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Detection of transient dopamine antioxidant radicals using electrochemistry in electrospray ionization mass spectrometry
Accepted Manuscript Title: Detection of transient dopamine antioxidant radicals using electrochemistry in electrospray ionization mass spectrometry Authors: Imran Iftikhar, Kholoud Mohammed Abou El-Nour, Anna Brajter-Toth PII: DOI: Reference:
S0013-4686(17)31505-0 http://dx.doi.org/doi:10.1016/j.electacta.2017.07.087 EA 29904
To appear in:
Electrochimica Acta
Received date: Revised date: Accepted date:
10-5-2017 13-7-2017 14-7-2017
Please cite this article as: Imran Iftikhar, Kholoud Mohammed Abou El-Nour, Anna Brajter-Toth, Detection of transient dopamine antioxidant radicals using electrochemistry in electrospray ionization mass spectrometry, Electrochimica Actahttp://dx.doi.org/10.1016/j.electacta.2017.07.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of transient dopamine antioxidant radicals using electrochemistry in electrospray ionization mass spectrometry
Imran Iftikhar1, Kholoud Mohammed Abou El-Nour2, Anna Brajter-Toth1,*
1
Department of Chemistry, University of Florida, Gainesville, Florida, 32611-7200, USA
2
Department of Chemistry, Faculty of Science, University of Suez Canal, Ismailia, 41522, Egypt
*Corresponding Author: Tel: +1352 392 7972/ Fax: +1352 392 4651 E-mail address: [email protected]
Abstract We describe formation and detection of transient radical intermediates and 2e-, 2H+ oxidation products (OPs) of dopamine (DA) by electrospray ionization (ESI) mass spectrometry (MS) which has not been reported previously. The results confirm formation of DA radicals by proton coupled electron transfer pathway. DA radicals and 2e-, 2H+ oxidation products that were identified are of interest because of their reported neurotoxicity. Using different solution compositions and MS detection conditions three different DA radicals were identified that have different stability. DA reactions with reactive oxygen species (ROS) relevant to DA reactivity in vivo are discussed. Detection of DA OPs by electrochemistry/mass spectrometry with a floated on-line electrochemical cell is also illustrated.
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Key wards Dopamine radicals; Electrochemistry; Electrospray Ionization; Mass Spectrometry; Neurotoxicity 1. Introduction In recent years major efforts have been directed at unraveling complex processes that contribute to neurodegenerative diseases.[1, 2] In these studies, surprisingly, endogenous neurotransmitters have been identified as potential sources of toxic products.[1-3] As a result, dopamine (DA), a significant catecholamine neurotransmitter in the brain,[2] has been investigated in some detail as a potential toxic agent. Based on these studies it has been proposed that in vivo reactions of DA with oxygen can generate toxic oxidation products (OPs). This can occur during oxidative stress, after DA release into cytosol from storage vesicles when DA reabsorption into the vesicles is not efficient. DA can be oxidized in the cytosol, where higher pH than in the vesicles facilitates the oxidation. Transient semiquinone radicals that were proposed to form during the oxidation have been proposed to be highly toxic even at low concentrations. However, the previously proposed toxicity of 2e-, 2H+ DA quinone OPs has been questioned.[3] Complex oxidations such as the oxidation of DA (a hydroquinone) can generate different intermediates and nucleophilic reaction products under different reaction conditions. Protoncoupled electron transfer (PCET)[4] reactions of DA forming DA radicals that can involve oxygen and reactive oxygen species (ROS) have been proposed for DA oxidations in vivo [3, 57]. These reactions have been difficult to study because of the limited availability of suitable methods. Limited information about DA semiquinone radicals has been obtained from electron spin resonance (ESR) studies in vitro.[6, 7] In ESR, unstable radical signals have been reported
2
that were attributed to unstable cyclized DA semiquinone.[3] Signals of longer-lived radical (t1/2 obs
= 79 s at pH 6.5) have also been detected and were attributed to another semiquinone.[7]
Chemical characterization of the radicals was not feasible by ESR. Other DA OPs have been detected and identified by electrochemistry on-line with mass spectrometry (EC/MS) during DA oxidation [8-10] and recently by NMR during chemical oxidation of DA.[11] Detection of DA radicals was not reported in these studies.[8-11] Based on prior work[12] we have postulated that under optimized conditions detection of DA radicals should be feasible by ESI MS. Detection of transient radical intermediates has been demonstrated in previous work in different ESI MS investigations. [12, 13] Detection of transient DA radical intermediates and OPs is demonstrated in this work. Characterization of DA radicals and DA OPs was possible by ESI MS. DA OPs were also detected under different conditions during fast oxidation of DA in EC/ESI MS. Additional merits of EC/ESI MS were realized in this work, providing kinetic information during electrochemical oxidation of DA under different conditions.[12, 14] Three DA radicals were detected by ESI MS. The results show that DA radicals are formed by proton-coupled electron transfer (PCET)[4] reactions pathways as a result of slow oxidation of DA in ESI MS with silica (SiO2) capillary as the electrospray (ES) emitter. Lower ionization energy observed during ESI with SiO2 emitter requires additional energy to be supplied for the detection of DA radicals and other OPs formed in low yields in ESI MS.[15, 16] Fast 2e-, 2H+ oxidation of DA was demonstrated during ESI MS with stainless steel (SS) emitter and in EC/ESI MS. In EC/ESI MS the SS ES emitter also acts as the working electrode in the online two electrode EC cell. The EC cell voltage is floated on the HV applied to the ES emitter during ESI. The applied EC cell voltage changes the effective voltage of the SS ES emitter
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during ESI. The results demonstrate that DA oxidation is rapid at the SS ES emitter during ESI and confirm that the rate of DA oxidation can be increased in EC/ESI MS by applying voltage to the EC cell. 2. Experimental 2.1. Materials Dopamine hydrochloride (98%) was purchased from Aldrich (St. Louis, MO). Formic acid (FA), glacial acetic acid (HAc) (99.9%), ammonium acetate (NH4Ac) and methanol (HPLC grade) were from Fisher (Pittsburgh, PA). All chemicals were used as received. Carrier solutions used in ESI MS were: (i) 50/49/1 vol% MeOH/H2O/FA, pH ~ 3, 1568 mS/cm, (ii) 50/49/1 vol% MeOH/H2O/HAc, pH ~ 4, 121.8 mS/cm, and (iii) 60/40/vol% MeOH/H2O/1 mM NH4Ac, pH ~ 6, 59.9 mS/cm.[12] Solution conductivity was determined at 250C using a conductivity probe calibrated in 0.01 M KCl (1413 mS/cm) and Vernier LabQuest computer interface. All solutions were freshly prepared with doubly deionized water. 2.2. Mass Spectrometry The APEX 4.7-T FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA) with a modified ESI ion source has been described in detail previously.[10, 17, 18] A cone shaped MS capillary inlet made of brass (500 divergence angle from 1.58 mm i.d. to 6.13 mm orifice diameter) was used.[10] The MS system is schematically illustrated in Fig. 1. Figure 1 Base peak intensity was similar using fused silica (100 µm i.d.) or cylindrical stainless steel (SS; 80 µm i.d.) capillaries as the electrospray (ES) emitter. A syringe pump (Cole Parmer 74900, Vernon Hills, Il) provided constant solution flow to the ES capillary. Using a silica ES emitter in the 20 to 100 µL/h flow rate range better signals were observed at 50 µL/h. Standard
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MS parameters were: hexapole storage time (HST), 0.5 s; ion transition time (ITT), 0.7 ms; skimmer voltage, 3.0–5.0 V; High voltage (HV) ~ 3kV; exit capillary voltage (Vc), ~ 40 V; number of scans, 20. The MS inlet capillary was heated to 1400 C. To induce ion dissociation in the capillary-skimmer region, exit capillary voltage was increased up to 90 V, which increased capillary-skimmer voltage bias to ~ 85 V. DA dimers were detected by increasing ion transition time from 0.7 ms to 1.0 ms. Mass spectra were obtained using Predator (version 1.2) and Modular ICR Data Acquisition and Analysis System (MIDAS) software[19] written at the National High Magnetic Field Laboratory (Tallahassee, Fl). Calculated exact masses were used to verify ion assignments. Additionally, relative intensities of isotopic peaks were used in ion assignments. The mass error was between _2 to +10 ppm. 2.3. On-Line Electrochemistry ESI MS (EC/ESI MS) Design and operation of the EC cell in the EC/ESI FTICR MS system with APEX 4.7-T FTICR mass spectrometer (Fig. 1) has been described.[10, 17, 18] The SS ES emitter capillary and a second SS capillary (5 and 4 cm long), separated by low volume Teflon tubing, formed the on-line EC cell, with SS counter and working electrodes. The SS working electrode additionally functioned as the ES emitter in EC/ESI MS. SS capillaries were attached to an adjustable xyz micropositioner (World Precision Instruments, Sarasota, Fl). The dc voltage was applied to the EC cell from a 9 V battery and was adjusted using a variable resistor (maximum 50 kΩ). The EC cell voltage was floated on the HV of the ES emitter. In EC/ESI MS solution flow rate was 30µL/h, which increased residence time of analyte at the SS WE. 3. Results and Discussion 3.1. DA Reactions in ESI MS with Silica ES Emitter Figure 2 5
Positive ion mode mass spectrum of DA obtained from pH 3 (50/49/1 vol% MeOH/H2O FA) carrier solution under standard ESI MS operating conditions, with silica (SiO2) capillary as the ES emitter, is shown in Fig. 2A. The mass spectrum shows an intense (10,000 cps) ion signal of [DA+H]+ (m/z 154) base peak together with the isotope ion peak. As shown in Fig. 2B, using higher capillary-skimmer voltage bias allows detection of new ion signals and under these conditions DA ion fragment (m/z 137) signal becomes the base peak. The new ion signals in Fig. 2B can be assigned to DA OPs (Scheme 1) discussed further below, 6-hydroxy dopamine 6-OHDA (m/z 170), p-topaminequinone PTQ (m/z 168), and 5,6dihydroxyindoline radical (DHIO•+ m/z 151). As shown in Fig. 2C, by (additionally) lowering the (standard) hexapole storage time (HST) from 0.6 to 0.3 s a new m/z 153 ion signal was detected that was assigned to DA semiquinone radical [DA]•+ (Scheme 1). Scheme 1 The ESI MS results in Figs. 2B and C confirm that DA ESI at SiO2 results in detection of DA radicals and other OPs. Oxidation reactions during ESI at SiO2 thus provide access to chemical/structural information not accessible by other methods about transient intermediates and OPs, with time resolution of ca. 3 ms required for ion transition from the ES interface to the ICR cell. In agreement with previous reports of outcomes of ESI under conditions that are unfavorable for analyte oxidations during ESI [12] formation of DA radicals was detected following ESI of DA at SiO2. DA oxidation is limited (is slow) under these conditions because of the lower effective voltage at the emitter due to its high resistance. ESI energy under these conditions is thus also lower, resulting in lower ion energy. As a result, in order to aid ion
6
dissociation for detection of DA radicals and DA OPs higher capillary-skimmer voltage bias or longer HST was required. The results in Fig. 2 point to the formation of two DA radicals (m/z 151 and 153) that have different stability. In previous ESR investigations of enzymatic and chemical oxidation of DA detection of DA radicals with different stability was justified by the structures proposed for the radicals.[3, 7] The reported ESR results are supported by this work. It is worth noting that high sensitivity realized using higher capillary-skimmer voltage bias was required for detection of DA radicals and DA OPs in ESI MS with SiO2. All subsequent structural assignments that were made were possible because of the high sensitivity and were made by exploiting the high resolution of FT ICR MS. Standard MS/MS methods, typically used for structural assignments, do not have the sensitivity and time resolution required for detection of the transient species present at low concentrations; consequently fragmentation products, required for structural assignment, would also not be detectable. Based on the results that were obtained by using different compositions of carrier solutions, limited oxidation of DA during ESI with SiO2 emitter was confirmed, independently of the carrier solution used. However, different OPs were detected in different carrier solutions, and at different pH. Using higher capillary-skimmer voltage bias and pH ~ 4 carrier solution m/z 152, 149, 134 and 123 ion signals were detected that were assigned to DA OP 5,6dihydroxyindoline DHIO (m/z 152) and its fragments [152-H2O]+ (134), [152-CH2NH]+ (m/z 123), and of DHI•+ radical (m/z 149) (data not shown). 3.2. DA Oxidation in ESI MS and EC/ESI MS with Stainless Steel (SS) ES Emitter Figure 3
7
Efficiency of ionization in ESI MS is determined by the experimental parameters that include the design of the ionization interface and the MS optics, as well as the MS operating conditions.[10, 13] In addition, ESI efficiency is impacted by the composition and pH of the carrier solution.[12, 20-25] In many cases this is an advantage of ESI MS method since small changes, in carrier solution composition and/or instrumental parameters, allow fine-tuning of the ESI efficiency. This is well-documented in EC/ESI MS where ESI efficiency can be improved by simply applying additional voltage to the SS ES emitter, which is used in EC/ESI MS as the on-line WE in the floated two electrode EC cell.[10, 26] Comparison of the results in Figs. 2A and 3A illustrates the impact of ES emitter material on ion signal distribution in ESI MS with SiO2 (Fig. 2A) and SS (Fig. 3A). Using SS emitter and pH 3 carrier solution, under the same MS conditions as in Fig. 2A where SiO2 ES emitter was used, m/z 152 ion signal of DA OP is the base peak, rather than the ion signal of DA (m/z 154). Oxidation of DA in ESI MS with SS is apparent from the results in Fig. 3A. ESI MS results with SS emitter in Fig. 3A show high intensity of DA (m/z 154) ion signal, relative to the intensity of DA OP (m/z 152) base peak, indicating high (ca. 80%) oxidation efficiency of DA in ESI MS at SS. Results from EC/ESI MS in Fig. 3B, where the SS emitter also acts as the WE in the on-line two electrode EC cell, show that after voltage is applied to the SS emitter/WE in the EC cell DA oxidation appears complete since DA (m/z 154) ion signal disappears. The results in Fig. 3C (and in Figs. 3F and I) were obtained after high capillary-skimmer voltage bias was used in ESI MS with SS. The results show that under these conditions only fragment ions were detected, consistent with high energy during ESI at SS resulting in extensive fragmentation of emitted ions when the capillary-skimmer voltage bias was increased. In
8
contrast, in ESI MS with SiO2 emitter, as shown by the results in Figs. 2B and C, higher capillary- skimmer voltage bias improved detection of DA radicals and DA OPs. Mass spectra of DA at pH 4 and 6 were obtained by ESI MS with SS emitter. As shown by the results DA OPs in ESI MS with SS emitter are easily detected at pH 4 and 6 under standard MS conditions. The results support DA oxidation during ESI at SS at pH 4 and 6 that is not detected under standard conditions in ESI MS with SiO2 (results not shown). Since in the mass spectra in Figs. 3D and G DA (m/z 154) ion signal is the base peak the results indicate less efficient oxidation of DA at pH 4 and 6 than at pH 3 (Fig. 3A). It is likely that lower oxidation efficiency of DA in ESI MS from these solutions is a result of lower conductivity of pH 4 (121.8 µS/cm) and 6 (59.9 µS/cm) solution than of the pH 3 solution (1568 mS/cm).[18, 20] Better ionization efficiency of DA at pH 6 than 4 is also indicated by the results in Figs. 3D and G, from higher intensity of DA (m/z 154) base peak ion signal at pH 6 than pH 4. This appears to be inconsistent with lower conductivity of pH 6 solution and typically low ESI efficiency at higher pH, when fewer [H+] ions are present in solution. It is therefore likely that ESI of preformed [DA+H]+ ions is not limited by solution conductivity but is, alternatively, determined by the efficiency of acid-base and other reactions during ESI.[12, 27] However, in ESI MS with SS emitter, as shown from the mass spectra in Figs. 3D and G, solution conductivity impacts oxidation efficiency of DA based on the relative intensities of DA and DA OP ion signals in the mass spectra. In the final outcome it is the ES emitter material that has the largest impact on DA oxidation efficiency during ESI, as shown by the results (Figs. 2A and 3A, D and G) which show low ion signals of DA OPs in ESI MS with SiO2 and high OP ion signals in ESI MS with SS.
9
In agreement with previously proposed 2e-, 2H+ oxidation pathways of DA in acidic aqueous solutions, [5, 8, 9, 28] at pH 3 and 4 DHIO (m/z 152) was the main DA OP that was detected in ESI MS at low pH (Scheme 2). In EC/ESI MS (Fig. 3B and E) ion signal of DHIO increased with increasing EC cell voltage, as expected. As discussed above, in EC/ESI MS complete oxidation of DA is indicated by the disappearance of DA (m/z 154) ion signal above 5 V at pH 4 (data not shown) and above 2 V at pH 3 (Fig. 3B). The results show that lower voltage is sufficient for DA oxidation at pH 3, consistent with higher conductivity of pH 3 solution discussed above. At pH 6 in ESI MS with SS emitter, DHI (m/z 150) was detected as the main OP (Scheme 2), consistent with the 2e-, 2H+ DA oxidation pathway in aqueous solutions at higher pH, [5, 9, 28]. In EC/ESI MS (Fig. 3H), DHI ion signal increased some over that in ESI MS ( Fog. 3G) but showed only a small dependence on EC cell voltage in contrast to the ion signal of DHIO (m/z 152) in Figs. 3B and E. DHI is formed by a complex pathway, and requires rapid oxidation of DA during ESI, and rapid cyclization of the initial DA OP DA quinone (DAQ; m/z 152) to DHIO, rapid subsequent oxidation of cyclic DHIO to DHI (Scheme 2). Since DHI was detected, these reactions must be fast during ESI of DA at pH 6. Scheme 2 3.3. Detection of DA Dimers in ESI MS and EC/ESI MS Figure 4 Proton-bound dimers can be detected when ESI in the positive ion mode is less efficient, resulting in proton sharing.[12] Often, under these conditions, radicals are also detected, and this has been interpreted as an indication of inefficient oxidations during ESI.[10, 12]
10
Since in acidic carrier solutions DA is present as [DA+H]+ (m/z 154) positive ion (pKa DA 8.92)[29] ESI of DA typically generates intense [DA+H]+ (m/z 154) ion signals even at low solution concentrations of DA.[10] Nevertheless DA dimers can be detected when DA ESI efficiency decreases.[10] In this work DA dimers were detected at pH 4 when DA ESI was least efficient, as determined from ion signal intensities discussed above. Using pH 4 carrier solution DA dimers (Scheme 3) were detected in ESI MS with SiO2 and SS emitters as shown in Figs. 4A and B. Scheme 3 Figure 5 Fig. 5 illustrates the distribution and intensity of dimer ion signals as a function of ion transition time (ITT), between the hexapole and the ICR cell, from the standard 0.7 to 1 ms using low capillary-skimmer voltage bias. As the results show, at longer ITT, collection of higher molecular weight ions becomes more efficient as the intensity of higher molecular weight dimer ion signals increases. Different distribution of ion signals after ESI of DA at SiO2 and SS in Fig. 4 is a result of different ionization processes at the two emitters. As shown in Fig. 4, in addition to protonbound DA dimers (DA---H+---DA), proton-bound dimers of DA OPs (OP-H+-OP) (Scheme 3) are also detected at longer ITT, allowing detection of DA OPs generated in low yield during ESI, as proton bound dimers with DA and as OP dimers. Figure 6 Fig. 6 shows the results of EC/ESI MS, when SS emitter also acts as the WE that illustrate the effect of EC cell voltage on ion signals of DA dimers. As expected, the results show that with increasing applied EC cell voltage DA oxidation efficiency increases and the intensity of DA-H+- DHIO (m/z 305) and DHIO-H+-DHIO (m/z 303) dimer ion signals increases while
11
ion signal intensity of [DA+H]+ (m/z 154), [DA-NH3+H]+ (m/z 137) and DA-H+-DA dimer (m/z 307) decreases. Figure 7 Efficiency of ESI of DA, and DA oxidation efficiency during ESI, is impacted by flow rate. As shown in Fig. 7 in ESI MS with SS emitter ion signal of m/z 307 DA dimer (DA---H+--DA) is highest at intermediate flow rates. However, longer contact time with the emitter, at low flow rates, improves DA oxidation efficiency, as shown in Fig. 7. Ion signal of the proton-bound dimer of DA OP, DHIO-H+-DHIO (m/z 303) (Scheme 3) is detected only at low flow rates. Under these conditions, however, DA fragmentation is also favored (Fig. 7). As shown in Fig. 7 DA fragmentation is also significant when ESI of DA is efficient at higher flow rates, when DA dimer ion signals are low. 3.4. DA Ion Formation during ESI and in the MS System The results discussed above confirm efficient ESI of DA from solutions containing preformed DA ions. This also means that during positive mode ESI of DA, the ES current is maintained efficiently by oxidations (at the ES emitter), preventing ion (negative) build up at the emitter. Generally the required oxidations during positive mode ESI involve oxidations of carrier solution components at the ES emitter. However, in case of DA, because of low DA oxidation potential of ca. 0.496 V vs SCE,[30, 31] DA oxidation was expected during ESI since the HV applied to the ES emitter was expected to be sufficient for DA oxidation at the emitter during ESI .[32, 33] As discussed above, large ion signals of DA OPs were detected in ESI MS with SS under standard MS conditions, confirming the expected oxidation of DA during ESI. In ESI MS with SiO2, however, only low ion signals of DA OPs were detected, and only after the capillary12
skimmer voltage bias was increased. This indicated lower ionization energy at the resistive SiO2, requiring additional energy for ion dissociation after ESI at SiO2. Since radicals were detected after ESI at SiO2, but not at SS, different oxidation pathways at SiO2 and SS were also indicated by the results during ESI of DA. Figure 8 The results summarized in Fig. 8 were obtained to gain insights into DA ESI processes at SiO2. As shown in Fig. 8, ion signals of DA (m/z 154) and DA OP (m/z 152) increased when the capillary-skimmer voltage bias was increased (in 40-45 V range) while m/z 137 fragment and radical (m/z 151) ion signals (not shown) remained low. Higher voltage bias thus improved sensitivity, confirming the formation of additional ions of DA after the higher voltage was applied, in agreement with improved dissociation of larger ion clusters that apparently formed during ESI at SiO2.[15] Additional increase in voltage (into 55-90 V range) resulted in increased fragmentation with m/z 137 DA fragment ion becoming the base peak (Fig. 8). Formation of m/z 137 ions by fragmentation of DA requires 310 kJ/mol [34, 35] while cleavage of the OH bond of DA quinone requires 338 kJ/mol.[36] As shown in Fig. 8, at high voltage bias DHIO•+ (m/z 151) radical ion signal was the base peak. Clearly OH bond cleavage at higher voltage bias, in this case of DHIO (cyclized DAQ) OH bond (Scheme 1), was efficient. High intensity of DHIO•+ ion signal under these conditions suggests good stability of the radical. Under the same conditions DHI•+ radical was not formed since DHI (m/z 150; Fig. 8) ion signal remained unchanged. As shown by the results in Figs. 2B and C, discussed above, detection sensitivity ESI MS with SiO2 was also improved by fine tuning of the HST. As shown in Fig. 9, at longer HST (up to ca. 0. 6 s) ion signals of DA and DA OPs increased when the capillary-skimmer voltage bias was 50 V. Longer HST thus improved sensitivity in ESI MS with SiO2, presumably as a result of 13
collisional dissociation in the hexapole of ion clusters and aggregates formed during ESI at SiO2. Still longer HST led to increased fragmentation and the m/z 137 fragment ion signal increased. The results showed that lower pressure in the hexapole than in the capillary-skimmer region favored the formation of m/z 137 DA fragment over m/z 151 DHIO•+ radicals. DHI (m/z 150) ion signal increased slowly with the increasing HST while the ion signal of the radical remained low. Figure 9 The main conclusion from the results is that ESI of DA at SiO2 lowered ion signal intensity, especially of the ion signals of DA OPs. Apparently, high resistance of SiO2 lowered ESI energy sufficiently to result in emission of larger aggregates in ESI MS with SiO2 than in ESI MS with SS which is significant in case of DA since DA and its OPs are known to aggregate easily by H-bonding. [37-42] As a result, detection of DA OPs and radicals the ESI MS with SiO2 required additional (collisional) energy for breaking down of the larger aggregates. As discussed above additional energy in ESI MS with SiO2 was supplied by increasing the capillaryskimmer voltage bias or the HST. Formation of DA radicals during ESI at SiO2, presumably by solution reactions at the SiO2 emitter during ESI, was verified by detection of low ion signals of the radicals (not shown) under standard MS conditions, without having to increase the capillary-skimmer voltage bias or the HST. The results also clearly showed that DHIO•+ (m/z 151), but not the DA•+ radicals, formed in the higher pressure capillary-skimmer region of the MS in ESI MS with SiO2. Formation of DA radicals was not detected in the hexapole where the pressure is low. DA radicals were not detected in ESI MS with SS. Proposed DA radical formation pathways are further discussed below. 3.5. DA Oxidation Pathways in ESI MS 14
Detection/identification of high yields of DA OPs after ESI of DA at SS confirmed rapid oxidation of DA in ESI MS with SS from water/methanol carrier solutions containing different electrolytes. Since DA OPs detected by ESI MS with SS were the same as those proposed for the 2e-, 2H+ oxidations of DA in aqueous solutions, [8-10, 28] DA oxidation during ESI at SS likely followed similar pathways. What was unique about ESI MS results with SS emitter was that they allowed detection of DA OPs during ESI at SS. This has been previously possible only in EC/ESI MS with the on-line EC cell after applying voltage to the cell, when the SS emitter also acted as the WE in the cell.[8-10] The proposed pathway of 2e-, 2H+ oxidation of DA during ESI in Scheme 2 was verifiable because of the high sensitivity in detection of DA OPs and their fragments and the high resolution of FTICR MS. DA OP assignment to DHIO was confirmed from its m/z 152 parent ion signal and the signals of fragment ions of DHIO of m/z 123 and 134.[9] Accelerated cyclization of the initial 2e-, 2H+ OP of DA at pH 6, as kcycl DAQ ~1 x 10-3 s-1, can accelerate the formation of DHIO (Scheme 2). [29] This in turn can accelerate oxidation of DHIO (E0’DHIO < E0’DA) to aminochrome (AC) (Scheme 2). AC m/z 150 ion signal was detected under these conditions. This assignment is supported by the m/z 150 of the parent ion and the detection of fragment of m/z 150 ion of m/z 122 that can form by loss of CO from AC.[9] In Scheme 2, AC is shown in equilibrium with DHI (m/z 149) since the m/z 149 ion signal consistent with the formation of DHI•+ was detected. The m/z 150 and 149 ions were not detected at pH 3 when kcycl DAQ ~1.5 x10 -4 -1
s .[29] Lower ESI voltage at resistive SiO2 can be expected to slow oxidations during at SiO2
during ESI and can be expected to result in different oxidation pathways during ESI at SiO2 and SS. As discussed above, DA radicals were detected only in ESI MS at SiO2 but not in ESI MS
15
with SS. Also AC (m/z 150) was detected at pH 4 following ESI at SiO2. In ESI MS with SS it was detected at pH 6 (Fig. 8). Slow apparent oxidation of DA during ESI at SiO2 means that water oxidation has to maintain the ES current at SiO2 during ESI of DA.[18] The expected (slow) 2e-, 2H+ oxidation of water (E0’= 1.51 V vs. SCE pH 4.3; 4e-, 4H+ process is much slower) generates hydrogen peroxide (reactive oxygen species; ROS), which is expected to contribute to generation of OH• radicals during ESI of DA. [43] As a result, DA can react with OH• ROS during ESI. DA reactions with ROS have been predicted to follow electron-proton transfer pathways, involving sequential electron-proton transfer (SEPT with 27.3% yield), proton coupled electron transfer (PCET with 9.7 % yield), as well as direct hydrogen atom (H•) transfer (HAT with 7.3% yield).[4] DA•+ radical is the main predicted DA OP in these oxidations. [DA]•+ (m/z 153) was detected by ESI MS with SiO2. Low ion signal and short HST required for [DA]•+ detection suggested low stability of the radical. Additionally, DA+(OH)• adducts have been predicted as products of DA oxidation in the electron-proton transfer pathways, following sequential electron-proton transfer pathway (RAF, 55.8% yield).[4] In ESI MS with SiO2 DA+(OH)• adducts 6OHDA and PTQ (Scheme 1) were detected as predicted. The results indicate that DA oxidation during ESI at SiO2 follows the electron-proton transfer pathways as well as the pathways predicted for in vivo DA oxidation[3] predicted by theory.[4] Competition, between oxidation and hydrogen transfer reactions in these pathways at different pH was confirmed by detection of DA+(OH)• adducts at lower pH, as 6OHDA (Scheme 1). ESI MS and EC/ESI MS results confirmed the differences in DA oxidation pathways at SS and SiO2 emitters. The results demonstrated the formation of ROS during slow oxidation of
16
DA in ESI MS with SiO2 and during collisions in the higher pressures capillary-skimmer region of the MS system. 3.6. Biological Significance of DA Oxidation Pathways in ESI MS The proposed toxic activity of DA in vivo has been linked to interactions of DA OPs, DA quinones and DA radical, with cellular components, leading to irreversible cellular damage and neurodegeneration. Based on the theoretical predications discussed above formation of DA radicals by rapid reactions with ROS is significant to DA reactivity in vivo.[4] However, in spite of many investigations, DA radicals have not been identified previously. Three DA radicals were identified in this work, allowing insights into radical stability, structure, and formation pathways that show apparent agreement with DA oxidation pathways proposed in vivo. The results indicate that the radicals form under different oxidation conditions (Scheme 1 and 2) and, additionally, indicated different stability of the radicals. DHIO•+ (m/z 151) radicals were shown as stable, while DA•+ (m/z 153) radicals were not as stable. Stability of DHI•+ (m/z 149) radicals is less clear due to low yields of the radicals that may result of low stability or complex formation pathway of the radical (Scheme 2). Formation of DHIO•+ and DHI•+ radicals requires cyclization of the initial DA OP DAQ (Scheme 1 and 2) that is favored at higher pH. Consequently, the radical formation could occur in vivo under conditions which favor slow oxidations of DA, during reactions with oxidizing agents such as ROS, such as the slow DA oxidation in ESI MS with SiO2. 4. Conclusions This work demonstrates the formation and detection of three radicals of DA and its OPs in ESI MS with SiO2. ESI at SiO2 favors slow oxidation of DA during ESI because of the resistance of SiO2 which can lower ESI voltage at the SiO2 emitter during ESI. Detection of
17
transient DA radicals and DA OPs was feasible in ESI MS with SiO2 because of the high sensitivity that was achieved by fine tuning MS operating conditions in ESI MS with SiO2. The results allowed identification of DA radicals and provided new information about the reaction conditions during DA oxidation that favor formation of DA radicals. The results show that DA radicals can form during slow oxidation of DA and in the higher pressure region of the MS system. The results provided experimental verification of the theoretically predicted proton coupled electron transfer oxidation pathway of DA that results in radical formation. The results also confirmed an alternative rapid 2e-, 2H+ oxidation pathway of DA that, based on the results, was followed in ESI MS of DA with SS emitter by the previously proposed solution pathways. Under these conditions formation of DA radicals was not detected. Rapid oxidation of DA by 2e-, 2H+ pathway, previously verified by EC/ESI MS, was confirmed in this work. Additionally, ESI MS results allowed identification of DA OPs formed at different pH. Sensitivity improvements that were demonstrated in this work in ESI MS were accomplished using a new approach that resulted in enhancement of ion dissociation in the MS system. The results illustrate the effect of the energies of ionization and collisions, as well as ion solvation and aggregate formation, on detection sensitivity in ESI MS. Improved sensitivity was required in this work in ESI MS with SiO2 emitter for detection of transient radicals and 2e-, 2H+ DA OPs. Sensitivity of ESI MS with SS emitter was sufficient for detection of DA OPs under standard MS operating conditions. Ion assignments were possible because of the high MS detection sensitivity and the high resolution of FTICR MS. The results provide new information about conditions favoring formation of a range of transient intermediates and products of DA oxidations that can be relevant to the biological reactivity of DA. In addition, the results provide information about the ESI processes that impact
18
sensitivity in ESI MS, of value in optimizing MS measurement sensitivity, and in the investigations of transient chemical reactions by MS. As the results demonstrate, relatively simple modifications of the MS operating conditions in ESI MS resulted in high detection sensitivity that was necessary for the detection of transient radicals and OPs. Simple change of the emitter material allowed access to new information. Previously, by using SS ES emitter as the WE in a floated on-line EC cell in EC/ESI MS, a simple approach, allowed SS emitter voltage to be easily fine-tuned as needed for improving detection sensitivity in EC/ESI MS by improving the ESI efficiency. This was exploited in this work for improving oxidation efficiency of DA in ESI MS with SS. The results provide evidence of DA oxidations that can generate different products during ESI and in the MS system and demonstrate which conditions can favor oxidations forming radicals in ESI MS, which has not been reported previously. It is of interest that DA oxidation pathways in ESI MS indicated by the results, while providing new information, are in excellent agreement with the previously proposed DA oxidation pathways, proposed from chemical and enzymatic as well as electrochemical studies in aqueous solutions. Acknowledgements The authors wish to thank Dr. John R. Eyler and Dr. Nick C. Polfer for providing access to the FT-ICR MS and for helpful technical guidance. The authors also thank Brooke E. Barnes and Alec G. Mackinnon for their help with the experiments.
19
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[17] D.W. Looi, J.R. Eyler, A. Brajter-Toth, Electrochemistry-electrospray ionization FT ICR mass spectrometry (EC ESI MS) of guanine-tyrosine and guanine-glutathione crosslinks formed on-line, Electrochimica Acta, 56 (2011) 2633-2640. [18] D.W. Looi, I. Iftikhar, A. Brajter-Toth, Electrochemical attributes of electrochemistry in tandem with electrospray mass spectrometry, Electroanal., 26 (2013) 1-9. [19] G.T. Blakney, C.L. Hendrickson, A.G. Marshall, Predator data station: A fast data acquisition system for advanced FT-ICR MS experiments, International Journal of Mass Spectrometry, 306 (2011). [20] L. Tang, P. Kebarle, Effect of the conductivity of the electrosprayed solution on the electrospray current - factors determining analyte sensitivity in electrospray mass-spectrometry, Analytical Chemistry, 63 (1991) 2709-2715. [21] M. Oss, A. Kruve, K. Herodes, I. Leito, Electrospray ionization efficiency scale of organic compounds, Analytical Chemistry, 82 (2010) 2865-2872. [22] R. Abburi, S. Kalkhof, R. Oehme, A. Kiontke, C. Birkemeyer, Artifacts in amine analysis from anodic oxidation of organic solvents upon electrospray ionization for mass spectrometry, European Journal of Mass Spectrometry, 18 (2012) 301-312. [23] J. Liigand, A. Kruve, I. Leito, M. Girod, R. Antoine, Effect of mobile phase on electrospray ionization efficiency, Journal of the American Society for Mass Spectrometry, 25 (2014) 1853-1861. [24] L.E. Sojo, N. Chahal, B.O. Keller, Oxidation of catechols during positive ion electrospray mass spectrometric analysis: Evidence for in-source oxidative dimerization, Rapid Communications in Mass Spectrometry, 28 (2014) 2181-2190. [25] J.F. Banks, S. Shen, C.M. Whitehouse, J.B. Fenn, Ultrasonically assisted electrospray-ionization for lc/ms determination of nucleosides from a transfer-rna digest, Analytical Chemistry, 66 (1994) 406-414. [26] T. Zhang, S.P. Palii, J.R. Eyler, A. Brajter-Toth, Enhancement of ionization efficiency by electrochemical reaction products in on-line electrochemistry/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, Anal. Chem., 74 (2002) 1097-1103. [27] B.M. Ehrmann, T. Henriksen, N.B. Cech, Relative importance of basicity in the gas phase and in solution for determining selectivity in electrospray ionization mass spectrometry, Journal of the American Society for Mass Spectrometry, 19 (2008) 719-728. [28] D.C.S. Tse, R.L. McCreery, R.N. Adams, Potential oxidative pathways of brain catecholamines, Journal of Medicinal Chemistry, 19 (1976) 37-40. [29] M.D. Hawley, Tatawawa.Sv, Piekarsk.S, R.N. Adams, Electrochemical studies of oxidation pathways of catecholamines, Journal of the American Chemical Society, 89 (1967) 447-&. [30] Y.Z. Song, Y. Song, Experimental and theoretical study of the electrochemical behavior of Nprotonated dopamine at Nafion multiwalled carbon nanotubes (MWNTs) modified glassy carbon (GCE) electrode, Canadian Journal of Chemistry-Revue Canadienne De Chimie, 84 (2006) 1084-1092. [31] H. Mohammad-Shiri, M. Ghaemi, S. Riahi, A. Akbari-Sehat, Computational and Electrochemical Studies on the Redox Reaction of Dopamine in Aqueous Solution, International Journal of Electrochemical Science, 6 (2011) 317-336. [32] A.T. Blades, M.G. Ikonomou, P. Kebarle, Mechanism of electrospray mass-spectrometry electrospray as an electrolysis cell, Analytical Chemistry, 63 (1991) 2109-2114. [33] G.J. Vanberkel, F.M. Zhou, Electrospray as a controlled current electrolytic cell - electrochemical ionization of neutral analytes for detection by electrospray mass-spectrometry, Analytical Chemistry, 67 (1995). [34] S.W. Benson, BOND ENERGIES, Journal of Chemical Education, 42 (1965) 502-&. [35] J.A. Kerr, Bond dissociation energies by kinetic methods, Chemical Reviews, 66 (1966) 465-&. [36] V.T. Varlamov, B.E. Krisyuk, A.V. Antonov, O-H bond dissociation energies in hydroquinones and 4hydroxyphenoxyl radicals and effect of solvation on the kinetics of reactions involving hydroquinones and semiquinone radicals, Russian Chemical Bulletin, 54 (2005) 2317-2324.
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[37] G. Alagona, C. Ghio, The effect of intramolecular H-bonds on the aqueous solution continuum description of the N-protonated form of dopamine, Chemical Physics, 204 (1996) 239-249. [38] J.D. Steill, J. Szczepanski, J. Oomens, J.R. Eyler, A. Brajter-Toth, Structural characterization by infrared multiple photon dissociation spectroscopy of protonated gas-phase ions obtained by electrospray ionization of cysteine and dopamine, Analytical and Bioanalytical Chemistry, 399 (2011) 2463-2473. [39] D.R. Dreyer, D.J. Miller, B.D. Freeman, D.R. Paul, C.W. Bielawski, Elucidating the Structure of Poly(dopamine), Langmuir, 28 (2012) 6428-6435. [40] M.E. Tessensohn, H. Hirao, R.D. Webster, Electrochemical Properties of Phenols and Quinones in Organic Solvents are Strongly Influenced by Hydrogen-Bonding with Water, Journal of Physical Chemistry C, 117 (2013) 1081-1090. [41] S.K. Callear, A. Johnston, S.E. McLain, S. Imberti, Conformation and interactions of dopamine hydrochloride in solution, Journal of Chemical Physics, 142 (2015). [42] A. Bouchet, M. Schutz, O. Dopfer, Competing Insertion and External Binding Motifs in Hydrated Neurotransmitters: Infrared Spectra of Protonated Phenylethylamine Monohydrate, Chemphyschem, 17 (2016) 232-243. [43] I.L. Kanev, A.Y. Mikheev, Y.M. Shlyapnikov, E.A. Shlyapnikova, T.Y. Morozova, V.N. Morozov, Are reactive oxygen species generated in electrospray at low currents?, Analytical Chemistry, 86 (2014) 1511-1517.
22
Figure 1. Schematic diagram of on-line electrochemistry ESI MS system (EC/ESI FTICR MS), showing the electrochemical (EC) cell. The EC cell consists of two SS capillaries joined by plastic tubing, with one SS capillary also serving as the ES emitter capillary. Different pressure regions are illustrated.
23
[DA+H] 154
100
+
(a)
75 50 25
Intensity (%)
0 100
[DA+H-NH3]
+
(b)
137
75
•+
[PTQ+H]+ 168 [6OHDA+H]+ 170
[DHIO]
50
151 154
25 0 137
100
(c)
75 50 [DA] 153
25 0 120
130
140
150
•+
160
170
180
m/z Figure 2. ESI MS of DA (100µM) in 50/49/1 vol %, MeOH/H2O/FA, pH~ 3, standard ESI MS conditions with capillary voltage (Vc) 40 V, hexapole storage time (HST) 0.6 s (A), Vc 90 V, HST 0.6 s (B), Vc 90 V, HST 0.3 s (C). Absolute base peak intensity: (A) 10k; (B) 7k: (C) 7k. Flow rate 50 µL/h.
24
100
100
+
[DHIO+H] 152
(a)
80
[DA+H] 154
80
60
60 [DA+H] 154
+
[152-CH2NH] 20
Intensity (%)
100
120
140
150
160
170
+
[DA+H-NH3] 20
123
0 110 100
152
(b)
40
[152-CH2NH]
20
130
+
[DHI+H] 150
+
123
0 110
60
+
40
+
[DA+H] 154
(g)
80
[DHIO+H] 152
+
40
100
+
(d)
120
130
140
(e)
150 152
160 154
137
0 170 110 100
80
80
80
60
60
60
40
40
120
130
0 110 100
120
(c)
130
140
150
160
170
80
170
154
150
137
20
0 110 100
123
160
40
20
123
150
(h)
123 20
140
120
130
140
150
160
100
137
(f)
0 170 110
80
120
130
140
150
160
170
160
170
137
(i)
80 +
60
60
[137-H2O] 119
+
[DA+H-NH3] 40
+
[152-H2O]
+
40
137
134
20
60
123 40 154
20
20
152 0 110
120
130
140
150
160
170
0 110
120
130
140
150
160
0 170 110
150
[150-CO] 122
154
119 120
130
140
150
m/z
Figure 3. ESI MS of DA (0.25 mM) ) at pH~ 3,4 and 6 respectively, standard ESI conditions, Vc 40 V, Flow rate 30 µL/h. HST 0.6 s, (A,D,G); EC/ESI MS, EC Cell Voltage 2.0 V, Vc 40 V (B,E,H); ESI MS, conditions same as (A,D,G), Vc 90 V (C,F,I). 25
100
154
(a)
[DA+DHIO+H] 305
80
[2DHIO+H] 303
60
+
[2DA+H] 307
+
+
40 152
Intensity (%)
20 0 120 100
140
160 154
180
200
220
240
260
280
307
60
305
152 137 303
20 0 120
320
(b)
80
40
300
140
160
180
200
220
240
260
280
300
320
m/z
Figure 4. ESI MS of DA (0.25 mM) in 50/49/1 vol.% in MeOH/H2O/HAc, pH ~ 4, Vc 40 V, HST 0.4 s, ITT 1.0 ms, silica ES emitter (A) stainless steel ES emitter (B).
26
m/z 154 m/z 301 m/z 303 m/z 305 m/z 307
100
Intensity (%)
80
60
40
20
0 -4
7.0x10
-4
-4
8.0x10
9.0x10
-3
1.0x10
Ion Transition Time (s)
Figure 5. Plot of DA and its dimers intensity vs ion transition time. Conditions as in Fig. 4 A.
27
m/z 152 m/z 303 m/z 307 m/z 137
80 70
Intensity (cps)
60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
EC Cell Voltage (V)
Figure 6. Effect of EC cell voltage on the intensity of DA ions. Conditions same as in 4 B.
28
m/z 154x10 m/z 152 m/z 137x3 m/z 303 m/z 307
300
250
Intensity (cps)
200
150
100
50
0 20
40
60
80
100
Flow Rate (uL/hr)
Figure 7. Effect of flow rate on the intensity of DA ions. Conditions same as in 4 B.
29
800
m/z 154x10 m/z 137x10 m/z 152 m/z 150 m/z 151
Intensity (cps)
600
400
200
0 30
40
50
60
70
80
90
Exit Capillary Voltage (Vc)
Figure 8.
Plot of DA ions intensity vs capillary voltage (Vc). 100 µM (DA) in 50/49/1 vol/% in MeOH/H2O/HAc , pH 4, HST 0.6 s, ITT 0.7 ms.
30
800
Intensity (a.u.)
600
m/z 154x10 m/z 137x10 m/z 152 m/z 150
400
200
0 0.2
0.4
0.6
0.8
1.0
Hexapole Storage Time (s)
Figure 9.
Plot of intensity vs hexapole storage time. DA (100 µM) in 50/49/1 vol/% in MeOH/H2O/HAc , pH 4, Vc 50 V, ITT 0.7 ms.
31
Scheme 1. Proposed DA oxidation pathways using silica ES emitter.
32
Scheme 2. Proposed DA oxidation pathways using a stainless steel ES emitter.
Scheme 3. Proposed proton bound dimers of DA and its oxidation product DHIO. 33
S0013-4686(17)31505-0 http://dx.doi.org/doi:10.1016/j.electacta.2017.07.087 EA 29904
To appear in:
Electrochimica Acta
Received date: Revised date: Accepted date:
10-5-2017 13-7-2017 14-7-2017
Please cite this article as: Imran Iftikhar, Kholoud Mohammed Abou El-Nour, Anna Brajter-Toth, Detection of transient dopamine antioxidant radicals using electrochemistry in electrospray ionization mass spectrometry, Electrochimica Actahttp://dx.doi.org/10.1016/j.electacta.2017.07.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of transient dopamine antioxidant radicals using electrochemistry in electrospray ionization mass spectrometry
Imran Iftikhar1, Kholoud Mohammed Abou El-Nour2, Anna Brajter-Toth1,*
1
Department of Chemistry, University of Florida, Gainesville, Florida, 32611-7200, USA
2
Department of Chemistry, Faculty of Science, University of Suez Canal, Ismailia, 41522, Egypt
*Corresponding Author: Tel: +1352 392 7972/ Fax: +1352 392 4651 E-mail address: [email protected]
Abstract We describe formation and detection of transient radical intermediates and 2e-, 2H+ oxidation products (OPs) of dopamine (DA) by electrospray ionization (ESI) mass spectrometry (MS) which has not been reported previously. The results confirm formation of DA radicals by proton coupled electron transfer pathway. DA radicals and 2e-, 2H+ oxidation products that were identified are of interest because of their reported neurotoxicity. Using different solution compositions and MS detection conditions three different DA radicals were identified that have different stability. DA reactions with reactive oxygen species (ROS) relevant to DA reactivity in vivo are discussed. Detection of DA OPs by electrochemistry/mass spectrometry with a floated on-line electrochemical cell is also illustrated.
1
Key wards Dopamine radicals; Electrochemistry; Electrospray Ionization; Mass Spectrometry; Neurotoxicity 1. Introduction In recent years major efforts have been directed at unraveling complex processes that contribute to neurodegenerative diseases.[1, 2] In these studies, surprisingly, endogenous neurotransmitters have been identified as potential sources of toxic products.[1-3] As a result, dopamine (DA), a significant catecholamine neurotransmitter in the brain,[2] has been investigated in some detail as a potential toxic agent. Based on these studies it has been proposed that in vivo reactions of DA with oxygen can generate toxic oxidation products (OPs). This can occur during oxidative stress, after DA release into cytosol from storage vesicles when DA reabsorption into the vesicles is not efficient. DA can be oxidized in the cytosol, where higher pH than in the vesicles facilitates the oxidation. Transient semiquinone radicals that were proposed to form during the oxidation have been proposed to be highly toxic even at low concentrations. However, the previously proposed toxicity of 2e-, 2H+ DA quinone OPs has been questioned.[3] Complex oxidations such as the oxidation of DA (a hydroquinone) can generate different intermediates and nucleophilic reaction products under different reaction conditions. Protoncoupled electron transfer (PCET)[4] reactions of DA forming DA radicals that can involve oxygen and reactive oxygen species (ROS) have been proposed for DA oxidations in vivo [3, 57]. These reactions have been difficult to study because of the limited availability of suitable methods. Limited information about DA semiquinone radicals has been obtained from electron spin resonance (ESR) studies in vitro.[6, 7] In ESR, unstable radical signals have been reported
2
that were attributed to unstable cyclized DA semiquinone.[3] Signals of longer-lived radical (t1/2 obs
= 79 s at pH 6.5) have also been detected and were attributed to another semiquinone.[7]
Chemical characterization of the radicals was not feasible by ESR. Other DA OPs have been detected and identified by electrochemistry on-line with mass spectrometry (EC/MS) during DA oxidation [8-10] and recently by NMR during chemical oxidation of DA.[11] Detection of DA radicals was not reported in these studies.[8-11] Based on prior work[12] we have postulated that under optimized conditions detection of DA radicals should be feasible by ESI MS. Detection of transient radical intermediates has been demonstrated in previous work in different ESI MS investigations. [12, 13] Detection of transient DA radical intermediates and OPs is demonstrated in this work. Characterization of DA radicals and DA OPs was possible by ESI MS. DA OPs were also detected under different conditions during fast oxidation of DA in EC/ESI MS. Additional merits of EC/ESI MS were realized in this work, providing kinetic information during electrochemical oxidation of DA under different conditions.[12, 14] Three DA radicals were detected by ESI MS. The results show that DA radicals are formed by proton-coupled electron transfer (PCET)[4] reactions pathways as a result of slow oxidation of DA in ESI MS with silica (SiO2) capillary as the electrospray (ES) emitter. Lower ionization energy observed during ESI with SiO2 emitter requires additional energy to be supplied for the detection of DA radicals and other OPs formed in low yields in ESI MS.[15, 16] Fast 2e-, 2H+ oxidation of DA was demonstrated during ESI MS with stainless steel (SS) emitter and in EC/ESI MS. In EC/ESI MS the SS ES emitter also acts as the working electrode in the online two electrode EC cell. The EC cell voltage is floated on the HV applied to the ES emitter during ESI. The applied EC cell voltage changes the effective voltage of the SS ES emitter
3
during ESI. The results demonstrate that DA oxidation is rapid at the SS ES emitter during ESI and confirm that the rate of DA oxidation can be increased in EC/ESI MS by applying voltage to the EC cell. 2. Experimental 2.1. Materials Dopamine hydrochloride (98%) was purchased from Aldrich (St. Louis, MO). Formic acid (FA), glacial acetic acid (HAc) (99.9%), ammonium acetate (NH4Ac) and methanol (HPLC grade) were from Fisher (Pittsburgh, PA). All chemicals were used as received. Carrier solutions used in ESI MS were: (i) 50/49/1 vol% MeOH/H2O/FA, pH ~ 3, 1568 mS/cm, (ii) 50/49/1 vol% MeOH/H2O/HAc, pH ~ 4, 121.8 mS/cm, and (iii) 60/40/vol% MeOH/H2O/1 mM NH4Ac, pH ~ 6, 59.9 mS/cm.[12] Solution conductivity was determined at 250C using a conductivity probe calibrated in 0.01 M KCl (1413 mS/cm) and Vernier LabQuest computer interface. All solutions were freshly prepared with doubly deionized water. 2.2. Mass Spectrometry The APEX 4.7-T FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA) with a modified ESI ion source has been described in detail previously.[10, 17, 18] A cone shaped MS capillary inlet made of brass (500 divergence angle from 1.58 mm i.d. to 6.13 mm orifice diameter) was used.[10] The MS system is schematically illustrated in Fig. 1. Figure 1 Base peak intensity was similar using fused silica (100 µm i.d.) or cylindrical stainless steel (SS; 80 µm i.d.) capillaries as the electrospray (ES) emitter. A syringe pump (Cole Parmer 74900, Vernon Hills, Il) provided constant solution flow to the ES capillary. Using a silica ES emitter in the 20 to 100 µL/h flow rate range better signals were observed at 50 µL/h. Standard
4
MS parameters were: hexapole storage time (HST), 0.5 s; ion transition time (ITT), 0.7 ms; skimmer voltage, 3.0–5.0 V; High voltage (HV) ~ 3kV; exit capillary voltage (Vc), ~ 40 V; number of scans, 20. The MS inlet capillary was heated to 1400 C. To induce ion dissociation in the capillary-skimmer region, exit capillary voltage was increased up to 90 V, which increased capillary-skimmer voltage bias to ~ 85 V. DA dimers were detected by increasing ion transition time from 0.7 ms to 1.0 ms. Mass spectra were obtained using Predator (version 1.2) and Modular ICR Data Acquisition and Analysis System (MIDAS) software[19] written at the National High Magnetic Field Laboratory (Tallahassee, Fl). Calculated exact masses were used to verify ion assignments. Additionally, relative intensities of isotopic peaks were used in ion assignments. The mass error was between _2 to +10 ppm. 2.3. On-Line Electrochemistry ESI MS (EC/ESI MS) Design and operation of the EC cell in the EC/ESI FTICR MS system with APEX 4.7-T FTICR mass spectrometer (Fig. 1) has been described.[10, 17, 18] The SS ES emitter capillary and a second SS capillary (5 and 4 cm long), separated by low volume Teflon tubing, formed the on-line EC cell, with SS counter and working electrodes. The SS working electrode additionally functioned as the ES emitter in EC/ESI MS. SS capillaries were attached to an adjustable xyz micropositioner (World Precision Instruments, Sarasota, Fl). The dc voltage was applied to the EC cell from a 9 V battery and was adjusted using a variable resistor (maximum 50 kΩ). The EC cell voltage was floated on the HV of the ES emitter. In EC/ESI MS solution flow rate was 30µL/h, which increased residence time of analyte at the SS WE. 3. Results and Discussion 3.1. DA Reactions in ESI MS with Silica ES Emitter Figure 2 5
Positive ion mode mass spectrum of DA obtained from pH 3 (50/49/1 vol% MeOH/H2O FA) carrier solution under standard ESI MS operating conditions, with silica (SiO2) capillary as the ES emitter, is shown in Fig. 2A. The mass spectrum shows an intense (10,000 cps) ion signal of [DA+H]+ (m/z 154) base peak together with the isotope ion peak. As shown in Fig. 2B, using higher capillary-skimmer voltage bias allows detection of new ion signals and under these conditions DA ion fragment (m/z 137) signal becomes the base peak. The new ion signals in Fig. 2B can be assigned to DA OPs (Scheme 1) discussed further below, 6-hydroxy dopamine 6-OHDA (m/z 170), p-topaminequinone PTQ (m/z 168), and 5,6dihydroxyindoline radical (DHIO•+ m/z 151). As shown in Fig. 2C, by (additionally) lowering the (standard) hexapole storage time (HST) from 0.6 to 0.3 s a new m/z 153 ion signal was detected that was assigned to DA semiquinone radical [DA]•+ (Scheme 1). Scheme 1 The ESI MS results in Figs. 2B and C confirm that DA ESI at SiO2 results in detection of DA radicals and other OPs. Oxidation reactions during ESI at SiO2 thus provide access to chemical/structural information not accessible by other methods about transient intermediates and OPs, with time resolution of ca. 3 ms required for ion transition from the ES interface to the ICR cell. In agreement with previous reports of outcomes of ESI under conditions that are unfavorable for analyte oxidations during ESI [12] formation of DA radicals was detected following ESI of DA at SiO2. DA oxidation is limited (is slow) under these conditions because of the lower effective voltage at the emitter due to its high resistance. ESI energy under these conditions is thus also lower, resulting in lower ion energy. As a result, in order to aid ion
6
dissociation for detection of DA radicals and DA OPs higher capillary-skimmer voltage bias or longer HST was required. The results in Fig. 2 point to the formation of two DA radicals (m/z 151 and 153) that have different stability. In previous ESR investigations of enzymatic and chemical oxidation of DA detection of DA radicals with different stability was justified by the structures proposed for the radicals.[3, 7] The reported ESR results are supported by this work. It is worth noting that high sensitivity realized using higher capillary-skimmer voltage bias was required for detection of DA radicals and DA OPs in ESI MS with SiO2. All subsequent structural assignments that were made were possible because of the high sensitivity and were made by exploiting the high resolution of FT ICR MS. Standard MS/MS methods, typically used for structural assignments, do not have the sensitivity and time resolution required for detection of the transient species present at low concentrations; consequently fragmentation products, required for structural assignment, would also not be detectable. Based on the results that were obtained by using different compositions of carrier solutions, limited oxidation of DA during ESI with SiO2 emitter was confirmed, independently of the carrier solution used. However, different OPs were detected in different carrier solutions, and at different pH. Using higher capillary-skimmer voltage bias and pH ~ 4 carrier solution m/z 152, 149, 134 and 123 ion signals were detected that were assigned to DA OP 5,6dihydroxyindoline DHIO (m/z 152) and its fragments [152-H2O]+ (134), [152-CH2NH]+ (m/z 123), and of DHI•+ radical (m/z 149) (data not shown). 3.2. DA Oxidation in ESI MS and EC/ESI MS with Stainless Steel (SS) ES Emitter Figure 3
7
Efficiency of ionization in ESI MS is determined by the experimental parameters that include the design of the ionization interface and the MS optics, as well as the MS operating conditions.[10, 13] In addition, ESI efficiency is impacted by the composition and pH of the carrier solution.[12, 20-25] In many cases this is an advantage of ESI MS method since small changes, in carrier solution composition and/or instrumental parameters, allow fine-tuning of the ESI efficiency. This is well-documented in EC/ESI MS where ESI efficiency can be improved by simply applying additional voltage to the SS ES emitter, which is used in EC/ESI MS as the on-line WE in the floated two electrode EC cell.[10, 26] Comparison of the results in Figs. 2A and 3A illustrates the impact of ES emitter material on ion signal distribution in ESI MS with SiO2 (Fig. 2A) and SS (Fig. 3A). Using SS emitter and pH 3 carrier solution, under the same MS conditions as in Fig. 2A where SiO2 ES emitter was used, m/z 152 ion signal of DA OP is the base peak, rather than the ion signal of DA (m/z 154). Oxidation of DA in ESI MS with SS is apparent from the results in Fig. 3A. ESI MS results with SS emitter in Fig. 3A show high intensity of DA (m/z 154) ion signal, relative to the intensity of DA OP (m/z 152) base peak, indicating high (ca. 80%) oxidation efficiency of DA in ESI MS at SS. Results from EC/ESI MS in Fig. 3B, where the SS emitter also acts as the WE in the on-line two electrode EC cell, show that after voltage is applied to the SS emitter/WE in the EC cell DA oxidation appears complete since DA (m/z 154) ion signal disappears. The results in Fig. 3C (and in Figs. 3F and I) were obtained after high capillary-skimmer voltage bias was used in ESI MS with SS. The results show that under these conditions only fragment ions were detected, consistent with high energy during ESI at SS resulting in extensive fragmentation of emitted ions when the capillary-skimmer voltage bias was increased. In
8
contrast, in ESI MS with SiO2 emitter, as shown by the results in Figs. 2B and C, higher capillary- skimmer voltage bias improved detection of DA radicals and DA OPs. Mass spectra of DA at pH 4 and 6 were obtained by ESI MS with SS emitter. As shown by the results DA OPs in ESI MS with SS emitter are easily detected at pH 4 and 6 under standard MS conditions. The results support DA oxidation during ESI at SS at pH 4 and 6 that is not detected under standard conditions in ESI MS with SiO2 (results not shown). Since in the mass spectra in Figs. 3D and G DA (m/z 154) ion signal is the base peak the results indicate less efficient oxidation of DA at pH 4 and 6 than at pH 3 (Fig. 3A). It is likely that lower oxidation efficiency of DA in ESI MS from these solutions is a result of lower conductivity of pH 4 (121.8 µS/cm) and 6 (59.9 µS/cm) solution than of the pH 3 solution (1568 mS/cm).[18, 20] Better ionization efficiency of DA at pH 6 than 4 is also indicated by the results in Figs. 3D and G, from higher intensity of DA (m/z 154) base peak ion signal at pH 6 than pH 4. This appears to be inconsistent with lower conductivity of pH 6 solution and typically low ESI efficiency at higher pH, when fewer [H+] ions are present in solution. It is therefore likely that ESI of preformed [DA+H]+ ions is not limited by solution conductivity but is, alternatively, determined by the efficiency of acid-base and other reactions during ESI.[12, 27] However, in ESI MS with SS emitter, as shown from the mass spectra in Figs. 3D and G, solution conductivity impacts oxidation efficiency of DA based on the relative intensities of DA and DA OP ion signals in the mass spectra. In the final outcome it is the ES emitter material that has the largest impact on DA oxidation efficiency during ESI, as shown by the results (Figs. 2A and 3A, D and G) which show low ion signals of DA OPs in ESI MS with SiO2 and high OP ion signals in ESI MS with SS.
9
In agreement with previously proposed 2e-, 2H+ oxidation pathways of DA in acidic aqueous solutions, [5, 8, 9, 28] at pH 3 and 4 DHIO (m/z 152) was the main DA OP that was detected in ESI MS at low pH (Scheme 2). In EC/ESI MS (Fig. 3B and E) ion signal of DHIO increased with increasing EC cell voltage, as expected. As discussed above, in EC/ESI MS complete oxidation of DA is indicated by the disappearance of DA (m/z 154) ion signal above 5 V at pH 4 (data not shown) and above 2 V at pH 3 (Fig. 3B). The results show that lower voltage is sufficient for DA oxidation at pH 3, consistent with higher conductivity of pH 3 solution discussed above. At pH 6 in ESI MS with SS emitter, DHI (m/z 150) was detected as the main OP (Scheme 2), consistent with the 2e-, 2H+ DA oxidation pathway in aqueous solutions at higher pH, [5, 9, 28]. In EC/ESI MS (Fig. 3H), DHI ion signal increased some over that in ESI MS ( Fog. 3G) but showed only a small dependence on EC cell voltage in contrast to the ion signal of DHIO (m/z 152) in Figs. 3B and E. DHI is formed by a complex pathway, and requires rapid oxidation of DA during ESI, and rapid cyclization of the initial DA OP DA quinone (DAQ; m/z 152) to DHIO, rapid subsequent oxidation of cyclic DHIO to DHI (Scheme 2). Since DHI was detected, these reactions must be fast during ESI of DA at pH 6. Scheme 2 3.3. Detection of DA Dimers in ESI MS and EC/ESI MS Figure 4 Proton-bound dimers can be detected when ESI in the positive ion mode is less efficient, resulting in proton sharing.[12] Often, under these conditions, radicals are also detected, and this has been interpreted as an indication of inefficient oxidations during ESI.[10, 12]
10
Since in acidic carrier solutions DA is present as [DA+H]+ (m/z 154) positive ion (pKa DA 8.92)[29] ESI of DA typically generates intense [DA+H]+ (m/z 154) ion signals even at low solution concentrations of DA.[10] Nevertheless DA dimers can be detected when DA ESI efficiency decreases.[10] In this work DA dimers were detected at pH 4 when DA ESI was least efficient, as determined from ion signal intensities discussed above. Using pH 4 carrier solution DA dimers (Scheme 3) were detected in ESI MS with SiO2 and SS emitters as shown in Figs. 4A and B. Scheme 3 Figure 5 Fig. 5 illustrates the distribution and intensity of dimer ion signals as a function of ion transition time (ITT), between the hexapole and the ICR cell, from the standard 0.7 to 1 ms using low capillary-skimmer voltage bias. As the results show, at longer ITT, collection of higher molecular weight ions becomes more efficient as the intensity of higher molecular weight dimer ion signals increases. Different distribution of ion signals after ESI of DA at SiO2 and SS in Fig. 4 is a result of different ionization processes at the two emitters. As shown in Fig. 4, in addition to protonbound DA dimers (DA---H+---DA), proton-bound dimers of DA OPs (OP-H+-OP) (Scheme 3) are also detected at longer ITT, allowing detection of DA OPs generated in low yield during ESI, as proton bound dimers with DA and as OP dimers. Figure 6 Fig. 6 shows the results of EC/ESI MS, when SS emitter also acts as the WE that illustrate the effect of EC cell voltage on ion signals of DA dimers. As expected, the results show that with increasing applied EC cell voltage DA oxidation efficiency increases and the intensity of DA-H+- DHIO (m/z 305) and DHIO-H+-DHIO (m/z 303) dimer ion signals increases while
11
ion signal intensity of [DA+H]+ (m/z 154), [DA-NH3+H]+ (m/z 137) and DA-H+-DA dimer (m/z 307) decreases. Figure 7 Efficiency of ESI of DA, and DA oxidation efficiency during ESI, is impacted by flow rate. As shown in Fig. 7 in ESI MS with SS emitter ion signal of m/z 307 DA dimer (DA---H+--DA) is highest at intermediate flow rates. However, longer contact time with the emitter, at low flow rates, improves DA oxidation efficiency, as shown in Fig. 7. Ion signal of the proton-bound dimer of DA OP, DHIO-H+-DHIO (m/z 303) (Scheme 3) is detected only at low flow rates. Under these conditions, however, DA fragmentation is also favored (Fig. 7). As shown in Fig. 7 DA fragmentation is also significant when ESI of DA is efficient at higher flow rates, when DA dimer ion signals are low. 3.4. DA Ion Formation during ESI and in the MS System The results discussed above confirm efficient ESI of DA from solutions containing preformed DA ions. This also means that during positive mode ESI of DA, the ES current is maintained efficiently by oxidations (at the ES emitter), preventing ion (negative) build up at the emitter. Generally the required oxidations during positive mode ESI involve oxidations of carrier solution components at the ES emitter. However, in case of DA, because of low DA oxidation potential of ca. 0.496 V vs SCE,[30, 31] DA oxidation was expected during ESI since the HV applied to the ES emitter was expected to be sufficient for DA oxidation at the emitter during ESI .[32, 33] As discussed above, large ion signals of DA OPs were detected in ESI MS with SS under standard MS conditions, confirming the expected oxidation of DA during ESI. In ESI MS with SiO2, however, only low ion signals of DA OPs were detected, and only after the capillary12
skimmer voltage bias was increased. This indicated lower ionization energy at the resistive SiO2, requiring additional energy for ion dissociation after ESI at SiO2. Since radicals were detected after ESI at SiO2, but not at SS, different oxidation pathways at SiO2 and SS were also indicated by the results during ESI of DA. Figure 8 The results summarized in Fig. 8 were obtained to gain insights into DA ESI processes at SiO2. As shown in Fig. 8, ion signals of DA (m/z 154) and DA OP (m/z 152) increased when the capillary-skimmer voltage bias was increased (in 40-45 V range) while m/z 137 fragment and radical (m/z 151) ion signals (not shown) remained low. Higher voltage bias thus improved sensitivity, confirming the formation of additional ions of DA after the higher voltage was applied, in agreement with improved dissociation of larger ion clusters that apparently formed during ESI at SiO2.[15] Additional increase in voltage (into 55-90 V range) resulted in increased fragmentation with m/z 137 DA fragment ion becoming the base peak (Fig. 8). Formation of m/z 137 ions by fragmentation of DA requires 310 kJ/mol [34, 35] while cleavage of the OH bond of DA quinone requires 338 kJ/mol.[36] As shown in Fig. 8, at high voltage bias DHIO•+ (m/z 151) radical ion signal was the base peak. Clearly OH bond cleavage at higher voltage bias, in this case of DHIO (cyclized DAQ) OH bond (Scheme 1), was efficient. High intensity of DHIO•+ ion signal under these conditions suggests good stability of the radical. Under the same conditions DHI•+ radical was not formed since DHI (m/z 150; Fig. 8) ion signal remained unchanged. As shown by the results in Figs. 2B and C, discussed above, detection sensitivity ESI MS with SiO2 was also improved by fine tuning of the HST. As shown in Fig. 9, at longer HST (up to ca. 0. 6 s) ion signals of DA and DA OPs increased when the capillary-skimmer voltage bias was 50 V. Longer HST thus improved sensitivity in ESI MS with SiO2, presumably as a result of 13
collisional dissociation in the hexapole of ion clusters and aggregates formed during ESI at SiO2. Still longer HST led to increased fragmentation and the m/z 137 fragment ion signal increased. The results showed that lower pressure in the hexapole than in the capillary-skimmer region favored the formation of m/z 137 DA fragment over m/z 151 DHIO•+ radicals. DHI (m/z 150) ion signal increased slowly with the increasing HST while the ion signal of the radical remained low. Figure 9 The main conclusion from the results is that ESI of DA at SiO2 lowered ion signal intensity, especially of the ion signals of DA OPs. Apparently, high resistance of SiO2 lowered ESI energy sufficiently to result in emission of larger aggregates in ESI MS with SiO2 than in ESI MS with SS which is significant in case of DA since DA and its OPs are known to aggregate easily by H-bonding. [37-42] As a result, detection of DA OPs and radicals the ESI MS with SiO2 required additional (collisional) energy for breaking down of the larger aggregates. As discussed above additional energy in ESI MS with SiO2 was supplied by increasing the capillaryskimmer voltage bias or the HST. Formation of DA radicals during ESI at SiO2, presumably by solution reactions at the SiO2 emitter during ESI, was verified by detection of low ion signals of the radicals (not shown) under standard MS conditions, without having to increase the capillary-skimmer voltage bias or the HST. The results also clearly showed that DHIO•+ (m/z 151), but not the DA•+ radicals, formed in the higher pressure capillary-skimmer region of the MS in ESI MS with SiO2. Formation of DA radicals was not detected in the hexapole where the pressure is low. DA radicals were not detected in ESI MS with SS. Proposed DA radical formation pathways are further discussed below. 3.5. DA Oxidation Pathways in ESI MS 14
Detection/identification of high yields of DA OPs after ESI of DA at SS confirmed rapid oxidation of DA in ESI MS with SS from water/methanol carrier solutions containing different electrolytes. Since DA OPs detected by ESI MS with SS were the same as those proposed for the 2e-, 2H+ oxidations of DA in aqueous solutions, [8-10, 28] DA oxidation during ESI at SS likely followed similar pathways. What was unique about ESI MS results with SS emitter was that they allowed detection of DA OPs during ESI at SS. This has been previously possible only in EC/ESI MS with the on-line EC cell after applying voltage to the cell, when the SS emitter also acted as the WE in the cell.[8-10] The proposed pathway of 2e-, 2H+ oxidation of DA during ESI in Scheme 2 was verifiable because of the high sensitivity in detection of DA OPs and their fragments and the high resolution of FTICR MS. DA OP assignment to DHIO was confirmed from its m/z 152 parent ion signal and the signals of fragment ions of DHIO of m/z 123 and 134.[9] Accelerated cyclization of the initial 2e-, 2H+ OP of DA at pH 6, as kcycl DAQ ~1 x 10-3 s-1, can accelerate the formation of DHIO (Scheme 2). [29] This in turn can accelerate oxidation of DHIO (E0’DHIO < E0’DA) to aminochrome (AC) (Scheme 2). AC m/z 150 ion signal was detected under these conditions. This assignment is supported by the m/z 150 of the parent ion and the detection of fragment of m/z 150 ion of m/z 122 that can form by loss of CO from AC.[9] In Scheme 2, AC is shown in equilibrium with DHI (m/z 149) since the m/z 149 ion signal consistent with the formation of DHI•+ was detected. The m/z 150 and 149 ions were not detected at pH 3 when kcycl DAQ ~1.5 x10 -4 -1
s .[29] Lower ESI voltage at resistive SiO2 can be expected to slow oxidations during at SiO2
during ESI and can be expected to result in different oxidation pathways during ESI at SiO2 and SS. As discussed above, DA radicals were detected only in ESI MS at SiO2 but not in ESI MS
15
with SS. Also AC (m/z 150) was detected at pH 4 following ESI at SiO2. In ESI MS with SS it was detected at pH 6 (Fig. 8). Slow apparent oxidation of DA during ESI at SiO2 means that water oxidation has to maintain the ES current at SiO2 during ESI of DA.[18] The expected (slow) 2e-, 2H+ oxidation of water (E0’= 1.51 V vs. SCE pH 4.3; 4e-, 4H+ process is much slower) generates hydrogen peroxide (reactive oxygen species; ROS), which is expected to contribute to generation of OH• radicals during ESI of DA. [43] As a result, DA can react with OH• ROS during ESI. DA reactions with ROS have been predicted to follow electron-proton transfer pathways, involving sequential electron-proton transfer (SEPT with 27.3% yield), proton coupled electron transfer (PCET with 9.7 % yield), as well as direct hydrogen atom (H•) transfer (HAT with 7.3% yield).[4] DA•+ radical is the main predicted DA OP in these oxidations. [DA]•+ (m/z 153) was detected by ESI MS with SiO2. Low ion signal and short HST required for [DA]•+ detection suggested low stability of the radical. Additionally, DA+(OH)• adducts have been predicted as products of DA oxidation in the electron-proton transfer pathways, following sequential electron-proton transfer pathway (RAF, 55.8% yield).[4] In ESI MS with SiO2 DA+(OH)• adducts 6OHDA and PTQ (Scheme 1) were detected as predicted. The results indicate that DA oxidation during ESI at SiO2 follows the electron-proton transfer pathways as well as the pathways predicted for in vivo DA oxidation[3] predicted by theory.[4] Competition, between oxidation and hydrogen transfer reactions in these pathways at different pH was confirmed by detection of DA+(OH)• adducts at lower pH, as 6OHDA (Scheme 1). ESI MS and EC/ESI MS results confirmed the differences in DA oxidation pathways at SS and SiO2 emitters. The results demonstrated the formation of ROS during slow oxidation of
16
DA in ESI MS with SiO2 and during collisions in the higher pressures capillary-skimmer region of the MS system. 3.6. Biological Significance of DA Oxidation Pathways in ESI MS The proposed toxic activity of DA in vivo has been linked to interactions of DA OPs, DA quinones and DA radical, with cellular components, leading to irreversible cellular damage and neurodegeneration. Based on the theoretical predications discussed above formation of DA radicals by rapid reactions with ROS is significant to DA reactivity in vivo.[4] However, in spite of many investigations, DA radicals have not been identified previously. Three DA radicals were identified in this work, allowing insights into radical stability, structure, and formation pathways that show apparent agreement with DA oxidation pathways proposed in vivo. The results indicate that the radicals form under different oxidation conditions (Scheme 1 and 2) and, additionally, indicated different stability of the radicals. DHIO•+ (m/z 151) radicals were shown as stable, while DA•+ (m/z 153) radicals were not as stable. Stability of DHI•+ (m/z 149) radicals is less clear due to low yields of the radicals that may result of low stability or complex formation pathway of the radical (Scheme 2). Formation of DHIO•+ and DHI•+ radicals requires cyclization of the initial DA OP DAQ (Scheme 1 and 2) that is favored at higher pH. Consequently, the radical formation could occur in vivo under conditions which favor slow oxidations of DA, during reactions with oxidizing agents such as ROS, such as the slow DA oxidation in ESI MS with SiO2. 4. Conclusions This work demonstrates the formation and detection of three radicals of DA and its OPs in ESI MS with SiO2. ESI at SiO2 favors slow oxidation of DA during ESI because of the resistance of SiO2 which can lower ESI voltage at the SiO2 emitter during ESI. Detection of
17
transient DA radicals and DA OPs was feasible in ESI MS with SiO2 because of the high sensitivity that was achieved by fine tuning MS operating conditions in ESI MS with SiO2. The results allowed identification of DA radicals and provided new information about the reaction conditions during DA oxidation that favor formation of DA radicals. The results show that DA radicals can form during slow oxidation of DA and in the higher pressure region of the MS system. The results provided experimental verification of the theoretically predicted proton coupled electron transfer oxidation pathway of DA that results in radical formation. The results also confirmed an alternative rapid 2e-, 2H+ oxidation pathway of DA that, based on the results, was followed in ESI MS of DA with SS emitter by the previously proposed solution pathways. Under these conditions formation of DA radicals was not detected. Rapid oxidation of DA by 2e-, 2H+ pathway, previously verified by EC/ESI MS, was confirmed in this work. Additionally, ESI MS results allowed identification of DA OPs formed at different pH. Sensitivity improvements that were demonstrated in this work in ESI MS were accomplished using a new approach that resulted in enhancement of ion dissociation in the MS system. The results illustrate the effect of the energies of ionization and collisions, as well as ion solvation and aggregate formation, on detection sensitivity in ESI MS. Improved sensitivity was required in this work in ESI MS with SiO2 emitter for detection of transient radicals and 2e-, 2H+ DA OPs. Sensitivity of ESI MS with SS emitter was sufficient for detection of DA OPs under standard MS operating conditions. Ion assignments were possible because of the high MS detection sensitivity and the high resolution of FTICR MS. The results provide new information about conditions favoring formation of a range of transient intermediates and products of DA oxidations that can be relevant to the biological reactivity of DA. In addition, the results provide information about the ESI processes that impact
18
sensitivity in ESI MS, of value in optimizing MS measurement sensitivity, and in the investigations of transient chemical reactions by MS. As the results demonstrate, relatively simple modifications of the MS operating conditions in ESI MS resulted in high detection sensitivity that was necessary for the detection of transient radicals and OPs. Simple change of the emitter material allowed access to new information. Previously, by using SS ES emitter as the WE in a floated on-line EC cell in EC/ESI MS, a simple approach, allowed SS emitter voltage to be easily fine-tuned as needed for improving detection sensitivity in EC/ESI MS by improving the ESI efficiency. This was exploited in this work for improving oxidation efficiency of DA in ESI MS with SS. The results provide evidence of DA oxidations that can generate different products during ESI and in the MS system and demonstrate which conditions can favor oxidations forming radicals in ESI MS, which has not been reported previously. It is of interest that DA oxidation pathways in ESI MS indicated by the results, while providing new information, are in excellent agreement with the previously proposed DA oxidation pathways, proposed from chemical and enzymatic as well as electrochemical studies in aqueous solutions. Acknowledgements The authors wish to thank Dr. John R. Eyler and Dr. Nick C. Polfer for providing access to the FT-ICR MS and for helpful technical guidance. The authors also thank Brooke E. Barnes and Alec G. Mackinnon for their help with the experiments.
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[17] D.W. Looi, J.R. Eyler, A. Brajter-Toth, Electrochemistry-electrospray ionization FT ICR mass spectrometry (EC ESI MS) of guanine-tyrosine and guanine-glutathione crosslinks formed on-line, Electrochimica Acta, 56 (2011) 2633-2640. [18] D.W. Looi, I. Iftikhar, A. Brajter-Toth, Electrochemical attributes of electrochemistry in tandem with electrospray mass spectrometry, Electroanal., 26 (2013) 1-9. [19] G.T. Blakney, C.L. Hendrickson, A.G. Marshall, Predator data station: A fast data acquisition system for advanced FT-ICR MS experiments, International Journal of Mass Spectrometry, 306 (2011). [20] L. Tang, P. Kebarle, Effect of the conductivity of the electrosprayed solution on the electrospray current - factors determining analyte sensitivity in electrospray mass-spectrometry, Analytical Chemistry, 63 (1991) 2709-2715. [21] M. Oss, A. Kruve, K. Herodes, I. Leito, Electrospray ionization efficiency scale of organic compounds, Analytical Chemistry, 82 (2010) 2865-2872. [22] R. Abburi, S. Kalkhof, R. Oehme, A. Kiontke, C. Birkemeyer, Artifacts in amine analysis from anodic oxidation of organic solvents upon electrospray ionization for mass spectrometry, European Journal of Mass Spectrometry, 18 (2012) 301-312. [23] J. Liigand, A. Kruve, I. Leito, M. Girod, R. Antoine, Effect of mobile phase on electrospray ionization efficiency, Journal of the American Society for Mass Spectrometry, 25 (2014) 1853-1861. [24] L.E. Sojo, N. Chahal, B.O. Keller, Oxidation of catechols during positive ion electrospray mass spectrometric analysis: Evidence for in-source oxidative dimerization, Rapid Communications in Mass Spectrometry, 28 (2014) 2181-2190. [25] J.F. Banks, S. Shen, C.M. Whitehouse, J.B. Fenn, Ultrasonically assisted electrospray-ionization for lc/ms determination of nucleosides from a transfer-rna digest, Analytical Chemistry, 66 (1994) 406-414. [26] T. Zhang, S.P. Palii, J.R. Eyler, A. Brajter-Toth, Enhancement of ionization efficiency by electrochemical reaction products in on-line electrochemistry/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, Anal. Chem., 74 (2002) 1097-1103. [27] B.M. Ehrmann, T. Henriksen, N.B. Cech, Relative importance of basicity in the gas phase and in solution for determining selectivity in electrospray ionization mass spectrometry, Journal of the American Society for Mass Spectrometry, 19 (2008) 719-728. [28] D.C.S. Tse, R.L. McCreery, R.N. Adams, Potential oxidative pathways of brain catecholamines, Journal of Medicinal Chemistry, 19 (1976) 37-40. [29] M.D. Hawley, Tatawawa.Sv, Piekarsk.S, R.N. Adams, Electrochemical studies of oxidation pathways of catecholamines, Journal of the American Chemical Society, 89 (1967) 447-&. [30] Y.Z. Song, Y. Song, Experimental and theoretical study of the electrochemical behavior of Nprotonated dopamine at Nafion multiwalled carbon nanotubes (MWNTs) modified glassy carbon (GCE) electrode, Canadian Journal of Chemistry-Revue Canadienne De Chimie, 84 (2006) 1084-1092. [31] H. Mohammad-Shiri, M. Ghaemi, S. Riahi, A. Akbari-Sehat, Computational and Electrochemical Studies on the Redox Reaction of Dopamine in Aqueous Solution, International Journal of Electrochemical Science, 6 (2011) 317-336. [32] A.T. Blades, M.G. Ikonomou, P. Kebarle, Mechanism of electrospray mass-spectrometry electrospray as an electrolysis cell, Analytical Chemistry, 63 (1991) 2109-2114. [33] G.J. Vanberkel, F.M. Zhou, Electrospray as a controlled current electrolytic cell - electrochemical ionization of neutral analytes for detection by electrospray mass-spectrometry, Analytical Chemistry, 67 (1995). [34] S.W. Benson, BOND ENERGIES, Journal of Chemical Education, 42 (1965) 502-&. [35] J.A. Kerr, Bond dissociation energies by kinetic methods, Chemical Reviews, 66 (1966) 465-&. [36] V.T. Varlamov, B.E. Krisyuk, A.V. Antonov, O-H bond dissociation energies in hydroquinones and 4hydroxyphenoxyl radicals and effect of solvation on the kinetics of reactions involving hydroquinones and semiquinone radicals, Russian Chemical Bulletin, 54 (2005) 2317-2324.
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[37] G. Alagona, C. Ghio, The effect of intramolecular H-bonds on the aqueous solution continuum description of the N-protonated form of dopamine, Chemical Physics, 204 (1996) 239-249. [38] J.D. Steill, J. Szczepanski, J. Oomens, J.R. Eyler, A. Brajter-Toth, Structural characterization by infrared multiple photon dissociation spectroscopy of protonated gas-phase ions obtained by electrospray ionization of cysteine and dopamine, Analytical and Bioanalytical Chemistry, 399 (2011) 2463-2473. [39] D.R. Dreyer, D.J. Miller, B.D. Freeman, D.R. Paul, C.W. Bielawski, Elucidating the Structure of Poly(dopamine), Langmuir, 28 (2012) 6428-6435. [40] M.E. Tessensohn, H. Hirao, R.D. Webster, Electrochemical Properties of Phenols and Quinones in Organic Solvents are Strongly Influenced by Hydrogen-Bonding with Water, Journal of Physical Chemistry C, 117 (2013) 1081-1090. [41] S.K. Callear, A. Johnston, S.E. McLain, S. Imberti, Conformation and interactions of dopamine hydrochloride in solution, Journal of Chemical Physics, 142 (2015). [42] A. Bouchet, M. Schutz, O. Dopfer, Competing Insertion and External Binding Motifs in Hydrated Neurotransmitters: Infrared Spectra of Protonated Phenylethylamine Monohydrate, Chemphyschem, 17 (2016) 232-243. [43] I.L. Kanev, A.Y. Mikheev, Y.M. Shlyapnikov, E.A. Shlyapnikova, T.Y. Morozova, V.N. Morozov, Are reactive oxygen species generated in electrospray at low currents?, Analytical Chemistry, 86 (2014) 1511-1517.
22
Figure 1. Schematic diagram of on-line electrochemistry ESI MS system (EC/ESI FTICR MS), showing the electrochemical (EC) cell. The EC cell consists of two SS capillaries joined by plastic tubing, with one SS capillary also serving as the ES emitter capillary. Different pressure regions are illustrated.
23
[DA+H] 154
100
+
(a)
75 50 25
Intensity (%)
0 100
[DA+H-NH3]
+
(b)
137
75
•+
[PTQ+H]+ 168 [6OHDA+H]+ 170
[DHIO]
50
151 154
25 0 137
100
(c)
75 50 [DA] 153
25 0 120
130
140
150
•+
160
170
180
m/z Figure 2. ESI MS of DA (100µM) in 50/49/1 vol %, MeOH/H2O/FA, pH~ 3, standard ESI MS conditions with capillary voltage (Vc) 40 V, hexapole storage time (HST) 0.6 s (A), Vc 90 V, HST 0.6 s (B), Vc 90 V, HST 0.3 s (C). Absolute base peak intensity: (A) 10k; (B) 7k: (C) 7k. Flow rate 50 µL/h.
24
100
100
+
[DHIO+H] 152
(a)
80
[DA+H] 154
80
60
60 [DA+H] 154
+
[152-CH2NH] 20
Intensity (%)
100
120
140
150
160
170
+
[DA+H-NH3] 20
123
0 110 100
152
(b)
40
[152-CH2NH]
20
130
+
[DHI+H] 150
+
123
0 110
60
+
40
+
[DA+H] 154
(g)
80
[DHIO+H] 152
+
40
100
+
(d)
120
130
140
(e)
150 152
160 154
137
0 170 110 100
80
80
80
60
60
60
40
40
120
130
0 110 100
120
(c)
130
140
150
160
170
80
170
154
150
137
20
0 110 100
123
160
40
20
123
150
(h)
123 20
140
120
130
140
150
160
100
137
(f)
0 170 110
80
120
130
140
150
160
170
160
170
137
(i)
80 +
60
60
[137-H2O] 119
+
[DA+H-NH3] 40
+
[152-H2O]
+
40
137
134
20
60
123 40 154
20
20
152 0 110
120
130
140
150
160
170
0 110
120
130
140
150
160
0 170 110
150
[150-CO] 122
154
119 120
130
140
150
m/z
Figure 3. ESI MS of DA (0.25 mM) ) at pH~ 3,4 and 6 respectively, standard ESI conditions, Vc 40 V, Flow rate 30 µL/h. HST 0.6 s, (A,D,G); EC/ESI MS, EC Cell Voltage 2.0 V, Vc 40 V (B,E,H); ESI MS, conditions same as (A,D,G), Vc 90 V (C,F,I). 25
100
154
(a)
[DA+DHIO+H] 305
80
[2DHIO+H] 303
60
+
[2DA+H] 307
+
+
40 152
Intensity (%)
20 0 120 100
140
160 154
180
200
220
240
260
280
307
60
305
152 137 303
20 0 120
320
(b)
80
40
300
140
160
180
200
220
240
260
280
300
320
m/z
Figure 4. ESI MS of DA (0.25 mM) in 50/49/1 vol.% in MeOH/H2O/HAc, pH ~ 4, Vc 40 V, HST 0.4 s, ITT 1.0 ms, silica ES emitter (A) stainless steel ES emitter (B).
26
m/z 154 m/z 301 m/z 303 m/z 305 m/z 307
100
Intensity (%)
80
60
40
20
0 -4
7.0x10
-4
-4
8.0x10
9.0x10
-3
1.0x10
Ion Transition Time (s)
Figure 5. Plot of DA and its dimers intensity vs ion transition time. Conditions as in Fig. 4 A.
27
m/z 152 m/z 303 m/z 307 m/z 137
80 70
Intensity (cps)
60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
EC Cell Voltage (V)
Figure 6. Effect of EC cell voltage on the intensity of DA ions. Conditions same as in 4 B.
28
m/z 154x10 m/z 152 m/z 137x3 m/z 303 m/z 307
300
250
Intensity (cps)
200
150
100
50
0 20
40
60
80
100
Flow Rate (uL/hr)
Figure 7. Effect of flow rate on the intensity of DA ions. Conditions same as in 4 B.
29
800
m/z 154x10 m/z 137x10 m/z 152 m/z 150 m/z 151
Intensity (cps)
600
400
200
0 30
40
50
60
70
80
90
Exit Capillary Voltage (Vc)
Figure 8.
Plot of DA ions intensity vs capillary voltage (Vc). 100 µM (DA) in 50/49/1 vol/% in MeOH/H2O/HAc , pH 4, HST 0.6 s, ITT 0.7 ms.
30
800
Intensity (a.u.)
600
m/z 154x10 m/z 137x10 m/z 152 m/z 150
400
200
0 0.2
0.4
0.6
0.8
1.0
Hexapole Storage Time (s)
Figure 9.
Plot of intensity vs hexapole storage time. DA (100 µM) in 50/49/1 vol/% in MeOH/H2O/HAc , pH 4, Vc 50 V, ITT 0.7 ms.
31
Scheme 1. Proposed DA oxidation pathways using silica ES emitter.
32
Scheme 2. Proposed DA oxidation pathways using a stainless steel ES emitter.
Scheme 3. Proposed proton bound dimers of DA and its oxidation product DHIO. 33