1024
Anal. Chem. 1986, 58, 1024-1028
Voltammetric Measurement of Tricyclic Antidepressants following Interfacial Accumulation at Carbon Electrodes Joseph Wang,* Mojtaba Bonakdar, and Cloud Morgan
Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
Controlled Interfacial accumulation of trlcyclic antidepressant drugs on carbon surfaces provides the bask for sensltlve and selective voltammetric measurements of these compounds. Cyclic voltammetry Is used to ald In the lnterpretatlon of results. Adsorptlon and &xtractlon/adsorptlonare shown to be the mechanlsms of preconcentration at the glassy carbon and carbon paste electrodes, respectively. The relative extent of accumulation depends on the structure of the antidepressant drug. The dlfferentlal pulse response Is evaluated with respect to electrolyte, electrode material, preconcentratlon perlod, concentratlon dependence, detection limlt, reproduciblllty, posslble interferences, and other variables. After 4 min of preconcentration, detectlon limits of 1.4 X lo-’ M trlmlpramlne, 1.5 X lo-’ M imipramine, and 1.7 X lo-’ M deslpramine are obtained. Transfer of the electrode, with the accumulated drug, to a blank solution Is used to ellmlnate interferences due to solution-phase electroactlve species. Applicability to direct measurements in physlological fluids Is Illustrated.
The tricyclic antidepressants desipramine, imipramine, and trimipramine are the most widely used drugs for the treatment of depression. These drugs are theorized to enhance noradrenergic activity through the blockade of norepinephrine reuptake in peripheral and central noradrenergic neurons. Their efficacy in alleviating depression or side effects depends upon their concentration in physiological fluids. Because of the low levels of tricyclic antidepressants in these fluids, highly sensitive techniques are required for their quantification. In addition, such techniques should fulfill the selectivity, speed, and simplicity requirements. Various methods for the measurements of tricyclic antidepressants have been used since 1960, as was reviewed by Scoggins et al. (1). These usually involve extraction with an organic solvent, followed by assay by gas chromatography, high-performance liquid chromatography, spectrofluorimetry, or radioimmunoassay. The utility of voltammetric techniques for trace measurements of tricyclic antidepressant drugs has not been explored, although amperometric detection was found useful for their monitoring in liquid chromatographic effluents (2, 3). While certain tricyclic antidepressants are electroinactive ( 4 ) ,the frequently used imipramine, trimipramine, and desipramine undergo oxidation through their amine moiety (3). The mechanism of the oxidation seems best described by a two-step, threeelectron ECE process ( 2 ) . These, and the growing use of differential pulse voltammetry in pharmaceutical analysis, have prompted our investigation to assess the suitability of voltammetric techniques for trace measurement of electroactive tricyclic antidepressants. The present paper describes the utility of differential pulse voltammetry for trace measurements of desipramine, imipramine, and trimipramine. We will show that the selectivity and sensitivity of such measurements can be improved substantially by preconcentrating the drugs into the electrode surface. The utility of such interfacial accumulation for trace
measurement of organic compounds has been demonstrated recently ( 5 , 6 ) . Unlike conventional stripping voltemmetry, where high sensitivity is achieved via electrodeposition, no charge-transfer reaction is involved in the accumulation. Most applications of this methodology are based on controlled adsorptive preconcentration at the electrode/solution interface. A mixed (adsorption/extraction) accumulation process is common using carbon paste (7,8)or wax impregnated graphite (9) electrodes. One of the compounds extensively investigated in these studies is the tranquilizer chlorpromazine (7-9). This, and other phenothiazines, has been shown to adsorb a t the surface of carbon electrodes and to extract into their binder (pasting liquid or wax) (7-11). The tricyclic antidepressants explored in the present work are structurally similar to phenothiazine tranquilizers (with the replacement of the sulfur by an ethylene linkage to produce a seven-membered central ring). This indicates that these drugs should exhibit similar surface activity. The interfacial and redox behaviors of several tricyclic antidepressants at glassy carbon and carbon paste electrodes are elucidated in the following sections, and the potential of the adsorption/extraction voltammetric procedure for trace measurement of these compounds is illustrated. Besides the clinical utility of these data, knowledge of the interfacial behavior of tricyclic antidepressants might provide a valuable insight into their interactions with biological membranes, Le., their mode of action. Structural representations of the compounds examined are shown in Figure 1.
EXPERIMENTAL SECTION Apparatus. The cell was a Bioanalytical Systems Model VC-2
voltammetric cell. The cell was joined to the working electrode, reference electrode (Ag/AgCl, Model RE-1, Bioanalytical Systems), and platinum wire auxiliary electrode through holes in its Teflon cover. A magnetic stirrer and a 1.2-cm-longstirring bar provided the convective transport during the preconcentration. In experiments involving the medium exchange, a second (measurement) cell was used. The carbon working electrodes included a glassy carbon disk (3 mm diameter, Bioanalytical Systems) and a “homemade”carbon paste (3 mm diameter), prepared by mixing graphite powder (Acheson 38) and Nujol oil (40% oil by weight). A fresh carbon paste surface was used daily, with the surface being smoothed on a computer card. The glassy carbon surface was polished daily with 0.05-bm alumina slurry, rinsed with copious amounts of deionized water, and allowed to air-dry. Differential pulse and cyclic voltammograms were recorded with EG&G Princeton Applied Research Models 364 and 264 polarographic analyzers, respectively. Reagents. Stock solutions (5 X lov4M) of the tricyclic antidepressants (Sigma) were prepared daily. Desipramine was dissolved in deionized water. The imipramine and trimipramine solutions were prepared by dissolving the compounds in ethanol and making up to volume with deionized water. The supporting electrolyte was 0.05 M phosphate buffer, prepared from a 1:4 mixture of KH2P0, and K2HP04,and adjusted to pH 9.0 with KOH. All solutions were prepared from deionized water and analytical grade reagents. The urine samples were obtained from a healthy volunteer. Normal Control Serum (Ortho Diagnostics, Lot No. 020A02) was dissolved in 5.0 mL of deionized water, according to the manufacturer’s recommendation. Samples were diluted (1 + 3) with the supporting electrolyte prior to use.
Q 1986 American Chemical Society 0003-2700/86/0358-1024$01.50/0
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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Table I. Cyclic Voltammetry Data for the Interfacial Accumulation of Tricyclic Antidepressants at the Glassy Carbon Electrodea CH I
surface
R2- CH I CH
drug desipramine imipramine
trimipramine Desipramine
R,=H
R,fH
Trimlpramine
R,=CH3
R2=CH3
lmipramlne
R,=CH3
R,fH
I
04
Q, KC
concn, mol/cm2
0.70 0.72 0.74
5.34 7.09 11.67
3.9 x 10-’O 5.2 X lo-’’ 8.6 X lo-’’
aConditions: as in Figure 2, except that 1-min (desipramine, imipramine) and 2-min (trimipramine)stirrings were employed.
Figure 1. Structural formulas of tricyclic antidepressants used in the present study.
0.8
E,,,, V
0
E,V
Figure 2. Repetitive cyclic voltammograms for 1 X M imipramine following 4 min of stirring at 0.0 V: carbon paste (A) and glassy carbon (B) electrodes dipped in a 0.05 M phosphate buffer solution (pH 9);scan rate, 50 mVIs.
Procedure. The preconcentration step was performed by immersing the working carbon electrode into a stirred 10-mL sample solution for a given time period. During this period, a 0.0-V potential was applied at the electrode. The stirring was then stopped, and the surface species was measured by applying a differential pulse ramp. In experiments involving medium exchange, the preconcentration proceeded at an open circuit; the electrode was then transferred to an electrolytic blank solution where the differential pulse ramp was applied. Following the potential scan, the electrode was held at + L O V for 2 min to clean it from the remaining accumulated species.
RESULTS AND DISCUSSION Figure 2 shows repetitive cyclic voltammograms for 1 X 10” M imipramine a t carbon paste (A) and glassy carbon (B) electrodes, recorded following 4-min stirring at 0.0 V. At both electrodes, the first scan (designated as 1)yields the larger current response, indicating accumulation of the analyte. Note the differences in the decrease of the current response in the subsequent scans: At the glassy carbon electrode (B), where the accumulation is soley by adsorption, rapid desorption of the reaction products results in a small, and stable, peak (corresponding to contribution of the solution species). In contrast, a gradual decrease in the response is observed at the carbon paste electrode (A), where the extractive/adsorptive accumulation and the redox reaction are followed by slow diffusion of the reaction product from the electrode interior back to the interface. Rapid desorption (or “back-extraction”) of the product are expected for reactions yielding a cation
radical. No peaks are observed in the cathodic branch, indicating an irreversible process. Similar anodic behavior was observed for desipramine, while trimipramine yielded a similar response a t the glassy carbon electrode and no response a t the carbon paste surface. Similar response characteristics were observed for voltammograms recorded after transferring the electrodes, with the accumulated drugs, to a pure electrolyte solution. Other antidepressants, not containing a nitrogen atom in their central ring (e.g., amitriptyline), did not yield a response a t the carbon electrodes. M solutions of the antidepressants and By use of 5 X the glassy carbon electrode, surface saturation was observed following 60 s (desipramine, imipramine) and 120 s (trimipramine). The response for the surface-adsorbed drugs under these conditions was used to estimate the surface coverage and scan rate dependence. The former can be measured for the quantity of charge consumed by the surface process. This, and additional cyclic vpltammetry data, is summarized in Table I. The trend in the surface coverage, trimipramine > imipramine > desipramine, is in agreement with the changes in hydrophobicity associated with the increased number of methyl substituents (Figure 1). The data of Table I were used to calculate the area occupied by an adsorbed drug molecule. Values of 19,32, and 43 nm2 were obtained for trimipramine, imipramine, and desipramine, respectively. While the adsorptive behavior is postulated to involve a planar orientation of the tricyclic ring system and the carbon surface (similar to that of phenothiazine compounds), it appears that minor differences in the side chain may result in an orientation change, especially in the case of trimipramine. The latter may result in a vertically oriented adsorbed layer, as indicated from the substantially lower area occupied by its molecules. Such change may exert profound effects on the resulting response. Throughout this study trimipramine exhibited an adsorptive stripping behavior that differs significantly from that of imipramine and desipramine. A simple explanation for the unique behavior of trimipramine is not available. Plots of log (peak current) vs. log (scan rate) for the surface-adsorbed antidepressants, over the 20-200 mV/s range, were linear (conditions as in Table I). These plots had slopes of 0.83,0.64, and 0.89 (correlation coefficients, 0.998, 0.997, 0.996) for trimipramine, imipramine, and desipramine, respectively. A slope of 1.00 is expected for an ideal reaction of surface species. The deviation from such behavior may be attributed to the ECE mechanism of the redox process. The spontaneous interfacial process can be utilized as an effective preconcentration step prior to the pulse voltammetric measurement. Figure 3 shows differential pulse voltammograms for 5 X lo-’ M desipramine (A), trimipramine (B), and imipramine (C), following 60-s adsorptive accumulation at the glassy carbon electrode. Also shown, as dotted lines, is the corresponding response without accumulation. The use of a short accumulation period results in peak current enhancements of 12 (trimipramine) and 4 (desipramine, imipramine). T h u s , while conventional measurements-without accumulation-do not permit convenient quantitation at the submicromolar concentration level, well-defined peaks are
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I
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
A
_I
C
l
1
I
L
EY
Flgure 3. Differential pulse voltammograms for 5 X M desipramine (A), trimipramine (E),and imipramine (C) solutions: preconcentration at the glassy carbon disk for 60 s at 0.0 V with 400 rpm stirring; scan rate, 5 mV/s; amplitude, 50 mV; supporting electrolyte, 0.05 M phosphate buffer (pH 9). The dotted lines represent the dlrect (0-min) response
/-
E,V
Figure 5. Voltammograms obtained after increasing the trimipramine concentration in 2 X M steps (a-e). Conditions are as in Figure 3.
300 ip,"A
100
100
2
4
,I"
Flgure 4. Dependence of the peak current on the preconcentration time for 5 X M desipramine (a),imipramine (V),and trlmipramine (0): glassy carbon (A) and carbon paste (8) working electrodes. Other conditions are as in Figure 3.
observed following the accumulation. The extent of accumulation, and accordingly the resulting response, is affected by the solution conditions. Various electrolytes such as phosphate buffers (pH 4, 7.4,9), ammonium chloride, potassium nitrate, borate buffer, and potassium chloride were examined. The degree of accumulation increased upon increasing the pH, as expected from the acidbase characteristics of these amines. Best results were obtained with a 0.05 M phosphate buffer (pH 9); this electrolyte was used throughout this study. Figure 4 shows the dependence of the voltammetric peak current on the preconcentration time for 5 X M desipramine, imipramine, and trimipramine. At the glassy carbon electrode (A), the imipramine and desipramine peaks increase rapidly with time at first and then level off. The trimipramine peak increases over the entire time range examined, yielding a 31-fold peak current enhancement at 5 min. This behavior of trimipramine is consistent with the cyclic voltammetric data summarized in Table I. At the carbon paste electrode (B), the desipramine and imipramine peaks gradually increase with preconcentrationtime. With 5-min preconcentration,the peak current enhancements for imipramine and desipramine are 6.5 and 2.9, compared to 4.3 and 5.2 at the glassy carbon electrode. (Trimipramine did not yield to response at the carbon paste electrode.) The different shapes of the time profiles observed at the glassy carbon and carbon paste electrodes reflect differences in the kinetics of the preconcentration process, adsorption vs. mixed extraction/adsorption (8). Throughout this study the glassy carbon electrode yielded improved signal-to-background characteristics and stability, compared to the carbon paste electrode, and thus was used for most of the quantitative work. While the above en-
C-M
xld
Flgure 6. Dependence of the peak current on the concentration of imipramine (a), desipramine (b), and trimipramine (c). Conditions are as in Figure 3.
hancements in sensitivity are important, the improvement in selectivity (described in the following sections) is the main advantage of the method. Forced convection during the preconcentration step affects the resulting peak current by increasing the rate of transport of antidepressant molecules to the electrode surface. For example, a 5-fold peak enhancement was obtained for a stirred (400 rpm) solution compared to a quiescent one (1 X lo4 M imipramine and trimipramine, at the carbon paste and glassy carbon electrodes, respectively). Such behavior indicates that convective mass transport is, to some degree, the limiting process. The differential pulse wave form yielded improved signal-to-backgroundcharacteristics over corresponding linear scan measurements and was used throughout. In order for the adsorptive accumulation process to possess significant analytical utility, it must exhibit a concentration dependence that is well-defined and reproducible. Figure 5 shows differential pulse voltammograms obtained after successive standard additions of trimipramine, each addition affecting a 2 X M increase in concentration; 60-s preconcentration periods were employed. These five measurements are part of eight concentration increments from 2 X M. The results of such calibration experto 1.6 x iments for several tricyclic antidepressants are summarized in Figure 6. For trimipramine (c) the peak current increases linearly with the concentration over the entire range examined (slope, 1603 nA KM-~; intercept, -53 nA; correlation coefficient, 0.998). Desipramine (b) and imipramine (a) exhibit a curvature in the response at concentrations higher than 6 X
ANALYTICAL CHEMISTRY, VOL. 58,NO. 6, MAY 1986
M. Such deviation from linearity poses no limitation, as the use of calibration plots allows quantitation over the entire concentration range. These profiles reflect the extent of surface coverage, as the peak current is a measure of the amount adsorbed. Accordingly, the different behavior of trimipramine is in agreement with the time dependence and cyclic voltammetry data, thus strengthening our hypothesis of different orientations. Detection limits were estimated from the signal-to-noise characteristics ( S I N = 3) of the differential pulse response for 1 X M of the drugs, following 4-min preconcentration (other conditions as in Figure 3). For desipramine, imipramine, and trimipramine, the detection limits are 1.7 X M, respectively. Thus, 30-50 M, 1.5 X M, and 1.4 x ng can be detected in the 10-mL solution used. In contrast, direct measurements did not yield a detectable response a t the concentration level employed. The detectability of the adsorptive voltammetric procedure compares favorably with that of techniques commonly used for measuring tricyclic antidepressants (1) and is adequate for routine monitoring in body fluids. The precision of the results has an important bearing on the utility of the method. The precision was estimated by six repeated measurements of various tricyclic antidepressants (conditions as in Figure 3, except that the concentration was 1.2 X M). These series yielded mean peak currents of 1.10 pA (desipramine), 0.98 pA (imipramine), and 1.02 pA (trimipramine), ranges of 1.01-1.21 pA (desipramine), 0.92-1.09 pA (imipramine), and 0.97-1.05 FA (trimipramine), and relative standard deviations of 6.9% (desipramine), 7.0% (imipramine), and 2.8% (trimipramine). A gradual decrease in the peak current (of approximately 1-3% between successive runs) was observed for desipramine and imipramine, indicating possible formation of an inhibiting film. Such changes do not affect the analytical utility of the method, as indicated from the relative standard deviations. Larger changes were observed a t the carbon paste electrode. For example, an analogous series for desipramine yielded a relative standard deviation of 10%. For real-life analytical applications, it is often the selectivity of the method that determines its practicality. An important feature of the adsorptive preconcentration approach is that the measurement step can be done in a solution that has a different (more ideal) composition than the sample solution (12, 13). By transfer of the electrode to a blank solution following the preconcentration step, a high degree of selectivity toward the surface species is obtained (as interferences due to solution-phase electroactive constituents are eliminated). Such medium exchange procedure permits the direct measurement of analytes in various real samples without any pretreatment of the sample (IO, 11,13,14). Figure 7 illustrates the advantages of the preconcentration/medium exchange approach for direct measurement of antidepressant drugs in body fluids. For this purpose, the diluted urine (A) and serum (B) samples were doped with micromolar concentrations of trimipramine. The voltammograms of these body fluids exhibit large anodic peaks that mask the trimipramine peaks of interest (curves b). In contrast, after exchanging to the pure supporting electrolyte (curves a), no response from solutionphase sample constituents is observed and the trimipramine peak can be detected. The effective correction of contributions from solution-phase species is indicated from the different current scales used. Two additional concentration increments of 3 X lo4 M trimipramine to the urine solution (used in Figure 7A) resulted in a linear increase of the peak obtained after medium exchange. Measurements of desipramine in urine, with and without medium exchange, yielded voltammograms similar to those shown in Figure 7A.
1027
0
Lb
i
b
I
0
0.3
0.6
E,V
0.3
0.6
Flgure 7. Voitammograms for diluted (1:3) urine (A) and normal control serum (B) samples, spiked with 3 X lo-' M (A) and 5 X lo-' M (B) trimipramine: (a)with medium exchange, (b) without medium exchange; preconcentration for 2 min; other conditions as in Figure 3.
While the medium exchange procedure corrects for interferences due to solution-phase electroadive species, adsorbable sample components (electroactive or electroinactive) may affect the response of interest. Under the experimental conditions employed, no interference from adsorbable oxidizable components was observed. This was indicated from the absence of peaks in voltammograms recorded following accumulation from urine samples and medium exchange. Uric acid, known to interact with carbon surfaces (14,15), does not accumulate from the basic solution (used in the present work) due to its ionization. This was evidenced from the elimination of the 5 X M uric acid peak after medium exchange (not shown). The possible interference of surface-active electroinactive components, which may inhibit the accumulation of the analyte, was also examined using a 2 X loW6M trimipramine solution of increasing albumin level (not shown; conditions as in Figure 3). A gradual decrease of the peak was observed upon successive additions of 20 ppm albumin (up to 20% depression a t 100 ppm). No further change in the trimipramine peak was observed upon increasing the albumin concentration up to 200 ppm. These data, as well as that of previous studies (10-14,26), indicate that absorptive stripping measurements of oxidazable compounds at carbon electrodes are less susceptible to interferences of organic surfactants compared to corresponding measurements of reducible species a t mercury surfaces. The former are thus more suitable for direct measurements in various body fluids.
CONCLUSION While no previous data are available regarding the voltammetric quantification of tricyclic antidepressants, the present work demonstrates that this can be successfully accomplished by coupling their interfacial and anodic behaviors a t various carbon electrodes. Such combination yields the desired sensitivity and selectivity, thus permitting measurements in various body fluids. A simple cleanup of such samples (e.g., extraction or protein precipitation) may be required in various situations. The preconcentration/medium exchange/voltammetric scheme can be easily accomplished by using a flow injection system (13),as desirable in the clinical laboratory. Larger extent of adsorption (i.e., higher sensitivity) may be obtained with the new quadrocyclic antidepressants. Besides its analytical utility, the reported interfacial behavior should shed some light on the interaction of these drugs with biosurfaces. Future work will examine the utility of various electrode coverages (dialysis membranes, polymeric coatings) to improve the stability and the feasibility of differentiation between the parent drugs and their metabolites (based on
1028
Anal. Chem. 1986, 58, 1028-1032
differences in their hydrophobicity and redox behaviors).
LITERATURE CITED P.;Norman, T. R.; Burrows, G. D. Clin. Chem. (Winston-Salem, N . C . ) 1980, 26, 5. (2) Suckow, R. R.; Cooper, T. B. J . Pharm. Sci. 1981, 70, 257. (3) "LCEC Application Note No. 40"; Bioanalytical Systems, Inc.: West Lafayette, IN. (4) Oelschlager, H. Bioelectrochem. Bioenerg . 1983, 10, 25. (5) Kalvoda, R. Anal. Chim. Acta 1982, 138, 11. (6) Wang, J. Am. Lab. (Fairfield, Conn.) 1985, 17(5),41. (7) Wang, J.; Freiha, E. A. Anal. Chem. 1984, 56, 849. (8) Wang, J.; Deshmukh, B. K.; Ronakdar, M J . Electroanal. Chem. 1985, 194, 339. (9) Jarbawi, T. 6.; Heinman, W. R.; Patrlarche, G. J. Anal. Chim. Acta 1981, 126, 57. (1) Scoggins, E. A.; Maguire, K.
(IO) Wang, J.; Freiha, B. A., Deshmukh, E. K. Bioelectrochem. Bioenerg., in press. Jarbawi, T. B.; Heineman, W. R. Anal. Chlm. Acta, in press. Wang, J.; Freiha, E. A. Anal. Chim. Acta 1983, 148, 79. Wang, J.; Freiha, E. A. Anal. Chem. 1983, 55, 1285. Chaney, E. N.; Baldwin, R. P. Anal. Chem. 1982, 5 4 , 2556. Wang, J.; Freiha, 6.A. Bioelectrochem. Bioenerg. 1984, 12, 225. (16) Wang, J.; Deshmukh, E. K.; Bonakdar, M. Anal. Lett. Part8 1985, I8 (89),1087.
(11) (12) (13) (14) (15)
RECEIVED for review October 25, 1985. Accepted December 16,1985. This work was supported by the National Institutes of Health (Grants GM30913-02 and RR08136-12) and the American Heart Association.
Alternating Current Voltammetry of Dopamine and Ascorbic Acid at Carbon Paste and Stearic Acid Modified Carbon Paste Electrodes Mark B. Gelbert' and D. J. Curran* Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01002
The Oxidation of dopamlne in acldlc and pH 7 solutions has been examined at the carbon paste electrode by ac and dc cycllc voltammetry. The results of the ac experiments at pH 7 suggest that polymerlratlon of the amlnochrome formed during the dopamine oxldatlon may occur. The stearate modlfled carbon paste electrode was used to study the ac and dc cyclic voltammetry of dopamine and ascorbic acid at pH 7. Separation of the dopamine and ascorbate waves was complete wlth the latter shifted to potentials positive of the former. I n solutlons containing both dopamine and ascorblc acid, the dopamine oxldatlon peak Is enhanced conslderably more than would be posslble due to ring closure of the amlne side chain. The mechanism is clearly a catalyzed EC type where the dopamine-quinone formed by the electrochemlcal reaction Is reduced back to dopamlne by the ascorbate ion. Thus, ascorblc acid Is seen In its common role as an antioxidant. The catalyzed dopamine peak is sultable for quantltatlve purposes when the concentration of ascorblc acid is held constant.
Electroanalytical techniques have been used for in vitro and in vivo studies of catecholamines, which have produced considerable information about these compounds in the CNS (1). The ability to distinguish among the various catecholamines, their precursors and metabolites, and ascorbic acid in vivo has been a major goal in electroanalytical research for some time. Work has been reported where the electrochemical method or the type of electrode used has been varied to accomplish this. Lane et al. employed semidifferential electroanalysis with carbon paste microelectrodes in the study of brain chemicals (2). Four distinguishable oxidation peaks were obtained from the caudate nucleus of the rat. The technique was latter applied by O'Neill and co-workers in an investigation of the rat stratium (3). Carbon fiber microelectrodes were first 'Present address: The Proctor & Gamble Co., Sharon Woods Technical Center, 11511 Reed Hartman Highway, Cincinnati, OH 45241.
reported by Gonon and co-workers ( 4 , s ) . They later electrochemically pretreated the electrodes at highly anodic potentials to achieve greater separation of the dopamine and ascorbic acid responses (6-10). Using differential normal pulse voltammetry and the treated electrodes, they achieved a peak separation of the two compounds of 190 mV (10). Ascorbic acid was the more easily oxidized and the sensitivity for dopamine was reported as 50 000 times that for ascorbic acid. Carbon electrodes of a different design were studied by Wightman and co-workers (11, 12). At their electrodes, a well-defined voltammetric response was obtained for dopamine and a draw-out response to ascorbic acid. Chemically modified electrodes have also been developed that separate the electrochemical responses of the catcholamines and ascorbic acid (13,14). A modified graphite paste electrode was prepared by mixing the Nujol paste with stearic acid (14). Electrostatic repulsion between the anionic carboxyl groups on the surface of the electrode and the ascorbate ion was considered to retard the rate of electron transfer and to be responsible for shifting the oxidation potential region of ascorbate to potentials more positive than that of dopamine. Alternating current voltammetry is a technique that is quite sensitive to the reversibility of the redox couple. Greater selectivity can be achieved relative to dc techniques by exploiting shifts in the peak potential and reduced sensitivity due to differing degrees of reversability between two given electroactive species. In the following work, the ac voltammetries of dopamine and ascorbic acid are explored at both the carbon paste electrode (CPE) and the stearic acid modified carbon paste electrode (MCPE). Cyclic voltammetry in the dc mode is used to confirm the results. Differences in the ac and dc results for dopamine at the CPE suggest the possibility of melanin formation. Results with the MCPE show complete separation of the dopamine and ascorbate waves and indicate a catalytic EC 'mechanism for the electrochemical oxidation of dopamine in the presence of ascorbate.
EXPERIMENTAL SECTION Apparatus. The ac voltammetric potentiostat with digital phase sensitive detection was built in-house (15). A block diagram of the instrument is shown in Figure 1. The analog signal from
0003-2700/86/0358-1028$01.50/0 1986 American Chemical Society
