Electrochemistry on line with mass spectrometry. Insight into biological

Electrochemistry On Line with Mass Slsectrometrv Insight into Biologital Redox Reactio; Kevin J. Volk Bristol- Myers Squibb Pharmaceutical Research Institute Wallingford, CT 06492

Richard A. Yost and Anna Brajter Tot h

-

University of Florida Department of Chemistry Gainesville, FL 3261 1

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-

0003-270019210364-21A/$02.50/0 0 1991 American Chemical Society

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 during 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, the transient nature of electrochemical reaction products re sulting 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-

itoring particular species in complex mixtures, techniques that 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 (I). EC/MS emerged from the 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 UV-vis spectroscopy, has 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 are 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 thermospray interfaces for analysis of solution species. Porous interfaces. O n - l i n e 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 l),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, and methane permeate the Teflon membrane 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, ( 1 ) . In later work Bruckenstein and Gadde were able to monitor ammonia

ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992

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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 CO, during a potential scan (7). A semipermeable silicone mem brane 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 (91, 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 a t catalytic electrode surfaces, is the driving

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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 ( ] I ) , 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 a t 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 responsi ble 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, 1 4 ) , the production of sample ions will depend on experimental parameters that affect the vaporization process, such as capillary diameter, tip temperature, and flow rates. Hambitzer and Heitbaum (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 generated products-it was possible to monitor the formation of dimers and trimers upon the electrooxidation of N ,N- 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 products obtained within 25 s of sampling while applying continuous cycling between -0.40 and +l.OV with a EiOO-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 monitor ing the chemistry of redox reactions has been driven by the realization that the reactions a t the electrodesolution 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), xanthine oxidase (19>,and cytochrome P-450 (20)have been similar to electrochemical products (and pathways). In studying such redox reactions, researchers 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 re quired derivatization followed by offline GC/MS analysis (21). Understandably, the conditions and the time required for derivatization were se vere 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 derivatiza tion (21). In addition to longer lived intermediates, products typically formed as a result of a series of hydrolysis reactions of electrochemically generated intermediates 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 electrochemistry/thermospray MS. One a t -

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 de vice that links the potentiostat and the mass spectrometer, permitting the data acquisition sequence of the mass spectrometer to begin a t the

tractive feature of an on-line EC/MS system is that it provides a n 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 parame ters. Increased dead volume not only will degrade the time resolution but

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Figure 3. Mechanism for the electrooxidation of N,N-dimethylaniline in neutral solution. Potentials are versus SCE. (Adapted from Reference 15.)

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INSTRUMEN 7ATION same time the potential pulse is applied to the electrochemical cell. Figure 5 illustrates the effect of a potential -step experiment 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 oxidation does not occur (-250 mV vs. a Pd reference electrode) t o a final potential of +800 mV, where oxidation occurs at the maximum rate. After 500ms, the potential was stepped back to the initial potential of -250 mV. The potential pulse resulted in the mass spectrometric profile of m / z 196 (the [M-HI- ion of the amination intermediate). Figure 5 clearly shows that the delay time before the mass spectrometer starts to respond to the formation 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 spectrometric hydrodynamic voltammograms. Cyclic voltammetry is a powerful electroanalytical tool in which the electrode potential can be rapidly scanned in search of potentials to initiate the redox 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 information as the electrode potential is changed. (Full- scan positive- and negative-ion mass spectra were obtained a t each potential.) An off-line cyclic voltammogram, in this case that of uric acid, indicates that a n oxidation process starts to occur a t -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 generated in this oxidation ([M-HI- at m/z 183), the mass spectrometric hydrodynamic voltammograms clearly indicate the formation of this intermediate as well as the final product, allantoin ([M - HI- at m/z 157). Most importantly, the EC/MS results provide crucial chemical information about each species formed in the redox reaction in close to real time. As can be seen in Figure 6, the mass spectrometric intensity of the starting material, uric acid, a t m / z 167 decreases dramatically (as a result of oxidation) as the electrode potential is increased. This result is expected because the high-efficiency (-80% a t 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 reaction occurs a t 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 material to a reactive intermediate can be followed by monitoring the intensity of the intermediate’s [M -HIion at m/z 183. This intermediate is unstable and disappears as a result of a hydration reaction (Figure 7). On-line chemical information from electrochemical reactions. One challenge in electrochemistry involves unraveling the complex homogeneous chemical reactions that often accompany heterogeneous electron transfer. (The reaction of uric acid is an excellent example.) These reactions can tremendously compli cate the analysis of products of bulk electrolysis, which has commonly been used to provide information about reaction pathways. Although judicious choice of solvents and electrolytes 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 - HI- ion of the amination intermediate at m/z 196. (Adapted from Reference 24.)

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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.)


erated intermediates react with the solvent. As a result, many products identified after bulk electrolysis have vastly different structures than the proposed electrochemical intermedi ates, particularly in aqueous electrochemistry. A simple mechanistic 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 much-needed 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,O or NH,) 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 elec tron transfer can be inferred from cy-

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Figure 7. Oxidativri pathway of uric acid i r i 0.1 M arrimonilcrri acetate. Molecular weights are shown for those intermediates and products that have been identified by ECiMS and ECIMSIMS. (Adapted from Reference 24.)


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INS7RUMEN7ArlON clic voltammetric experiments. A totally reversible system should have a ipa/ipcratio (Le., the ratio of anodic to cathodic peak currents) of 1. In Figure 6 the ipa/iPcratio is much greater than one, which indicates that the electrochemically generated species has undergone a chemical reaction t h a t 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 intermediate structures. These intermediates and other products resulting from the nucleophilic addition reactions of uric acid illustrated 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 electrochemically. Although the electrochemistry of uric acid is fairly straightforward, the subsequent solution reactions and rearrangements are quite complex and require t h e separation power of MS/MS. Figure 7 illustrates the oxidation pathway of uric acid based on EC/MS and EC/MS/MS results and demonstrates the capabilities of EC/MS for on-line characterization of dynamic reactions. Each species produces either an [M + HI' ion or an [M - HI- ion in the thermospray mass spectrum, thus facilitating molecular weight confirmation. In addition, a totally unexpected compound, parabanic acid, was observed

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with the EC/MS system. The formation 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 MSlMS Ideally, the chemical information generated via EC/MS should simplify characterization of complex redox reactions. However, when EC/MS is done without on-line chromatographic separation (to obtain the best time resolution), the resulting mass spectrum is a mixture of molecular, adduct, and fragment ions of unreacted starting material, inter mediates, thermal decomposition products, and final products. Although chemical analysis of the resulting mixture may appear to be a problem, most LC/MS interfaces such as thermospray generally provide primarily molecular weight in formation in the form of [M + HI' and [M-HI- 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 fragmentation simplifies the interpretation of results, the molecular weight information provided by these techniques is not sufficient evidence for compound identification. To obtain the necessary structural information for the identification of reactants and products formed in redox reactions and subsequent chemical reactions, MS/MS can be used to

HPLC pump

-robe

Figure 8. Instrumental setup for on-line EC/MS/MS. (Adapted with permlsslon from Reference 31 .)

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produce s t r u c t u r a l l y significant product ions by collisionally activated 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 individual components in the often complex electrolysis mixture. The capability to obtain structural information on each component in a n electrolysis mixture without chromatographic separation is a unique feature of EC/MS/MS (26, 27). The system developed 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 positive- and negative-ion mass spectra must be considered. This is especially true for compounds that produce only an [M + HI' or [M - HIion. Compounds that produce both are ideal for the identification of unknown reaction intermediates and products because t h e 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 [M + HI' ion of the imine amine intermediate (MW 183) identified in Figure 7; this product spectrum is a n excellent example of the structural information that can be obtained by MS/MS. The 184' ion initially fragments to 167' ion (loss of NH,), which corresponds to the protonated primary diimine intermediate (MW 166). Additional import a n t structural information is obtained when consecutive neutral losses occur, such as the confirmation by successive losses of CONH, CONH, and CO of three CO groups of the int ermediate. Clearly, important substructure information about key electrochemical intermediates is provided by EC/MS/MS. As is evident from Figure 9a and 9b, the product spectra of an [M + HI' ion and of its complementary [M - HI- ion will generally result from different fragmentation pathways, partly because of t h e sites of protonation ([M + HI' ion) and deprotonation ([M - HI- ion) and the stability of the resultant ions. Indeed, the positive and negative ion product spectra provide complementary structural information. For example, only the [M + HI' ion fragments with a loss of NH,, a result that indicates the presence of a primary amine. The use of EC/MS/MS allowed positive identification of the intermediates and products shown in Figure 7, despite the complexity of the

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INSTRUMEN TATION reactions following the electrochemical oxidation of uric acid. Positiveand negative-ion normal mass spectra were extremely helpful in final identifications because they provided molecular weight information; how ever, structural assignments r e quired MS/MS. Although the structure elucidation power of MS/MS is well documented (26, 27), MS/MS can also be used to confirm the presence of proposed products of electrochemical reactions if standards are available. Such confirmation can be accomplished by comparing the pro duct spectra of authentic standards with those of electrochemically generated products (24-25,223-31).Each compound produces a unique fragmentation pattern under MS/MS conditions that is characteristic of its structure, analogous to an IR fingerprint or an E1 spectrum. EC/MS studies for modeling enzymatic reactions Electrochemical methods have been used successfully to investigate the redox chemistry of biologically signif-

istry of catecholransmitters (32, 33) ery and co-workers on nothiazine tranquilizer drugs (34, 351, electrochemical methods are well suited to provide insight into redox and related chemical reactivity of compounds of biological interest. Adams' studies have provided insights into the ways in which catecholamines and the intermediates formed during electrooxidation might be involved in certain types of neurochemical behavior. McCreery's work indicates that the decay rate of phenothiazine radical cations formed during electrooxidation depends on the structure of the radical. Because

ciated with enzymes cannot as yet be precisely duplicated using this method, similarities remain. Based on the early work of Kissinger and co-workers (22, 23), Getek and Korfmacher studied acetaminophen conjugate formation during electrooxidation (36).In this work, the reaction of acetaminophen with glutathione and cysteine after electrochemical oxidation was monitored by on-line EC/MS using a thermospray interface. Mixing the electrolyzed acetaminophen with either glutathione or cysteine resulted in the formation of the acetaminophen conjugates on the time scale of 1 s (36).The conjugation of acetaminophen and glutathione is suspected to occur after in vivo oxidation of acetaminophen to a reactive species (37). Dryhurst and co-workers have also shown that there is considerable similarity between the pathways of the electrochemical and peroxidase catalyzed oxidation of uric acid (18). To provide a partial picture of the reaction products, it was necessary to combine electrochemical measure chromatography, LC, /MS following sample derivatization. However, these techniques could provide only limited information about unstable intermedia t e s a n d t h e dynamic solution reactions that follow the initial elec-

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nite insight into the mechanism and structural factors that mlght influence metabolite formation from the radical. In addition, Dryhurst and co-workers have investigated the electrochemistry of many purines and correlated the electrochemical reaction products with the enzymatic product profile (18). In electrochemical studies of biological molecules, a n electrode is poised at a suitable potential and is used to simulate the redox enzyme. Although the unique selectivity asso28 A

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 1 , JANUARY 1,1992

trochemical and enzymatic oxidation. The capability to simulate an enzymatic reaction with EC/MS was recently demonstrated and correlated with the information obtained using a n enzyme reactor on line with MS (31). An enzyme reactor with covalently bound horseradish peroxidase was used for the on-line enzyme/MS studies. The amount of time needed for the substrate, in this case uric acid, to react with the enzyme was varied by changing the flow rate of the mobile phase through the enzyme reactor. Because thermospray is a flow-rate-dependent technique, a tee was placed after the enzyme column and make-up flow from another HPLC pump was used so that the thermospray interface always operated a t a constant combined flow rate. Comparison of the thermospray mass spectra obtained from t h e peroxidase - catalyzed oxidation with the electrochemical oxidation of uric acid showed that the intermediates and products were generally t h e same, although their relative abundances were different (31).The product/intermediate intensity ratios [5 hydroxyhydantoin + amine + HI' (134+/184') and [alloxan monohydrate - CO, + acetate]-/ [imine amine - HI- (175-/182-) can in fact indicate the degree of completeness of the reactions that follow the initial oxidation process. For example, elec-

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trochemical oxidation a t +0.50 V is characterized by a 134+/184+intensity ratio of 0.30 and a 175-/182- intensity ratio of 0.25, whereas enzymatic oxidation for 1 min yields a 134'1184' intensity ratio of 1.8 and a 175-/ 182- intensity ratio of 0.45. These intensity ratios indicate that the chemical reactions of the electrochemical oxidation intermediates have proceeded to a greater extent in the enzymatic oxidation, leading to greater relative abundances of the final products. In fact, the enzymatically generated intermediates have a much longer residence time in solution (reaction time of -1 min in the reactor) than do the electrochemical intermediates (500 ms); this accounts for the different intensity ratios. During the 1-min enzymatic reaction period, some of the imine alcohol and imine amine can decay to the bicyclic imidazolone as well as to the final products (Figure 7). Greater relative abundances of the 139- and the 141' ions, which correspond to the [M - HI- and [M + HI' of the bicyclic imidazolone, indicate that this in fact occurs. Because the electrochemical and enzymatic thermospray mass spectra are virtually identical, both reactions must follow similar reaction pathways. Therefore, it is reasonable to postulate that the structures of the intermediates and products formed and identified by EC/TSP/MS/MS

reflect those of the enzymatically generated compounds of the same molecular weights. Hence, the observation of the imine alcohol and the imine amine intermediates during the peroxidase - catalyzed oxidation supports the formation of a similar primary intermediate in the enzymatic and electrochemical oxidation of uric acid. Advantages and limitations of the thermospray interface for EC/MS Successful analysis by EC/MS requires that the component of interest in the condensed phase be transformed into gas-phase ions. In thermospray, heat is applied, which quickly vaporizes the LC eluent and ionizes the analyte (Figure 2). This heating process has two major consequences for on-line EC/MS work. First, reaction rate constants for the disappearance of intermediates cannot be readily compared with values obtained by room-temperature methods. The rapid temperature changes that occur during the thermospray vaporization process can alter the kinetics of the reactions responsible for product formation. Second, although the thermospray technique is generally known as a "soft" ionization technique, some thermal decomposition can occur and complicate mass spectral interpretation of a redox reaction if the analysis is performed in a flow injection mode. Temperature

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SWAGELOK Tube Fittings conditions during thermospray vaporization change in < 1 s from 25 “C to > 200 “C. If thermal decomposition is suspected, it is often helpful to clarify the situation by using HPLC to separate electrochemically generated species prior to their encounter with the thermospray interface (EC/HPLC/ MS) (25). The thermospray LC/MS interface normally generates backpressures of -900 psi at a flow rate of 1.5 mL/min. Because an additional 1000-2000 psi of backpressure can be added to the system by placing an HPLC column between the electrochemical cell and the thermospray interface, the cell must be capable of withstanding operating pressures as high as 2000-3000 psi. I n Figure 10 we can see how EC/HPLC/MS can be used to separate and identify electrochemical oxidation products of 6 - thiopurine (6-TP). Five peaks are detected eluting off the HPLC column. (Several electrochemical products thermally decompose to yield ions of the same mass - to - charge ratio.) These peaks correspond to purine -6 - sulfinic acid (RSO,H), which thermally decomposes within the thermospray probe to purine ([M+HI+ at m/z 121) before detection; hypoxanthine ([M + HI’ a t m / z 137); purine-6-sulfinamide (RSONH,, [M+H]+at m/z 184); purine-6-sulfonic acid (RSO,H, [M+H]+ at m / z 201); and 6-thiopur i n e d i s u l f i d e ( 6 - MP d i s u l f i d e , [ M + HI+ a t m / z 3 0 3 ) . RSO,H, RSO,H, and 6-MP disulfide form in solution following electron transfer (25). Hypoxanthine and RSONH, originate in solution from 6-thiopurine sulfenic acid, RSOH, which is a 2e- oxidation product of 6-TP and which decomposes rapidly before separation. Purine is a decomposition product of RSONH, and RS0,H. Hypoxanthine is also a decomposition product of RSONH,; h o w e v e r , RSONH,, unlike RSO,H, can still be detected by MS despite the decomposition. Extensive thermal decomposition of the disulfide is also clear from the results in Figure 10. These products were identified by comparing the mass spectra and chromatographic retention times of electrochemically generated products with those of standards (25). EC/HPLC/MS can be especially valuable in providing information about reaction pathways, because individual products can be monitored as a function of applied potential. In contrast to bulk electrolysis, in which results can sometimes be misleading

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because unstable compounds can decompose during long electrolysis times, and cyclic voltammetry, in which peaks are sometimes masked by background discharge, the products generated at the electrodesolution interface are monitored by ECIHPLC. Therefore, when used in combination with MS, W - v i s spectrophotometry, or other electroanalytical techniques, EC/HPLC can be a n important addition to the arsenal of techniques used to characterize redox reactions. Fast atom bombardment and electrochemistry Bartmess and co-workers recently introduced electrochemically assisted fast atom bombardment (FAB) in an attempt to overcome the compounddependent nature of static FAB (38). This approach h a s potential for EC/MS work because it demonstrates that electron transfer reactions can occur on the FAB probe tip, directly inside the ion source of the mass spectrometer. However, the supporting matrix (typically glycerol) is quite different from that used in conventional aqueous electrochemistry. In Bartmess’ work, electrooxidation was used to assist the ionization of 1- bromohexadecane in a glycerol matrix. Without activation, 1-bromohexadecane did not produce a response by FAB. In the two-electrode configuration of electrochemically assisted FAB, however, there is very little control of the working electrode potential and, because very large potentials (k15 V) are applied, virtually all analytes can react, making it difficult to interpret the results of the electrochemical process. Unlike the thermospray system, there is no delay time required for transport of material into the instrument for electrochemically assisted FAB, because electrochemical studies are carried out inside the mass spectrometer. Although further work is needed, these studies, along with recent results from Anderson and House (39),indicate that electrochemically assisted FAB may have an important impact on future EC/MS work. Future directions Although recent developments have concerned the use of thermospray and FAB interfaces for the analysis of nonvolatile compounds, the semipermeable membrane and porous frit interfaces will continue to provide important information about catalytic mechanisms by monitoring gaseous products. EC/MS should prove especially useful in understanding


complex biological redox processes. When the cost of mass spectrometric instrumentation precludes the routine use of EC/MS and EC/HPLC/MS techniques, on-line EC/HPLC (with UV-vis or electrochemical detection) is an alternative approach for characterizing individual processes that cannot be easily analyzed by cyclic voltammetry or coulometry. By using on-line EC/HPLC and EC/HPLC/MS, the processes responsible for product for mation can often be separated as a function of electrode potential. The evolution of new LC/MS interfaces such as continuous-flow FAB and electrospray should provide new and unique opportunities for EC/MS interfacing. For example, the electrospray and continuous -flow FAB inter faces are capable of handling high molecular weight components and can be expected to dramatically extend the range of compounds studied and the problems addressed by EC/MS; furthermore, they may minimize the chemical transformations that can arise from the high temperatures employed in the thermospray interface. Indeed, preliminary experiments under way at Bristol-Myers Squibb indicate the potential of the electrospray interface for EC/MS. With further acceptance, more applications using EC/MS to study the redox chemistry of biomolecules and to model enzymatic reactions are expected. In addition, various types of mechanistic studies can be performed using stable isotope labeling. For synthetic work, EC/MS can be used for monitoring, screening, and identifying compounds produced via electrosynthesis. Advantages include small sample size requirements, mechanistic insights, and rapid analysis. Clearly, the on-line marriage between electrochemistry and MS is a promising new analytical method for solving important problems. The authors acknowledge the assistance of Mike Lee in our early studies and Mike Freund for the review of the manuscript. The research a t the University of Florida was sponsored in part by the National Science Foundation (RAY); the U.S. Army Chemical Research, Development, and Engineering Center (ABT, RAY); the National Institutes of Health (ABT); the University of Florida Interdisciplinary Center for Biotechnology Research (ABT); the Division of Sponsored Research at the University of Florida (AEiT, RAY); and a Merck-Dohme graduate fellowship (KJV).

References (1) Bruckenstein, S.; Gadde, R. R. J. Am. Chem. SUC.1971,93, 793. ( 2 ) Blakley, C . R.; Vestal, M. L. Anal. Chem. 1983,55, 750. (3) Kuwana, T.; Winograd, N. Electruana-

lytical Chemistry; Marcel Dekker: New York, 1974;Vol. 7,Chapter 1. (4)Chang, H.; Johnson, D. C.; Houk, R. S. Tr. A. C. 1989,8,328. ( 5 ) Bittins-Cattaneo, B.; Cattaneo, E.; Konigshoven, P.; Vielstich, W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1991; Vol. 17. (6)Bruckenstein, S.;Gadde, R. R. J. Electroanal. Chem. 1974,50,163. (7)Bolzan, A. E.; Iwasita, T.; Vielstich, W. J. Electrochem. SOC.1987,134,3052. ( 8 ) Pinnick, W. J.; Lavine, B. K.; Weisenberger, C. R.; Anderson, L. R. Anal. Chem. 1980,52,1102. (9)McFadden, W. H. Techniques in Combined Gas Chromatography/Mass Spectrome t v ; John Wiley and Sons: New York, 1973. (10)Brockman, T. J.; Anderson, L. B. Anal. Chem. 1984,56,207. (11)Hartung, T.; Baltruschat, H. Langmuir 1990,6,353. (12)Covey, T. R.; Bruins, A. P.; Henion, J. D. Org. Mass Spectrom. 1988,23,178. (13)Alexander, A. J.; Kebarle, P. Anal. Chem. 1986,58,470. (14)Parker, C. E.;Smith, R. W.; Gaskell, S. J.; Bursey, M. M. Anal. Chem. 1986, 58,1661. (15)Hambitzer, G.; Heitbaum, J. Anal. Chem. 1986,58,1067. (16)Mizoguchi, T.;Adams, R. N. J. Am. Chem. SOC.1962,84,2058. (17)Galus, Z.;Adams, R. N. J. Am. Chem. SOC.1962,84,2061. (18)Dryhurst, G.;Kadish, K. M.; Scheller, F.; Renneberg, R. Biological Electrochemistv,Academic Press: New York, 1982. (19)McKenna, K.; Brajter-Toth, A. J. Electroanal. Chem. 1987,233,49. (20)Andrews, P.A.; Pan, S-S.; Bachur, N. R. J. Am. Chem. SOC.1986,108,4158. (21)Brajter-Toth, A.; Dryhurst, G. J. Electroanal. Chem. 1981,122,205. (22)Miner, D. J.; Rice, J. R.; Riggin, R. M.; Kissinger, P. T. Anal. Chem. 1981, 53, 2258. (23)Rice, J. R.; Kissinger, P. T. Biochem. Biophys. Res. 1982,104,1312. (24)Volk, K. J.; Yost, R. A.; Brajter-Toth, A. Anal. Chem. 1989,61,1709. (25)Volk, K. J.; Yost, R. A.; Brajter-Toth, A. J. Chromatogr. 1989,474,231. (26)Rudewicz, P.; Straub, K. M. Anal. Chem. 1986,58,2928. (27)Perchalski, R. J.; Yost, R. A.; Wilder, B. J. Anal. Chem. 1982,54,1466. (28)Volk, K. J.; Childers-Peterson, T. E.; McKenna, K.; Kraske, P. J.; BrajterToth, A. In Redox Chemistry and Intetfacial Behavior of Biological Molecules; Dryhurst, G.; Niki, K., Eds.; Plenum Press: New York, 1988. (29)Volk, K. J.; Lee, M. S.; Yost, R.A.; Brajter-Toth, A. Anal. Chem. 1988, 60, 720. (30)Volk, K.J.; Yost, R. A.; Brajter-Toth, A. J. Electrochem. SOC.1990,137,1764. (31)Volk, K.J.; Yost, R. A.; Brajter-Toth, A.J. Pham. Biomed. Anal. 1990,8,205. (32)Hawley, M. D.; Tatawawadi, S. V.; Piekarski, S.; Adams, R. N. J. Am. Chem. SOC.1967,89,447. (33)Adams, R. N. Anal. Chem. 1976,48, 1126 A. (34)McCreery, R. L. J. Pharm. Sci. 1977, 66,357. (35)Neptune, M.; McCreery, R. L. J. Oq. Chem. 1978,43,5006. (36)Getek, T.A.; Korfmacher, W. A.; McRae, T.A.; Hinson, J.A. J. Chromatogr. 1989,474,245.

(37)Potter. D.: Hinson. J. Mol. Pharmacol. . 1986,30,33.' (38)Bartmess. J. E.:P h i b s . L. R. Anal. . Chem. 1987,59,2012. (39)House, S.D.; Anderson, L. B. Presented at the 177th Meeting of the Electrochemical Society, Montreal, Canada, 1990;paper 585. .

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Kevin J. Volk, research investigator at the Bristol- Myers Squibb Pharmaceutical Research Institute, received his B.S. degree in chemistry from the University of Central Florida in 1985 and his Ph.D. in analytical chemistry from the University of Florida in 1989. His research interests include the use of hyphenated analytical methods to provide information concerning biological systems, trace mkture analysis, and peptide sequencing.

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@ Richard A. Yost, professor of chemistry at the University of Florida, received his B.S. degree in chemistry from the University of Arizona and his Ph.D. in analytical chemistry fromMichigan State University in 1979. His research interests center on the instrumentation, findamentals, and applications of M S / M S . Having descended from a long line of electrochemists, he finds it intriguing to be investigating electrochemistry via MS/MS.

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J Anna Brajter-Toth, associate professor of chemistry at the University of Florida, received her M.S. degreefrom the University of Warsaw and her Ph.D. from Southern Illinois University in 1979. Her research interests include development of new instrumental approaches for the investigation of redox processes relevant to biological reactivity and the development of new practical electrode suflaces.

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