Unexpected Reduction of Iminoquinone and Quinone Derivatives in Positive Electrospray Ionization Mass Spectrometry and Possible Mechanism Exploration
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Unexpected reduction of iminoquinone (IQ) and quinone derivatives was first reported during positive electrospray ionization mass spectrometry. Upon increasing spray voltage, the intensities of IQ and quinone derivatives decreased drastically, accompanying the increase of the intensities of the reduction products, amodiaquine (AQ) and phenol derivatives. To gain more insight into the mechanism of such reduction, we explored the experimental factors that are influential to corona discharge (CD). The results show that experimental parameters that favor severe CD, including metal spray emitter, using water as spray solvent, sheath gas with low dielectric strength (e.g., nitrogen), and shorter spray tip-to-mass spectrometer inlet distance, facilitated the reduction of IQ and quinone derivatives, implying that the reduction should be closely related to CD in the gas phase.
KeywordsIminoquinone Quinone Reduction Corona discharge Electrospray ionization
Redox modifications of analytes generally occur during electrospray ionization mass spectrometry (ESI MS). Solution-phase electrochemical reaction and gas-phase corona discharge (CD) are commonly deemed as “culprits” for the phenomena. An ESI source can be viewed as an electrolytic cell [1, 2, 3], in which oxidation reaction occurs in the positive mode and reduction reaction occurs in the negative mode. Van Berkel’s group has done extensive investigations over the past decades [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]. In addition, analyte redox can also be induced by the massive oxidative or reductive species, e.g., OH, O, H, N, HO2, N2 +, N+, O3, which are generated via CD in the gas phase [15, 16, 17, 18]. Since CD occurs in both positive and negative polarities, oxidation or reduction of analytes will also occur in both ionization modes for ESI.
In conventional ESI MS, there exists a striking asymmetry between the incidence of oxidation and reduction reaction . Oxidation reactions occur more frequently than reduction reactions. Electrochemistry-induced oxidations include electrolysis of analyte molecules (peptide [20, 21], metallo porphyrins [4, 14], reserpine , amodiaquine (AQ) , steroid sulfates , hydroquinones , and isochromene ), solvent molecules [20, 24] as well as ESI electrodes [7, 8, 21, 25, 26]. CD-induced oxidations include oxygenation of hydroquinone , stearic acid , phosphorothioate oligonucleotides , peptide , and protein . However, only a few reduction reactions were reported during ESI process. Gianelli et al. reported Cu (II) reduction during positive ESI mode, which was attributed to charge transfer between Cu (II) complexes and the solvent molecules in the gas phase, and electrochemical reaction during ESI process was excluded by deuterated methanol . Gu et al. reported the reduction of CH3CN to CH3CH2NH2 in positive ESI , and electrolysis of water was believed to be responsible for that reduction. However, there was no sufficient evidence to illustrate that the water electrolysis occurred at the electrode/solution interface rather than in the gas phase. Furthermore, the reduction of diquat and paraquat dication to monovalent cation  and 1,6-dichloro-1,4-benzoquinone to phenol were also reported during ESI MS . However, the underlying mechanism remained unexplored.
Phenolic compounds are readily oxidized during ESI, and therefore are commonly used to investigate the electrolysis performance of ESI [7, 35, 36] or as the redox buffer  during ESI MS. However, unexpected reduction of iminoquinone (IQ, oxidative product of AQ) and quinone derivatives was recently observed in positive ESI MS in our study. Though quinone reduction was reported in some traditional ionization sources, such as electron ionization (EI) , fast atom bombardment (FAB) , secondary ion mass spectroscopy (SIMS) , and atmosphere pressure chemical ionization (APCI) , it was for the first time reported in positive ESI MS. To gain more insight into the mechanism of this unusual reduction in positive ESI MS, we investigated the effect of experimental parameters that relate to CD in the gas phase on that reduction, including solvent composition, sheath gas, spray emitter material, and spray tip-to-mass spectrometer inlet distance.
Materials and Reagents
HPLC grade methanol (CH3OH) was purchased from Honeywell Burdick & Jackson Inc. (Morristown, NJ, USA). AQ, 1,4-benzoquinone (1,4-BQ), methyl-p-benzoquinone (MBQ), 1,4-naphthoquinone (1,4-NQ), and 1,4-anthraquinone (1,4-AQ) were obtained from Sigma-Aldrich Chemical Co. Ltd. (St. Louis, Missouri, USA). Glutathione (GSH) was purchased from Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). Ammonium acetate (NH4Ac) and hydrofluoric acid (HF) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). All these reagents were used without any further purification. Distilled water (18.2 MΩ) was produced by Milli-Q system (Millipore Inc., Bedford, MA, USA).
To achieve stable spray with the home-built ESI source, the top 5 mm of the fused silica capillary was etched using the method introduced by Kelly .
All MS experiments were carried out using a Thermo LTQ mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The mass spectrometer conditions were as follows: S lens voltage, 42% (positive mode); capillary temperature, 275 °C. Ion injection time was set as 10 ms and all signals were averaged by three microscans.
Results and Discussion
Incidental Discovery of Iminoquinone Reduction in Positive ESI MS
However, what was the “culprit” that led to the unexpected reduction of IQ when the spray voltage was raised from 3 to 5 kV (10–11 min)? It is well known that ionization efficiency affects the signal intensities of analytes in the mass spectrometer. An intuitive speculation was that the signal variations of AQ, SIQ, and IQ were attributable to their ionization efficiencies during the electrospray process. This possibility was excluded by the following experiment. Before the AQ solution was electrochemically oxidized, the spray voltage was adjusted from 3 to 5 kV. It showed that the signal intensity of AQ barely changed (Supplementary Figure S2), stating that the signal variations of AQ, SIQ, and IQ were not induced by their ionization efficiencies at the given spray voltages.
Electrochemical reaction in the liquid phase and CD reaction in the gas phase are known as two common reasons for analyte redox during ESI MS. However, the possibility of electrochemistry-induced reduction could be excluded from two aspects. First, electrochemical reduction reaction is well recognized to occur only in negative ESI mode. Second, electrochemical reaction occurs at the solution/electrode interface, which requires time for sample solution with electrode contact to pass through the fused silica capillary. Therefore, electrochemistry-induced redox should be observed with a time delay, as discussed already in this article.
Effect of Mass Spectrometer Parameters on Iminoquinone Reduction
Gianelli et al. reported the reduction of Cu (II) to Cu (I) in positive ESI mode and attributed it to CD . To explore whether CD is responsible to the IQ reduction in positive ESI mode, we investigated the effect of the following experimental parameters related to CD on that reduction, including spray solvent composition, sheath gas, spray emitter material, and the spray tip-to-mass spectrometer inlet distance.
Reduction of Quinone Derivatives in Positive ESI MS
The above results indicate the effect of CD on IQ reduction during ESI MS. In addition, we tested whether other quinonoid compounds could experience similar reduction during ESI MS. BQ, MBQ, 1,4-NQ, and 1,4-AQ, which are well-defined reversible redox species, were chosen as the test compounds.
However, not all quinonoid compounds experienced such reduction during positive ESI MS. 1,4-NQ-GSH and 1,4-AQ-GSH could not be reduced no matter what experimental parameters were adjusted (Supplementary Figure S11). This might be related with the reduction potential of different quinone species. The species with higher reduction potential may be more readily reduced during electrospray process, as implied by the reduction potentials of BQ, MBQ, 1,4-NQ, and AQ being –0.851, –0.928, –1.029, and –1.259 V, respectively .
Unexpected reductions of IQ and quinone derivatives during ESI MS were reported for the first time. The reductions were further investigated and the experimental results suggested that it was closely related with CD in the gas phase. Through adjusting experimental parameters that strengthened CD, e.g., improving spray voltage, using water as spray solvent, using metal spray emitter, or shortening the spray tip-to-mass spectrometer distance, IQ and quinone derivatives were more readily reduced. This finding implies that greater attention should be paid during analyzing readily reductive species with ESI MS, such as quinonoid compounds, as well as readily oxidative species.
The authors are grateful for financial supports from the National Natural Science Foundation of China (21475121, 21665003), the Guangxi Natural Science Fund Project (no. 2016GXNSFBA380140).
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