrometers do not show a peak at m/z 32 for the superoxide radical-anion because their ion-transfer optics are deliberately designed to suppress the transmission of low-mass ions. Thus, we were unable to record a peak for the m/z 32 ion on a Quattro Ultima or Synapt instruments; however, an intense signal was observed at m/z 32 on a Waters ZQ instrument (Supplementary Figure S2). Moreover, the intensity of the m/z 32 signal increased as the capillary voltage was raised.
Owing to the proton-abstracting properties of the superoxide radical-anion, any sufficiently acidic gaseous molecule is expected to undergo deprotonation under negative-ion HePI conditions [22, 33, 34]. However, we have previously shown that compounds with low acidities form superoxide radical-anion adducts, rather than deprotonate . A closer scrutiny revealed that the spectra from both 1,4-hydroquinone and resorcinol showed small peaks at m/z 142 ([M + 32]–•), indicating that both of these compounds form superoxide radical-anion adducts (Fig. 2b and d). In addition, when the hydroquinone adduct was mass-isolated and subjected to collision-induced dissociation, in the recorded spectrum a small peak appeared at m/z 32, confirming that the peak at m/z 142 is indeed an adduct of neutral hydroquinone with a species of 32-Da mass (Fig. 3b). Therefore, we concluded that O2 –• adduct formation is the prerequisite to initialize the oxidative ionization pathway. Furthermore, the spectrum for the fragmentation of the O2 –• adduct of 1,4-hydroquinone-d 4 (m/z 146) showed peaks for losses of HO-O• and H2O2 instead of DO-O• and D2O2, which supported our hypothesis that elements of H2O2 are eliminated from the adduct during the oxidative process (Figs. 3a and 4).
We propose that the superoxide radical-anion acts as a base and abstracts a hydroxyl proton from 1,4-hydroquinone (m/z 109) to generate the hydroperoxyl radical (the conjugate acid of O2 –•) and 4-hydroxyphenolate anion. The two entities produced in this way constitute an ion-radical complex (Structure 2, Fig. 4). Within the ion-radical complex, the hydroperoxyl radical then roams over to the free hydroxyl group and abstracts a hydrogen atom (H•) to form hydrogen peroxide and a semiquinone radical-anion (m/z 108) (Structure 3, Fig. 4, Supplementary T3); this step is also supported by the fragmentation of m/z 109 to give an m/z 108 product ion (Supplementary Figure S3).
To demonstrate the significance of the O2 –• for the oxidative ionization process, the enclosed HePI source was engulfed with sulfur hexafluoride (SF6), a powerful electron scavenger [35, 36]. A selective-ion recording (SIR) experiment conducted on ions generated from a methanolic solution of 1,4-hydroquinone by ESI at a capillary voltage of 3.20 kV showed that a dramatic initial decrease of the intensities of both m/z 108 and 109 signals occurs upon introduction of SF6 to the source. However, the SF6 had a more dramatic effect on the attenuation of the intensity of the m/z 108 signal than that of the m/z 109 ion (Supplementary Figure S4). Evidently, SF6 reduces the abundance of the O2 –• in the source. Consequently, the oxidative ionization process is suppressed, and the intensity of the peak at m/z 108 sharply decreases (Supplementary Figure S4). This result also supports the notion that m/z 108 and 109 ions originate primarily from two distinct mechanisms.
In contrast to the useful “plasmometer” properties of 1,4-hydroquinone demonstrated in the SYNAPT ion source under ESI conditions, the other two dihydroxybenzene isomers were found to be much less efficient. The efficacy of 1,4-hydroquinone as plasmometer compound is primarily due to its high electron affinity and strong reducing properties [50, 51, 52]. Because the m/z 108 ion could also be generated by collision-induced homolytic dissociation of the m/z 109 ion (Supplementary Figure S3), post-ionization ion activation must be maintained as low as possible. Under low ion-activation conditions (low sampling-cone voltages, and low collision energies in the trap and transfer cells), both catechol and resorcinol displayed no marked differences in their ESI mass spectra recorded with changing capillary voltages (Supplementary Figure S5a, b). More importantly, the relative intensities of the m/z 108 and 109 peaks did not change much in a nanospray SYNAPT source as long as discharges were avoided, for example keeping the electrical field differences low by not placing the capillary tip too close to the cone orifice (Supplementary Figures S5, S6). The latter finding indicates that the nanospray source is much closer to “true” ESI conditions over a wide range of capillary voltages than is the high-flow ESI source, if discharges can be avoided.
In order to make a generalization on the classes of compounds that undergo reductive oxidation, HePI mass spectra from several dihydroxybenzene derivatives were acquired. It was found that many, but not all, dihydroxybenzene derivatives can undergo oxidative ionization. For example, many substituted 1,4-hydroquinones (Supplementary Figure S7) and catechols (Supplementary Figure S8) also undergo oxidative ionization under HePI conditions. The presence of additional functional groups on the benzene ring of dihydroxybenzenes exerts a considerable effect on the relative intensities of the (M – 2)–• and (M – 1)– peaks. For example, a carbaldehyde group in substituted catechols strongly assists the formation of the corresponding (M – 2)–• species relative to unsubstituted catechol (Supplementary Figure S8a, b), whereas a cyano group in the 3 position exerts only a moderate effect (Supplementary Figure S8c). With derivatives of resorcinols, the presence or absence of substituent groups matter little because 1,3-quinones are less commonly encountered (Supplementary Figure S9). The 1,3-isomer is considered an avatar of the so-called non-Kekule species; however, its diradical form has been reported [53, 54, 55]. Furthermore, additional methyl substituents on the benzene ring in dihydroxybenzenes wield a much smaller, or negligible, effect in the same respect (Supplementary Figures S7a, b, S9a, b, S10a). Evidently, electron-withdrawing groups with π–bonds conjugated to the aromatic ring stabilize the phenoxide anion formed in the first step of oxidative ionization to such an extent that the second step (the formation of an M – 2 radical- anion) is disfavored.
Certain phenolic compounds undergo oxidative ionization due to their interaction with the superoxide radical-anion generated in the atmospheric-pressure HePI and APCI sources. Under ESI conditions, oxidative ionization occurs only when the ion source has a certain degree of plasma character. One particular analyte, 1,4-hydroquinone, can be used to effectively gauge the degree of “plasmaticity” of an ion source. A contrast can be drawn between analytes that prefer to undergo oxidative ionization versus those that preferentially deprotonate: the former prefer to lose elements of hydrogen peroxide (34 Da) from their O2 –• adducts upon CID, whereas the latter preferentially lose a hydroperoxyl radical.
The authors thank Sathis Weerasinghe and Concorde Specialty Gases for providing the sulfur hexafluoride. They also thank Bristol-Myers Squibb Pharmaceutical Company (New Brunswick, NJ) for the donation of the Waters Quattro Ultima mass spectrometer.
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