Anal. Chem. 1997, 69, 5067-5072
An Electrochemical Cell for On-Line Electrochemistry/Mass Spectrometry Merle Corazon S. Regino and Anna Brajter-Toth*
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200
A new electrochemical flow-through cell has been developed for on-line electrochemistry/mass spectrometry (EC/MS) that allows easy access to the working electrode for resurfacing or replacement and that can withstand back pressures of ∼2000 psi. Simple off-line hydrodynamic electrochemical methods have been developed to determine the cell conversion efficiency. The methods can be used to determine the number of moles of analyte transferred from the cell to the LC/MS interface and thereby estimate the sensitivity in on-line EC/MS. The results that have been obtained show that the mobilephase composition may influence the cell conversion efficiency. The highest conversion efficiency in the flowthrough cell (∼32%) was obtained using 90/10 CH3OH/ H2O as the mobile phase, while the lowest conversion efficiency (∼1.8%) was observed with an aqueous mobile phase. Using different mobile phases, the electrochemical flow-through cell was tested in thermospray and particle beam (PB) EC/LC/MS. Conversion efficiencies obtained with the cell on-line in EC/PB/MS are in agreement with the efficiency determined by off-line hydrodynamic electrochemical methods. New insights into nucleophilic reactions of a quinone formed in the electrochemical oxidation of dopamine were obtained online by EC/PB/MS using the new cell. Electrochemistry on-line with mass spectrometry (EC/MS) is a powerful technique for the direct on-line investigation of electrontransfer reactions.1-16 The method can provide chemical information in close to real time (∼500 ms) about the intermediates and (1) Brukenstein, S.; Gadde, R. R. J. Electroanal. Chem. 1974, 50, 163. (2) Pinnick, W. J.; Lavine, B. K.; Weisenberger, C. R.; Anderson, L. B. Anal. Chem. 1980, 52, 1102. (3) Brockman, T. J.; Anderson, L. B. Anal. Chem. 1984, 56, 207. (4) Ren, H.; Szpylka, J.; Anderson, L. B. Anal. Chem. 1996, 68, 243-249. (5) Skou, E.; Munk, J. J. Electroanal. Chem. 1994, 367, 93-98. (6) Wasmus, S.; Samms, S. R.; Savinell, R. F. J. Electrochem. Soc. 1995, 142, 1183-1189. (7) Hambitzer, G.; Heitbaum, J. Anal. Chem. 1986, 58, 470. (8) Volk, K.; Yost, R.; Brajter-Toth, A. Anal. Chem. 1989, 61, 1709. (9) Volk, K.; Yost, R.; Brajter-Toth, A. Anal. Chem. 1992, 64, 21A-33A. (10) Jones, J. A.; Yost, R. Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31-June 5, 1992; p 1711. (11) Regino, M.; Brajter-Toth, A. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996; p 177. (12) Zhou, F.; Van Berkel, G. J. Proceedings of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29-June 4, 1994; p 1002. (13) Zhou, F, Van Berkel, G. J. Anal. Chem. 1995, 67, 3643-3649. (14) Bond, A. M.; Colton, R.; D’Agostino, A.; Downard, A. J.; Trager, J. C. Anal. Chem. 1995,67, 1691-1695. (15) Thompson, M. Electrochemical and Mass Spectrometric Investigations of Biological Molecules. Ph.D. Thesis, University of Florida, 1993. S0003-2700(96)01261-9 CCC: $14.00
© 1997 American Chemical Society
products formed in the electrode reaction.9 Since the inception of EC/MS different types of liquid (LC) interfaces including thermospray (TSP),7-9 particle beam (PB),10,11 and electrospray (ES)12-14 have been used to facilitate solution (of analyte) transfer from the flow-through electrochemical cell to the mass spectrometer. Time resolution of ∼500 ms in EC/TSP/MS has allowed Volk and co-workers8,9 to detect short-lived intermediates in the biologically relevant electrochemical oxidations of purines. The various commercial and home-built electrochemical cells that have been designed for on-line EC/LC/MS7-9 share the disadvantage of a working electrode that is difficult to access, preventing facile resurfacing or modification of the electrode surface. A good cell should facilitate removal of the working electrode from the cell for surface modification. A thin-layer cell design, with high working electrode area-to-cell volume ratio, is required to ensure high conversion efficiency of the cell and good sensitivity in on-line EC/MS in the determination of products of the electrode reactions. The cell geometry is also important for maintaining an uniform solution flow and an uniform current at the working electrode. In this paper, we describe a thin-layer flow-through electrochemical cell that was designed to allow easy access to the working electrode; the cell can withstand the high back pressures encountered during on-line EC/LC/MS. The cell consists of a working electrode that can be removed for resurfacing or replacement or can be chemically modified outside of the cell. A reproducible thin-layer flow-through channel in the cell is obtained by tightning the threaded Nylon bolt, which houses the working electrode, into a threaded Nylon cell block, opposite the auxiliary electrode (Figure 1). No apparent effects on responses from a cylindrical working electrode design were observed. Simple off-line electrochemical hydrodynamic methods have been developed to characterize the thin-layer cell, including the determination of the cell conversion efficiency. MS experiments on-line validated the cell performance predicted by the off-line electrochemical measurements. A new application of the cell in on-line EC/MS with a PB liquid interface is illustrated. EXPERIMENTAL SECTION Materials. Potassium ferricyanide was obtained from Mallinckrodt (St. Louis, MO), and uric acid (2,6,8-trioxypurine) and dopamine (3-hydroxytyramine) were obtained from Sigma (St. Louis, MO). Ammonium acetate (NH4OAc), glacial acetic acid (HOAc), and HPLC-grade methanol were from Fisher (Pittsburgh, PA). All chemicals were used as received. (16) Baczynskyj, L.; Althaus, J.; Voigtlander, V. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 1216, 1996; p 262.
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Figure 1. Cross section of the electrochemical cell: working electrode (W), auxiliary electrode (A); reference electrode (R).
Aqueous solutions of 1 mM K3Fe(CN)6 and 1 mM uric acid (UA) were prepared in 0.1 M NH4OAc. For the determination of the working electrode area, 1 mM K3Fe(CN)6 in 0.10 M KCL was used. Dopamine (DA) solutions (∼0.66 mM) were prepared in 0.2 M NH4OAc/HOAc. The pH*17 of DA solutions was determined in 100% methanol and 90/10 CH3OH/H2O with a pH meter. Electrochemical Methods. A Model 173 PAR potentiostat (EG&G Princeton Applied Research, Princeton, NJ) was used for EC/MS, which was performed at a constant potential. A Bioanalytical Systems electrochemical analyzer (BAS-100, Bioanalytical Systems, Inc., West Lafayette, IN) was used in off-line experiments. Electrochemical Cell. The design of the electrochemical cell is shown in Figure 1. The inverted T-shaped cell body is made from Nylon and has a cylindrical central chamber with 0.13 cm inlet and outlet holes. Stainless steel (0.0254 cm i.d.) and Teflon tubing (0.0254 cm i.d.), with Swagelok fittings (1/8 P-NPT), joins the cell to the pump and the LC interface, respectively. The cell is able to tolerate a back pressure of ∼2500 psi. The pyrolytic graphite working (WE) electrode (3 × 3 mm, Electrosynthesis, Lancaster, NY) was sealed in a threaded Nylon bolt with epoxy (Dexter Corp., Pittsburgh, CA) and was connected to a copper wire with a conducting silver epoxy (Epoxy Technology Inc., Billerica, MA). The outside diameter of the Nylon bolt housing the WE was 3/8 in. o.d., with a thread size of 3/8-16 UNC. The WE was polished and resurfaced on a 600-grit silicon carbide paper (Mark V Laboratory, East Granby, CT) with a metallographic polishing wheel (Buehler Ecomet I) to produce a rough pyrolytic graphite (RPG) WE. The electrode areas of ∼0.07-0.13 cm2 were determined by chronocoulometry18 with a potential step from +0.400 to 0.000 V vs SCE, with Do K3Fe(CN)6 ) 7.63 × 10-6 cm2/s.19 The WE was resurfaced after every 5060 experiments. Palladium (∼1 mm diameter wire) auxiliary (AE) and quasireference20 (R) electrodes were sealed in threaded Nylon bolts with epoxy. The AE was positioned in the cell block directly (17) Bates, R. Determination of pH Theory and Practice; Wiley: New York, 1973. (18) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980. (19) von Stackelberg, M; Pilgram, M.; Toome, V. Z. Electrochem. 1953, 57, 342350. (20) Sawyer, D. T.; Sobkowiak, A.; Roberts, J. L., Jr. Electrochemistry for Chemists; Wiley: New York, 1995.
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opposite the WE in a perpendicular orientation (Figure 1) to minimize the solution resistance.21 The flow-through thin-layer channel serves as a salt bridge to the R electrode, which was positioned downstream. The volume of the thin-layer cell is controlled by first electrically shorting the WE and the AE and then loosening the threaded bolt by a fourth of a revolution. The channel that is produced has a measured diameter of 400 µm. The total cell volume is 66 µL measured from the channel entrance to exit (0.127 cm in diameter, 4.10 cm long). The calculated response time in EC/MS, to a pulse of concentration, determined by the volume of the cell and the tubing that connects the cell to the LC/MS interface, with the tubing volume of ∼8 µL, is ∼839 ms at a flow rate of 2.0 mL/min (1.68 s at 1.0 mL/min). A longer response time may be caused by a nonideal geometry of the WE in the thin-layer cell. Determination of the Cell Conversion Efficiency. The rate of Fe(CN)63- to Fe(CN)64- conversion (mol/s) in the thin-layer cell was determined from the WE steady-state current during electrolysis of continuously flowing Fe(CN)63-. The flow rate of Fe(CN)63- (mol/s) was determined from the flow rate and the concentration of Fe(CN)63-. The conversion efficiency was determined from the ratio of the moles converted per moles pumped. The flow rate of continuously flowing Fe(CN)63- was 1.0-4.0 mL/min, and the cell was polarized by linear sweep voltammetry (scan rates 1-100 mV/s). The cell conversion efficiency was also determined by flow injection analysis (FIA) from the integrated FIA peak areas, from the background-corrected peaks of UA and DA. A Spectra Physics SP8700 (Thermo Separation Products, Fremont, CA) solvent delivery system with a Rheodyne (Model 7125) injector, fitted with a 100-µL sample loop, was used in FIA experiments. UA was injected (100 µL) into 0.1 M NH4OAc mobile phase (1.0 mL/min). The response of UA was measured at a constant potential in the potential range between -0.5 and 0.8 V vs Pd. DA was injected (20 µL) into 100% methanol or into 90/10 CH3OH/H20 mobile phase (flow rate 0.4 mL/min). The response of DA was determined at +0.3 V vs Pd. All measurements were done in triplicate. On-Line EC/MS. The on-line EC/MS system was equipped with a Hewlett-Packard 1050 Series HPLC pump with a 20 or 100µL injector loop for PB and TSP MS, respectively. In on-line EC/ MS experiments, the flow-through electrochemical cell was positioned between the HPLC pump and the ion source of the mass spectrometer. A Vestec Thermospray LC/MS system (Model 201 Vestec Corp., Houston TX with a Vector/One Data System) was used for EC/TSP/MS, with the filament and discharge electrode off. The tip temperature (T2) was optimized at 1.0 mL/min at 198210 °C at a source block temperature of 340 °C. Positive ion spectra were obtained in the m/z range from 141 to 184 at a rate of 0.3 scans/s. A Hewlett-Packard 5989A quadrupole mass spectrometer with a Hewlett-Packard 59980B particle beam interface (Palo Alto, CA) was used for EC/PB/MS. Positive chemical ionization (PCI) mass spectra were obtained with methane as the reagent gas at the ion source pressure of 2.7 × 10-4 Torr. The desolvation chamber temperature and the nebulizer He pressure were 60 °C and 54 psi, respectively. Mass spectra were acquired at a rate of 1.0 (21) Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry; Marcel Dekker, Inc.: New York, 1984.
Table 1. Electrochemical Cell Performance at Different Flow Ratesa flow rate, (mL/min)
limiting current × 105 (A)
no. of mol reacted/s × 1010
conversion efficiency (%)
diffusion layer thickness, (µm)
4.0 2.0 1.5 1.0
4.39 3.10 2.91 2.61
4.55 3.21 3.02 2.71
0.68 0.96 1.20 1.62
240 340 370 410
a Electrode area, 0.11 cm2; scan rate is 10 mV/s; supporting electrolyte, 0.1 M NH4OAc.
Figure 2. Hydrodynamic cyclic voltammograms of 1 mM Fe(CN)63at 1 (- - -) and 8 (s) mV/s in 0.1 M NH4OAc; working electrode area, 0.10 cm2; flow rate, 1.0 mL/min. Inset is the average plot of potential vs log((iL - i)/i) at 1, 2, 4, 6, and 8 mV/s (slope, 57.96 ( 0.58 mV).
scan/s in the m/z range of 100-200. RESULTS AND DISCUSSION Electrochemical Off-Line Characterization of the FlowThrough Thin-Layer Cell. Hydrodynamic voltammograms of 1 mM Fe(CN)63-/4- obtained at a flow rate of 1 mL/min in the flowthrough thin-layer cell at the voltammetric scan rate of 1-8 mV/s are shown in Figure 2. A constant steady-state current that is observed indicates that in this flow rate and scan rate range the rate of mass transport of Fe(CN)63-(convective) to the WE is greater than the rate of electron transfer of Fe(CN)63-. Logarithmic analysis (insert, Figure 2) of the i-E curves gave a slope of 58.0 ( 0.6 mV, indicating Nernstian behavior of Fe(CN)63- at the WE. At a flow rate of 1.0 mL/min, at a scan rate greater than 10 mV/s, the rate of electron transfer exceeds the rate of mass transfer and a peak-shaped voltammogram is obtained (data not shown). The steady-state response in Figure 2 confirms thin-layer behavior of the cell under the experimental conditions (1.0 mL/ min; 1-8 mV/s). Thus, under these conditions, transport of analyte in the cell is able to keep up with or exceed the rate of the electrode reaction. The limiting current, iL, depends on the solution flow rate in the cell.18,20 The results that were obtained at different flow rates are summarized in Table 1. The results show an increase in iL with an increase in flow rate; iL follows a dependence on solution velocity, V (cm/s), of V0.38 in agreement with previous reports.22,23 Diffusion layer thickness, do, that can be determined from iL18 is also shown in Table 1. (At the cylindrical, WE do is an average (22) Weber, S. J. Electroanal. Chem. 1983, 145, 1-7. (23) Hirata, Y.; Lin, P. T.; Novotny, M.; Wightman, R. M. J. Chromatogr. 1980, 181, 287.
of several parameters related to the solution flow). The do values in Table 1 show that under the experimental conditions when a steady-state response is observed in the flow-through cell, do approaches the thickness of the thin-layer channel between the WE and AE (∼400 µm). Diffusion layer thickness can be used to measure the relative efficiency of (convective) mass transport.18,20 The thickness is expected to decrease with an increase in flow rate as verified by the experimental results in Table 1. Diffusion time through the thin-layer channel can be estimated (t ) do2/2D); at a flow rate of 1.0 mL/min, t is ∼80 s. At this flow rate, the residence time in the cell (cell volume divided by flow rate) is 0.24 s. Since the diffusion time is greater than the residence time in the channel, the cell conversion efficiency, defined as the fraction of analyte introduced into the cell that undergoes the electrochemical reaction, is low (Table 1). Nevertheless, the conversion efficiency, which determines the number of moles of analyte that reach the LC/MS interface, is sufficiently high for sensitive detection by MS on-line. At higher flow rates, the conversion efficiency decreases (Table 1) because of the shorter residence time in the cell. Under the conditions when the concentration of the unreacted analyte in the flow-through cell approaches zero,20 maximum (100%) conversion efficiency of the cell is attained (then do ) ADo/ u). Such efficiency can be obtained (at u ) 1.0 mL/min practical for EC/TSP/MS, with a maximum possible area of the WE of A of 0.64 cm2) when do is ∼3.7 µm. In other words, to reach 100% conversion efficiency, with the experimental u and A, channel thickness in the cell must decrease from 400 to 3.7 µm. This will decrease the diffusion time of the analyte in the cell to ∼7.1 ms with the diffusion in the cell becoming significantly shorter than the residence time in the cell. Cell Efficiency in FIA. Figure 3b shows a hydrodynamic electrochemical response of UA in FIA which was used to characterize the flow-through cell off-line. The plot shows moles of UA reacted vs the WE potential in the flow-through cell, at a flow rate of 1.0 mL/min (this flow rate was used in EC/TSP/ MS). As shown by the results, steady-state current of UA develops at potentials positive of ∼0.35 V,9 past the voltammetric peak (Figure 3a) recorded in a stationary solution at the same RPG WE. The average cell conversion efficiency determined from the steady-state region is ∼1.85%. The agreement between the cell conversion efficiency obtained with UA (by FIA) and that obtained with continuously flowing Fe(CN)63- (Table 1) indicates that the conversion efficiency is independent of the analyte. Similar hydrodynamic electrochemistry results obtained for DA demonstrate, however, that the conversion efficiency depends on the composition of the mobile phase. For example, in 100% CH3Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Table 2. Intermediates and Products of Uric Acid Oxidation by EC/TSP/MSa m/z (% RA)
positive ions identified
141(31) 143(7) 158(10) 159(22) 169(100) 176(1) 177(2) 184(2)
[bicyclic imidazolone +H]+ [alloxan + H]+ [2-oxo-4-imino-5-ureidoimidazolidine + H]+ [allantoin + H]+ [uric acid + H]+ [allantoin + NH4]+ [5-hydroxyhydantoin-5-carboxamide + NH4]+ [imine amine + H]+
aTip temperature, 210 °C; source temperature, 340 °C; flow rate of 0.1 M NH4OAc mobile phase, 1.0 mL/min.
Figure 3. (a) Cyclic voltammogram of 1 mM uric acid in 0.1 M NH4OAc. Rough pyrolytic graphite WE, Pd R; scan rate 100 mV/s. (b) FIA of uric acid in 0.1 M NH4OAc. FIA conditions: flow rate, 1.0 mL/ min; 100 µL of 1 mM uric acid injected.
Figure 4. EC/TSP/MS of uric acid in 0.1 M NH4OAc at 0.7 V vs Pd. Flow rate, 1.0 mL/min; tip temperature, 210 °C; source temperature, 340 °C. Asterisks indicate oxidation intermediates and products that have been identified.
OH as the mobile phase, the conversion efficiency is 6 ( 4% while in 90/10 CH3OH/H2O the efficiency is 32 ( 9%. In an aqueous mobile phase, in the absence of an analyte, high residual current slowly decays to a constant value which may reflect high initial activity of the electrode which decreases with time.11,24,25 When the mobile phase contains methanol, the residual i-t response is significantly smaller resulting in higher conversion efficiency. EC/TSP/MS. When the electrochemical oxidation of UA is monitored by EC/TSP/MS multiple intermediates and products are observed as shown in Figure 4. The [M + H]+ ion of UA at m/z 169 is observed as the base peak during the oxidation when the block temperature is 340 °C. The intermediates identified by EC/TSP/MS during different stages of the oxidation8 at 0.7 V, and the final products, are summarized in Table 2. (24) Austin, D. S.; Polta, J. A.; Polta, T. Z.; Tang, A. P.; Cabelka, T. D.; Johnson, D. C. J. Electroanal. Chem. 1984, 108, 227-48. (25) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A-597A.
5070 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
In agreement with previous results, the bicyclic imidazolone ([M + H]+ at m/z 141), which results from both ammonolysis and hydrolysis of a diimine,8 is the most abundant intermediate. The imine amine [M + NH4]+ at m/z 184,8 whose relative mass spectral abundance varies strongly with tip temperature, and the 5-hydroxyhydantoin-5-carboxamide ([M + NH4]+ at m/z 177),8 are the least abundant intermediates. The [M + H]+ ions of the products, alloxan (m/z 143), which originates from the diol, and 2-oxo-4-imino-5-ureidoimidazolidine (m/z 158), which results from ammonolysis of the bicyclic imidazolone, were also detected.8 Allantoin ([M + H]+ at m/z 159 and [M + NH4]+ at m/z 1768) was the most abundant product. The low abundance of the m/z 158 product relative to the abundance of allantoin (m/z 159 plus 176) points to a low yield of ammonolysis reactions relative to hydrolysis as expected at this tip temperature.8 The results show that the relatively low conversion efficiency in the new flow-through cell does not influence the distribution of the intermediates and products of the electrode reaction in comparison to the results with the high-efficiency commercial cell.8 The results confirm that the efficiency is high relative to the sensitivity of EC/TSP/MS, allowing detection of the intermediates and products of UA oxidation. EC/PB/MS. PB LC/MS interface can be used to enhance MS sensitivity for nonaqueous samples. Figure 5 illustrates the EC/PB/mass spectrometric hydrodynamic voltammograms of 0.66 mM DA in 0.2 M NH4OAc/HOAc injected into 90/10 CH3OH/H2O (pH* 6.3) mobile phase. A cyclic voltammogram at the same RPG WE obtained in a stationary solution (Figure 5) shows that the electrochemical oxidation of DA begins at ∼+0.1 V and reaches completion at ∼+0.20 V. Oxidation of DA can be followed by EC/PB/MS by monitoring the decrease in the abundance of the [M + H]+ ion of DA at m/z 154 (Figure 5). The EC/PB/MS oxidation of DA begins at somewhat lower potentials and reaches steady state at ∼+0.20 V. As shown in Figure 5, the intensity of the 154+ ion of DA drops by ∼28% as a result of the EC/PB/MS oxidation. This apparent conversion efficiency of DA is in agreement with the conversion efficiency determined by off-line methods by FIA. Similarly, the intensity of the [(M + H)+ - NH3] ion of DA at m/z 137 (not shown) decreases by ∼28%. In the potential range of -0.1 to 0.0 V, where a decrease in the intensity of the m/z 154 ion of DA occurs, the intensity of the
Figure 6. Proposed pathway for the EC/PB/MS oxidation of DA in 0.2 M NH4OAc/HOAc buffer (pH* 6.3) in 90/10 CH3OH/H2O .
Figure 5. EC/PB/MS of DA in 90/10 CH3OH/H2O with selected ion monitoring of intermediates and products of DA oxidation; 20 µL injection of 0.66 mM DA in 0.2 M NH4OAc/HOAc; flow rate, 0.4 mL/ min; desolvation temperature, 60 °C; He pressure, 54 psi. (Top) Cyclic voltammogram of 0.66 mM DA in 0.2M NH4OAc/HOAc buffer pH* 6.3 in 90/10 CH3OH/H2O on RPG WE, Pd R. Scan rate, 100 mV/s.
ion at the m/z 152 increases (Figure 5). The increase in the intensity of the ion at m/z 152 supports the formation of DA o-quinone in a two-electron oxidation of DA.26-28 At potentials positive of 0.0 V, however, the intensity of the m/z 152 ion decreases, indicating the disappearance of the o-quinone, in agreement with its known reactivity.26-28 The hydrodynamic EC/PB/MS profile of ions at m/z 154 and 152 (Figure 5) also shows that the ratio of DA/DA o-quinone does not change at potentials positive of 0.0 V. Kinetically this means that the reaction(s) of DA o-quinone, which follow the electrode reaction that generates the o-quinone, do not alter the redox equilibrium of DA/DA o-quinone. This indicates that the reactions are slow under the experimental conditions. Ammonolysis of DA o-quinone can occur via a reaction with ammonia generated from the NH4OAc buffer.8 A proposed pathway is illustrated in Figure 6. According to this pathway, the [M + H]+ ion at m/z 167 (Figure 5) may be due to semiquinone V, which can form from aminodopamine IV. Facile oxidation may explain the low abundance of aminodopamine (IV, [M + H]+ m/z 169, Figure 5) relative to that of other intermediates. Intracyclization of V, in favor of its availability for the IV/V redox equilibrium,29,30 may explain the low abundance of the m/z 167 (26) Hawley, M.; Tatawawadi, S.; Piekarski, S.; Adams, R. N. J. Am. Chem. Soc. 1967, 89, 447. (27) Sternson, A. W.; McCreery, R.; Feinberg, B.; Adams, R. N. J. Electroanal. Chem. 1973, 46, 313. (28) Dryhurst, G.; Kadish, K.; Scheller, F.; Renneberg, R. Biological Electrochemistry; Academic Press: New York, 1982; Vol. 1. (29) Blank, C. L.; McCreery, R. L.; Wightman, R. M.; Chey, W.; Adams, R. N.; Reid, J. R.; Smissman, E. E. J. Med. Chem. 1976, 19, 178. (30) Blank, C. L.; Kissinger, P. T.; Adams, R. N. Eur. J. Pharmacol. 1972, 19, 391.
ion of V compared to the final product (VIII) ion at m/z 150. The appearance of the ion at m/z 150 (Figure 5) verifies the formation of VIII, the final oxidation product, which is the same as the product formed when sufficient unprotonated DA o-quinone undergoes intramolecular 1,4-Michael addition.27 The EC/MS results indicate that the electrochemical oxidation at potentials more positive than ∼0.8 V, apparent from the cyclic voltammogram in Figure 5, does not influence the abundance of the intermediates and has only a small effect on the abundance of the final product. This indicates that the oxidation(s) in this potential range has(have) a low product yield, possibly as a result of slow kinetics of formation of the product(s). The processes were not investigated further. Based on the sum of the peak areas of the ions, of the intermediates, and of the products that have been identified, relative to the peak area of the total ion profile, conversion efficiency is ∼13.5% for the intermediates and products. Therefore, intermediates and products may be ionized less efficiently than the parent compound. However, the results show that the sensitivity of the cell in combination with EC/PB/MS allows the detection of the intermediates and products of DA oxidation.
CONCLUSIONS The versatility of the new electrochemical thin-layer flowthrough cell for on-line EC/MS has been verified under different conditions including continuous flow of analyte, FIA, EC/TSP/ MS, and EC/PB/MS. The cell was used to demonstrate the broad scope of potential analytical applications of EC/MS, including in the investigations of redox reactions in nonaqueous and mixed nonaqueous/aqueous solvents. The off-line electrochemical methods that have been developed to characterize the cell conversion efficiency have provided a quantitative approach for estimating the sensitivity in on-line EC/MS. Furthermore, the results demonstrate the sensitivity of on-line EC/PB/MS for nonaqueous samples and illustrate an application of EC/PB/MS in the studies of nucleophilic reactions that follow the electrochemical oxidation of DA. Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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ACKNOWLEDGMENT This paper is dedicated to Prof. Z. Kublik on the occasion of his 75th birthday. We thank Dr. Maurice V. Thompson for his initial work on the design of the cell and Dr. John P. Toth for helpful discussions and for access to the mass spectrometers used in this research.
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Received for review December 12, 1996. October 6, 1997.X
Accepted
AC961261N
X
Abstract published in Advance ACS Abstracts, November 15, 1997.
