Anal. Chem. 1991, 63,845-847
or DDC procedures given in the literature for determining inorganic arsenic species by GC (3, 4).
LITERATURE CITED (1) Cullen, W. R.; Reimer, K. J. Chem. Rev. 1989, 89, 713. (2) Beckermann, B. Anal. Chim. Acta 1982, 135, 77. (3) Dix, K.; Cappon, C. J.; Toribara, T. Y. J . Chromatogr. Scl. 1987, 2 5 , 164. (4) Daughtrey. E. H.; Fitchett, A. W.; Mushak, p. Anal. Chim. Acta 1975, 7 9 , 199. ( 5 ) Neeb, R . Pure Appl. Chem. 1982. 5 4 , 847. (6) Schaller, H.; Neeb, R . Fresenius' 2. Anal. Chem. 1986, 323, 473. (7) Schaller. H.: Neeb. R . Fresenius' Z . Anal. Chem. 1987, 327, 170.
a45
Tavlarldis, A.; Neeb, R. Fresenius' 2.Anal. Chem. 1978, 292, 135. Mok, W. M.; Shah, N. K.; Wai, C. M. Anal. Chem. 1986, 58, 110. Mok, W. M.; Wai, C. M. Anal. Chem. 1987, 5 9 , 233. SchneMer, H.; Neeb, R. Fresenlus' 2.Anal. Chem. 1978, 293, 11. Aggarwal, S. K.; Kinter, M.; Wills, M. R.; Savory, J.; Herold, D. A. Anal. Chem. 1990, 6 2 , 111. (13) Tseng, W. P. Environ. Health Perspect. 1977, 19, 109. (8) (9) (10) (11) (12)
RECEIVED for review May 29, 19%. Accepted January 24, 1991. This material is based upon work supported by the Idaho Science Foundation under EPSCoR Program Of the Grant No. RII-8902065.
Amperometric High-Performance Liquid Chromatographic Detection of NADH at a Base-Activated Glassy Carbon Electrode Eugene J. Eisenberg* and Kenneth C. Cundy Department of Drug Delivery, Sterling Research Group, Great Valley, Pennsylvania 19355
INTRODUCTION In recent years, there has been increasing interest in the electrochemical oxidation of dihydronicotinamide adenine dinucleotide (NADH) in aqueous solutions ( I d ) , both in the direct study of the NAD+/NADH redox couple in the electron-transport chain and in the area of analytical applications. Among these applications is the indirect determination of components involved in NAD+/NADH-coupled enzymatic oxidations (levels of substrates and inhibitors, enzyme activity, etc.). As a consequence, a considerable amount of research has been directed toward development of an analytical procedure for the detection of NADH. Liquid chromatography with electrochemical detection (LC-EC) is in itself a very sensitive and selective technique for the determination of many important analytes. Coupled with postcolumn enzymatic reactions involving the NAD+/NADH redox couple, it can be a powerful tool in the determination of many dehydrogenase substrates and inhibitors. However, to date there are few examples of the use of electrochemical detection for this purpose (6, 7). At high anodic potentials, NADH undergoes a two-electron oxidation in aqueous solution to produce nicotinamide adenine dinucleotide (NAD+). The slow heterogeneous electron transfer a t solid electrodes consequently requires high overpotentials to achieve sufficient sensitivity. But at high operational potentials, a large number of solutes as well as mobile-phase constituents and impurities become electroactive, and as a result, the advantages of the technique, namely its selectivity and sensitivity, cannot be realized. The most common approach to the detection of compounds with nonideal redox behavior is the use of an appropriate catalyst that can improve electron-transfer kinetics, either via homogeneous catalysis, e.g., redox mediators in solution (phenazine methosulfate (7)), or via heterogeneous catalysis on the surface of a solid electrode. Since the use of redox mediators in HPLC mobile phases or as a postcolumn additive can adversely affect reproducibility, sensitivity, and HPLC separation parameters or be too expensive for routine applications, electrode modifications appear to be more suitable for use in LC-EC. Considerable work has been directed toward the surface modification of glassy carbon electrodes, commonly used in LC-EC systems. Specific chemical modification of a surface by attachment of appropriately selected electrocatalysts (6,8),physicochemical modification through polishing (9, IO), and general electrode conditioning by chemical or electrochemical oxidation of the surface ( 1 , 11-13) have proven to be effective in improving electrode response for a number 0003-2700/91/0363-0845$02,50/0
of compounds. Numerous examples of electrode oxidation have been reported, most of which used the application of alternating oxidizing and reducing potentials of various magnitude and duration. However, to date only a few techniques were shown to be suitable for application to the HPLC environment, with its relatively high organic content and high flow rates. Most of the above-mentioned techniques are time consuming and limited to voltammetric measurements in static solutions with modified surfaces requiring frequent and lengthy regeneration. I t has recently been reported that the ferricyanide redox couple (12) and dopamine (13) approached nearly ideal response kinetics at a glassy carbon electrode anodically activated in basic solution. In addition to being simple and reliable, this method of activation also greatly lowered the electrode capacitance in comparison to other modes of activation. As a consequence of developing sensitive and selective analytical procedures for the HPLC detection of bile acids, which can be measured by their enzymatic reaction with NAD+ to yield NADH, the utility of a glassy carbon electrode subjected to electrochemical base activation of the surface has been examined for measurement of NADH in LC-EC systems. The electrochemical behavior of activated and untreated electrodes and characteristics such as sensitivity and dynamic range are compared and the stability of the activated electrode is discussed in the present study.
EXPERIMENTAL SECTION Apparatus. The HPLC apparatus consisted of a Hitachi L-6OOO HPLC pmp, 655-40A autosampler, a Zorbax CN column (50 X 6 mm, 3 u) (Mac-Mode Analytical, Chadds Ford, PA), postcolumn enzyme reactor (IMER, Chrompack Inc., Raritan, NJ) loaded with 3a-hydroxysteroid dehydrogenase, and a Bioanalytical Systems Model LC-IBI 17AT dual amperometric detector (BioanalyticalSystems, West Lafayette, IN) equipped with MFlOOO glass carbon electrode in conjunction with Ag/AgCl (saturated KCl) reference electrode. The same electrode with a jumper connector was used in conjunction with Ag/AgCl (saturated KC1) reference electrode and a platinum wire auxiliary electrode for cyclic voltammetry. The voltammetry measurements were performed on a Bioanalytical Systems Model CV-27 cyclic voltammetric apparatus. Reagents. Unless stated otherwise, the HPLC mobile phase was a 0.5 mM NAD+ solution in 3070 methanol405 M phosphate buffer (pH 7.5). A 0.05 M phosphate buffer solution, adjusted to pH 7.5, was used as the supporting electrolyte in the voltammetry experiments. 3a-Hydroxysteroid dehydrogenase (EC 1.1.1.50,27 units/mg of protein) was obtained from Sigma. NAD+ 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 8,APRIL 15, 1991
and NADH, disodium salt, grade I were obtained from Boehringer Mannheim, 1 M NaOH was obtained from Baker, and other chemicals were supplied by Fluka and were of the highest grades available. Enzyme Immobilization. A new IMER cartridge (10 X 2.1 mm) (Chrompack) was connected to an HPLC pump, and 1mL of 3n-hydroxysteroid dehydrogenase (16 units/mL) in 0.05 M NaHCO, was recirculated through the cartridge for 1 h at room temperature (0.5 mL/min flow rate). Electrode Preparation. A glassy carbon electrode was polished with a 0.5-pm alumina suspension from BAS electrode polishing kit, rinsed with water, sonicated in methanol for 2 min, and dried under a stream of nitrogen. The electrode was then activated according to Anjo et al. (13)in an electrochemical cell with 1 M NaOH electrolyte for 5 min at a potential of +1.2 V. Following activation,the electrode was rinsed with distilled water and installed into a thin-layer flow cell of an amperometric HPLC detector. R E S U L T S AND DISCUSSION A dominant process during electochemical oxidation of glassy carbon has been reported to be the formation of a homogeneous film of graphitic oxide phase (11, 12). The structural composition of the film has not yet been characterized. It contains up to 20% oxygen by weight and is composed of a variety of compounds including single aromatic rings and polymers. The film formed during the electrochemical base activation was slightly dull in its appearance and could not be removed even by 5-min sonication in methanol or acetonitrile. In fact, its removal required intensive repolishing. Although its catalytic action is apparently due to a combination of factors including electrochemical mediation by quinone type redox moieties in the graphitic oxide film, we feel that it is probably mostly due to the increased adsorption of the redox couple. A very similar voltammetric behavior has been observed for catechols at a base-activated electrode (13)and an electrode surface modified with dispersed alumina particles whose catalytic activity can be attributed almost entirely to increased adsorption (9, IO). Voltammetry of NADH at Modified Electrodes. Following base activation, the electrode was immediately immersed in a 0.5 mM NADH solution. The cyclic voltammograms at 50 mV s-l observed for a supporting electrolyte solution with and without NADH at both untreated and treated electrodes are presented in Figure 1. In the former case, the oxidiation of NADH consists of a broad uncatalyzed, irreversible wave having a maximum a t a peak potential of approximately +0.85 V vs AgfAgCl. With the pretreated glassy carbon surface, the anodic wave became much more defined and shifted down to +0.50 V. Figure 1A shows the cyclic voltammograms for modified electrode in the presence and absence of NADH. A large increase in the oxidation wave was observed on addition of NADH to the electrolyte solution. The voltammogram for the treated electrode also shows a reduction wave a t -1.2 V on a reversed scan. This wave suggests regeneration of NAD+ and apparently corresponds to reduction of NAD+ electrochemically adsorbed on the electrode surface. The waves remained unchanged on continuously repeated cyclic scanning from -1.6 to +1.2 V for 1 h. LC-EC w i t h t h e Modified Electrode. Chromatograms of NADH at pretreated and untreated electrodes are presented in Figure 2. NADH was produced as a result of the postcolumn enzymatic oxidation of cholic acid in the presence of NAD+. At the operating potential of +0.8 V vs AgfAgCl, a high response was obtained for the activated electrode, whereas no signal could be detected for the untreated electrode. When data from cyclic voltammetry are compared with data obtained under LC-EC conditions in a thin-layer detector cell, different observations are to be expected. The actual applied
A
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Flgure 1. Cyclic vottammograms of electrochemically baseactivated (A) and untreated (B) glassy carbon electrodes for 0.05 M phosphate buffer (pH 7.5) with 0.5 mM NADH (-) and without NADH (---); po-
tential scan rate of 50 mV/s.
CHOLIC ACID
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Flgure 2. Chromatograms of cholic acid (100 pmol on column) detected via enzymatic NADH production at +0.8 V vs Ag/AgCI at (A) base-activated and (B) untreated glassy carbon electrodes. Mobile phase, 0.1 mM NAD in 30/70 methano110.05 M phosphate buffer (pH 7.5); flow rate 1.5 mL/min; temperature 30 O C .
potentials in LC-EC usually have to be set higher then those observed for the anodic wave in cyclic voltammography, in order to compensate for higher ohmic resistance of the thin layer (14, 15). This difference in the potentials may vary depending on the cell design, supporting electrolyte concen-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 8, APRIL 15, 1991
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Figure 3. Hydrodynamic voltammograms for enzymatically generated NADH (1 nmol on column) at a base-activated (0)and untreated (0) glassy carbon electrode. Conditions are in the text.
tration, etc. The hydrodynamic voltammograms (analyte peak current vs potential profiles) allow more accurate analysis of electrode behavior under actual chromatographic conditions. Figure 3 demonstrates the hydrodynamic voltammograms for the activated and untreated electrodes. The voltammogram for the electrochemically treated electrode reaches a wellformed plateau, indicating improved electron-transfer kinetics, whereas for the untreated glassy carbon a defined plateau is never reached. This behavior generally correlates with data from cyclic voltammograms. With cholic acid of increasing amounts (0.3-300 pmol on column) as an analyte, a linear calibration plot was obtained with correlation coefficients of better than 0.990. Since the conversion efficiency of the immobilized enzyme under the HPLC conditions is approximately 30%, this range corresponds to 0.1-100 pmol of NADH. The detection limit, defined as the amount of an analyte injected that produced a signal-to-noise ratio of 2, was 0.3 pmol of cholic acid (100 fmol of NADH). The detection limits reported for the enzymatic determination of bile acids obtained by fluorometric (16)or electrochemical detection methods (6) were significantly higher (3 and 20 pmol, respectively). In practical LC-EC applications, the use of different mobile phases may be necessary. To demonstrate the stability of the base-activated glassy carbon electrode in various media, three different mobile phases, consisting of 0.05 M phosphate buffer (pH 7.5), buffer-methanol (70:30), or buffer-acetonitrile (85:15), were used. To test the stability of the response at +0.8 V, 1 0 - ~ L aliquots of NADH in the corresponding mobile phase (1pg/mL) were injected every 20 min over a 24-h period. The amperometric detector was connected in series with a fluorometer (Hitachi F-1000, excitation 325, emission 460, sensitivity set a t 1). The fluorometric response was used to compensate for variability in the injection size as well as for changes in NADH concentration over the time period. The ratio of amperometric to fluorometric response for each injection was plotted vs time. The catalytic activity over the study period (24 h) remained virtually unchanged for the aqueous mobile phase and declined slightly for the mobile phases with methanol and acetonitrile. The relative standard deviations for the peak ratios were 2.570, 7.8%, and 11.5% over the complete series of 72 measurements. Figure 4 dem-
0
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TIME AFTER TREATMENT, MIN
Figure 4. Response of the baseactivated glassy carbon electrode in different mobile phases over a 4-h period. The ratio of the amperometric to fluorometric response for each injection is plotted vs time period during which the activated electrode was subjected to continuous use. (0) 0.05 M phosphate buffer (pH 7.5); (A)buffer-methanol (70:30), and (0) buffer-acetonitrile (85: 15). Other conditions are described in the text.
onstrates a typical response of the activated electrode over a 4-h period. Treated electrodes continued to exhibit improved response during several days of uninterrupted operation, and their performance could easily be restored with a 5 min of reactivation in 1 M NaOH a t +1.2 V.
CONCLUSIONS Several previous studies have demonstrated the utility of the base-activated glassy carbon electrode for the detection of electroactive species with slow electron-transfer kinetics. However, these investigations were limited to cyclic voltammetry in static solution. In this work, we have explored the suitability of an electrochemically baseactivated glassy carbon electrode as a sensor for NADH in LC-EC systems. The treated electrode was compatible with mobile phases commonly used in HPLC. It can provide analytically convenient, selective, highly sensitive, and stable detection of NADH, thus following the chromatographic determination of numerous chemical entities involved in NAD+ NADH-coupled oxidation processes. The devised strategy appears to have broad applicability, and its further development is in progress.
LITERATURE CITED (1) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 4 7 , 1337. (2) Moiroux, J.; Elving, P. J. J . Am. Chem. Soc. 1980, 702, 6533. (3) Blankspoor, R. L.; Miller, L. L. J . Electroanal. Chem. 1984, 777, 231. (4) Matsue, T.; Chang, H.-C.; Uchida, 1.; Osa, T. Tetrahedron Lett. 1988, 29 (13),1551. (5) Ravichadran, K.; Baldwin, R. P. Anal. Chem. 1983, 55, 1782. (6) Marco-Varga, G. J . Chromatogr. 1987,408, 157. (7) Karnada, S.;Maeda. M.; Tsuji, A. J . Chromatogr. 1982, 239, 773. (8) Bartalits, L.; Nagy, G.; Pungor, E. Anal. Lett. 1984, 77(B1), 14. (9) Zak. J.; Kuwana, T. J . Am. Chem. Soc. 1982, 704, 5514 (10) Wang, J.; Freiha, B. Anal. Chem. 1984, 56, 2266. (11) Kepley, L. J.; Bard, A. J. Anal. Chem. 1988, 6 0 , 1459. (12) Beilby, A.; Carlsson, A. J . Electroenal. Chem. Znterfacial Ekctrochem. 1988, 248, 283. (13) Anjo, D. M.; Kahr, M.; Khodabakhsh, M. M.; Nowinski, S.; Wagner, M. Anal. Chem. 1989, 67, 2603. (14) Mefford, I. N. Methods Blochem. Anal. 1985, 3 1 , 221. (15) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980;p 413. (16) Hayashl. M. J . Chromatogr. Biomed. Appl. 1985, 338, 195.
RECEIVED for review July 9,1990. Accepted January 24,1991.
