Electrochemistry On Line with Mass Spectrometry Insight into Biological Redox Reactions Kevin J. Volk Bristol-Myers Squibb Pharmaceutical Research Institute Wallingford, CT 06492
Richard A. Yost and Anna Brajter-Toth University of Florida Department of Chemistry Gainesville, FL 32611
The on-line combination of MS and electrochemistry is a direct and sensitive method that provides chemical information about electrochemically generated species with excellent time resolution. This combination offers the capability to directly monitor reactants, short-lived intermediates, and products of electrochemical reactions as a function of electrode potential, with high selectivity for individual components. Because a mixture of re-
actants, intermediates, and products is often generated, the use of MS/MS can provide structural confirmation of intermediates and products without loss of time resolution. To provide chemical information in real time about the species formed d u r i n g electrochemical reactions, several problems must be overcome. For example, the liquid environment of electrochemical methods poses a unique difficulty in interfacing with a gas-phase method such as MS. In addition, t h e t r a n s i e n t n a t u r e of electrochemical reaction products resulting from chemical reactions that may occur in solution makes excellent time resolution a requirement for real-time analysis. Detection and identification of solution species (as opposed to gaseous products) present real challenges. Although researchers have long recognized the utility of MS for mon-
INSTRUMENTATION 0003-2700/92/0364-21 A/$02.50/0 © 1991 American Chemical Society
itoring particular species in complex mixtures, techniques t h a t take full advantage of the combination of electrochemistry and MS were not developed until recently. A new technique that couples electrochemistry on line with MS, known as EC/MS, makes it possible to detect electrochemically generated species without being limited to gaseous products, as was the case in earlier studies (1). EC/MS emerged from t h e development of commercial LC/MS interfaces such as thermospray (2). Chemical information about electrochemical reactions can be obtained by other spectroscopic techniques. For example, spectroelectrochemistry, in which the progress of electrochemical reactions can be monitored by transmission U V - v i s spectroscopy, h a s been a particularly popular method (3). However, the inherent selectivity and sensitivity of MS allow monitoring and identification of individual species in mixtures that may be too complex for analysis by other on-line methods. These capabilities a r e of particular interest in the study of redox reactions of biologically active compounds, because electron transfer reactions are frequently coupled to fast chemical reactions. The on-line coupling of electrochemistry with MS is the subject of two recent literature reviews (4, 5).
Monitoring electrochemical reactions Two types of interfaces have been used to join EC and MS: porous interfaces for analysis of gaseous products and thermo spray interfaces for analysis of solution species.
P o r o u s i n t e r f a c e s . On-line EC/MS proved elusive until Bruckenstein and Gadde (1) successfully combined these two analytical techniques two decades ago. In this pioneering work, a porous electrode was placed on line with a mass spectrometer (Figure 1), which allowed the analysis of volatile intermediates and products generated during an electrochemical reaction. A porous Teflon membrane acted as the inlet by allowing volatile components to pass through it while it served as a barrier to the liquid phase. Because small molecules such as 0 2 and methane permeate t h e Teflon m e m b r a n e more readily t h a n do larger molecules such as the solvent or larger organics, the lower molecular weight components were enriched. In the original work MS was used to monitor the electrochemical generation of oxygen from aqueous HC10 4 (1). In later work Bruckenstein and Gadde were able to monitor ammonia
ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992 · 21 A
INSTRUMENTATION production via electrochemical reduction of nitrate (6). Bolzan and coworkers used a similar system to study the electrochemical oxidation of glucose and monitored production of C 0 2 during a potential scan (7). A semipermeable silicone membrane was used by Anderson and coworkers to study the reduction of dibromocyclohexane to cyclohexane (8). Because silicone membranes have been used to separate permeable organics from the impermeable carrier gas in GC/MS (9), it was assumed that the electroactive reactants and products would be partitioned into
and diffuse through the membrane and would be detected by MS (8). However, because of the selective nature of the silicone membranes, only certain reactants and products permeated the membrane (8, 10). The early work with semipermeable membrane or porous frit interfaces clearly demonstrated the potential benefits and insights t h a t could be obtained with on-line combinations of electrochemistry with MS. The need to better understand the mechanisms and kinetics of electron transfer, particularly at catalytic electrode surfaces, is the driving
Figure 1. Porous frit interface for the analysis of volatile products. (Adapted from Reference 1.)
Figure 2. Thermospray LC/MS interface. 22 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992
force that continues to advance research with such interfaces (11), and the chemical systems that have been studied by this approach have been tabulated (5). However, determination of only the volatile components of electrochemical reactions limits the number of systems that can be studied; most electrochemical processes that occur at the electrode-solution interface do not produce volatile products. Thermospray interfaces. It was not until the invention of the thermospray interface by Vestal and Blakley (2) that MS could be routinely used for the determination of polar and nonvolatile compounds in solution, such as the typical products of electrochemical reactions. The thermospray LC/MS interface rapidly volatilizes LC eluent by simple direct resistive heating of a metal capillary within the vaporizer probe (Figure 2). When this vaporization process occurs in the vacuum of a mass spectrometer, a jet of vapor consisting of a mist of fine particles and solvent droplets is created. Although the fundamental processes of thermospray ionization are not completely understood, at least two different mechanisms may be responsible for ion formation (12): direct ion evaporation of preformed ions from the droplets and chemical ionization (CI) of neutral sample molecules by ions produced from the ammonium acetate buffer. Direct ion evaporation may occur for any molecule ionized in aqueous solution. Because gas-phase ion-molecule reactions such as CI have been shown to influence the thermospray process (13, 14), the production of sample ions will depend on experimental p a r a m e t e r s that affect the vaporization process, such as capillary diameter, tip temperature, and flow rates. H a m b i t z e r a n d H e i t b a u m (15) were the first to successfully use a thermospray LC/MS interface with an electrochemical cell on line with the mass spectrometer. These experiments demonstrated the potential for on-line detection of electrochemically g e n e r a t e d products—it was possible to monitor the formation of dimers and trimers upon the electrooxidation of /V,/V-dimethylaniline (DMA) at a Pt electrode (Figure 3) with a time resolution of —10 s between generation in the electrochemical cell and detection by MS. Figure 4 shows the mass spectrum of DMA a n d p r o d u c t s o b t a i n e d within 25 s of sampling while applying continuous cycling between —0.40 and +1.0 V with a 500-mV/s sweep
rate (15). The products observed by EC/MS during the oxidation of DMA are consistent with the mechanism proposed by Adams and co-workers (16, 17). Probes of redox reaction chemistry
Development of methods for monitoring the chemistry of redox reactions has been driven by the realization that the reactions at the electrode solution interface can provide useful information about enzymatic redox reactions, leading to insights about biological reaction pathways. For example, the products identified (and the reaction pathways deduced) for peroxidase (18), x a n t h i n e oxidase (19), and cytochrome P-450 (20) have been similar to electrochemical products (and pathways). In studying such redox reactions, r e s e a r c h e r s have been most successful when using electrochemical methods in conjunction with other methods such as HPLC, molecular spectroscopy, and MS. Because each technique provides only limited information, a combination of these techniques is often required (18). Prior to the development of LC/MS interfaces, analysis of mixtures of polar and nonvolatile compounds produced in electrochemical reactions of compounds of biological interest required derivatization followed by offline GC/MS analysis (21). Understandably, the conditions and the time required for derivatization were severe limitations when using GC/MS to identify electrochemically generated reaction intermediates. The use of MS was possible only after the intermediates were detected by spectroelectrochemistry following fast electrolysis in a thin-layer cell and then rapidly frozen, freeze-dried, and converted to volatile products through derivatization (21). In addition to longer lived i n t e r m e d i a t e s , products typically formed as a result of a series of hydrolysis reactions of electrochemically generated i n t e r m e d i a t e s could be identified. Kissinger and co-workers have exploited the power of HPLC connected on line with an electrochemical cell to obtain insights into redox reactions (22, 23). This approach was recently used in an attempt to understand the nature of a key intermediate in the redox activation of mitomycin C, a powerful quinone-containing antitumor agent that alkylates DNA and RNA upon reductive activation by cytochrome P-450 (20). Time resolution of electrochemi s t r y / t h e r m o s p r a y MS. One at-
tractive feature of an on-line EC/MS system is that it provides an opportunity to attain sufficient time resolution to detect short-lived intermediates and the potential for real-time analysis. The delay time and hence the dead volume between the electrochemical cell and the mass spectrometer are therefore crucial p a r a m e ters. Increased dead volume not only will degrade the time resolution but
also will decrease the probability of detecting important short-lived intermediates. The time resolution of an EC/MS system can be determined using a potential-step experiment (24). This experiment requires a triggering device that links the potentiostat and the mass spectrometer, permitting the data acquisition sequence of the mass spectrometer to begin at the
Figure 3. Mechanism for the electrooxidation of /V.W-dimethylaniline in neutral solution. Potentials are versus SCE. (Adapted from Reference 15.)
Figure 4. Mass spectrum of /v,A/-dimethylaniline and its oxidation products. (Adapted from Reference 15.) ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992 · 23 A
INSTRUMENTATION same time the potential pulse is ap plied to the electrochemical cell. Figure 5 illustrates the effect of a p o t e n t i a l - s t e p e x p e r i m e n t on the mass spectrometric intensity of an intermediate with m/z 196 produced during the oxidation of 9-methyluric acid. In this experiment, a solution of 9-methyluric acid was continuously pumped through the electrochemical cell and into the mass spectrometer. A square wave was applied for 500 ms from an initial potential where oxida tion does not occur (-250 mV vs. a Pd reference electrode) to a final potential of +800 mV, where oxidation occurs at the maximum rate. After 500 ms, the potential was stepped back to the ini tial potential of -250 mV. The poten tial pulse resulted in the mass spec trometric profile of m/z 196 (the [M - H]~ ion of the amination inter mediate). Figure 5 clearly shows that the delay time before the mass spec trometer starts to respond to the for mation of this intermediate is 520 ms. This is a dramatic improvement over the 10-s time resolution obtained with the system used by Heitbaum and Hambitzer ( 15). Mass s p e c t r o m e t r i c h y d r o d y namic voltammograms. Cyclic voltammetry is a powerful electroanalytical tool in which the electrode potential can be rapidly scanned in search of potentials to initiate the re dox reaction. Once started, the redox reaction may be probed further with electroanalytical techniques. Infor
mation provided by these techniques includes reaction potential, number of electrons transferred in the redox reaction, and reaction kinetics. Figure 6 illustrates how EC/MS can be used to obtain chemical infor mation as the electrode potential is changed. (Full-scan positive- and negative-ion mass spectra were ob tained at each potential.) An off-line cyclic voltammogram, in this case that of uric acid, indicates that an oxidation process starts to occur at —0.0 V and reaches a maximum rate at ~ +0.40 V. Although additional electrochemical experiments can be used to characterize the stability of the imine alcohol intermediate gen erated in this oxidation ([M - H]~ at m/z 183), the mass spectrometric hydrodynamic voltammograms clearly indicate the formation of this inter mediate as well as the final product, allantoin ([Μ - Η Γ at m/z 157). Most importantly, the EC/MS results pro vide crucial chemical information about each species formed in the re dox reaction in close to real time. As can be seen in Figure 6, the mass spectrometric intensity of the starting material, uric acid, at m/z 167 decreases dramatically (as a re sult of oxidation) as the electrode po tential is increased. This result is ex pected because the high-efficiency ( - 8 0 % at a flow rate of 2.0 mL/min) coulometric cell used in this study
depletes most of the reactant (24). A steady-state situation develops for all ion intensities with potentials > +0.40 V because of the hydrodynamic flow of reactant. The plateau region of the mass spectrometric hydrodynamic voltammograms corre sponds to the potential past the cyclic voltammetric peak where the reac tion occurs at the maximum rate. This expected behavior allows for easy correlation between off-line electrochemical d a t a and on-line EC/MS results. The conversion of the starting ma terial to a reactive intermediate can be followed by monitoring the inten sity of the intermediate's ΓΜ - H]~ ion at m/z 183. This intermediate is unstable and disappears as a result of a hydration reaction (Figure 7). On-line chemical information from e l e c t r o c h e m i c a l r e a c t i o n s . One challenge in electrochemistry in volves unraveling the complex ho mogeneous chemical reactions that often accompany heterogeneous elec tron transfer. (The reaction of uric acid is an excellent example.) These reactions can tremendously compli cate the analysis of products of bulk electrolysis, which h a s commonly been used to provide information about reaction pathways. Although judicious choice of solvents and elec trolytes can simplify this process, many reactive electrochemically gen-
Figure 5. Potential-step/MS experiment with 9-methyluric acid. (a) Initial potential -250 mV, step potential 800 mV, and final potential -250 mV. Pulse duration 500 ms. (b) Relative intensity of the [M - H]~ ion of the amination intermediate at m/z 196. (Adapted from Reference 24.)
Figure 6. Mass spectrometric hydrodynamic voltammograms of uric acid. Each data point represents the average of triplicate injections of uric acid at each potential. Off-line cyclic voltammogram (scan rate 200 mV/s) of uric acid is shown for comparison. (Adapted from Reference 24.)
24 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY1,1992
erated intermediates react with the solvent. As a result, many products identified after bulk electrolysis have vastly different structures than the proposed electrochemical intermediates, particularly in aqueous electrochemistry. A simple m e c h a n i s t i c analysis is often sufficient to determine the origin of many products of electron transfer reactions, but certain subsequent chemical reactions and rearrangements may be so complex that it is difficult or impossible
to determine the origin of many of the final products. The information generated from on-line EC/MS experiments can provide m u c h - n e e d e d information in such cases. As shown in Figure 7, a 2e~, 2H + electrochemical oxidation of uric acid generates a diimine intermediate; any nucleophilic species present in solution (e.g., H 2 0 or NH :i ) can react with this unstable intermediate. The thermospray LC/MS interface uses an ammonium acetate
buffer for ionization. As a result, in an EC/MS system using a thermospray interface, amination or ammonolysis reactions can occur because ammonia can act as a nucleophile (25). Although those additional nucleophilic reactions can complicate the interpretation of the EC/MS results, they can also provide additional evidence for the presence of a reactive intermediate. Chemical processes following electron transfer can be inferred from cy-
Imine alcohol MW184
Uric acid MW168
Imine amine MW 183
Bicyclic imidazolone MW140
5-Hydroxyhydantoin5-carboxamide MW159
Alloxan monohydrate MW160
5-Hydroxyhydantoin MW 116
2-Oxo-4-imino5-ureidoimidazolidine MW 157
Allantoin MW158
Parabanic acid MW 114
Oxamic acid MW89
Figure 7. Oxidation pathway of uric acid in 0.1 M ammonium acetate. Molecular weights are shown for those intermediates and products that have been identified by EC/MS and EC/MS/MS. (Adapted from Reference 24.)
ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992 · 25 A
INSTRUMENTATION clic voltammetric experiments. A to tally reversible system should have a inJir,r ratio (i.e., the ratio of anodic to pa
pc
*
>
cathodic peak currents) of 1. In Fig ure 6 the ipJipc ratio is much greater than one, which indicates that the electrochemically generated species has undergone a chemical reaction that prevents the corresponding quantitative reduction of the product on the return sweep. Detection and identification of such species by MS provide evidence for t h e product structure and for the primary inter mediate structures. These i n t e r m e d i a t e s a n d o t h e r products resulting from the nucleophilic addition reactions of uric acid il lustrated in Figure 7 can be easily monitored and characterized by an EC/MS system. On-line chemical identification is of particular interest because not all intermediates and products can be detected electrochem ically. Although the electrochemistry of uric acid is fairly straightforward, the subsequent solution reactions and rearrangements are quite com plex a n d r e q u i r e t h e s e p a r a t i o n power of MS/MS. Figure 7 illustrates the oxidation pathway of uric acid based on EC/MS and EC/MS/MS re sults and demonstrates the capabili ties of EC/MS for on-line character ization of dynamic reactions. Each species produces either an [M + H] + ion or an [M - H]~ ion in the thermo spray mass spectrum, thus facilitating molecular weight confirmation. In addition, a totally unexpected com pound, parabanic acid, was observed
Thermospray probe
EC cell
with the EC/MS system. The forma tion of parabanic acid from 5-hydroxyhydantoin requires a second oxida tion step, and it is unclear how this reaction occurs in a flowing system. Identifying electrochemical reaction products by MS/MS Ideally, the chemical information generated via EC/MS should sim plify c h a r a c t e r i z a t i o n of complex redox r e a c t i o n s . However, w h e n EC/MS is done without on-line chro matographic separation (to obtain the best time resolution), the result ing mass spectrum is a mixture of molecular, adduct, and fragment ions of unreacted starting material, inter m e d i a t e s , t h e r m a l decomposition products, and final products. Al though chemical analysis of the re sulting mixture may appear to be a problem, most L C / M S interfaces such as thermospray generally pro vide primarily molecular weight in formation in the form of [M + H] + and [M - H]~ ions. If the interface produced abundant fragment ions for the individual mixture components, the mass spectra of the electrolysis mixtures would be almost impossible to interpret. Although lack of frag mentation simplifies the interpreta tion of results, the molecular weight information provided by these tech niques is not sufficient evidence for compound identification. To obtain the necessary structural information for the identification of reactants and products formed in re dox reactions and subsequent chemi cal reactions, MS/MS can be used to
HPLC pump
Detection Thermospray source Ionization
Mass analysis
Dissociation
Mass analysis
Figure 8. Instrumental setup for on-line EC/MS/MS. (Adapted with permission from Reference 31.) 26 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992
produce structurally significant product ions by collisionally acti vated dissociation (CAD) of selected precursor ions. MS/MS provides not only structural information but also an additional separation stage that makes it possible to identify individ ual components in the often complex electrolysis mixture. The capability to obtain structural information on each component in an electrolysis mixture without chromatographic separation is a unique feature of EC/MS/MS (26, 27). The system de veloped in our laboratory for such studies is shown in Figure 8. To fully characterize the interme diates and products formed following an electrochemical reaction, both the p o s i t i v e - and n e g a t i v e - i o n m a s s spectra must be considered. This is especially true for compounds that produce only an [M + H] + or [Μ - H]~ ion. Compounds t h a t produce both are ideal for the identification of un known reaction intermediates and products because the molecular weight is confirmed, as illustrated in Figure 9. Figure 9a shows the product spectrum of a positive ion at m/z 184, which corresponds to the LM + HJ+ ion of the imine a m i n e i n t e r m e d i a t e (MW 183) identified in Figure 7; this product spectrum is an excellent example of the structural information that can be obtained by MS/MS. The 184 + ion initially fragments to 167 + ion (loss of NH 3 ), which corresponds to the protonated primary diimine inter mediate (MW 166). Additional impor t a n t structural information is ob t a i n e d when consecutive n e u t r a l losses occur, such as the confirmation by successive losses of CONH, CONH, and CO of three CO groups of the in termediate. Clearly, i m p o r t a n t s u b s t r u c t u r e information about key electrochem ical i n t e r m e d i a t e s is provided by EC/MS/MS. As is evident from Fig ure 9a and 9b, the product spectra of an ΓΜ + H] + ion and of its comple mentary [M - H]~ ion will generally result from different fragmentation p a t h w a y s , p a r t l y because of t h e sites of protonation ([M + H] + ion) and deprotonation ([M - H ] " ion) and the stability of the r e s u l t a n t ions. Indeed, the positive and nega tive ion product s p e c t r a provide complementary structural informa tion. For example, only the [M + H] + ion fragments with a loss of NH :j , a result t h a t indicates the presence of a primary amine. The use of E C / M S / M S allowed positive identification of the inter mediates and products shown in Fig ure 7, despite the complexity of the
