ANALYTICAL SCIENCES MARCH 2000, VOL. 16 2000 The Japan Society for Analytical Chemistry 293 Surface Improvement of Glassy Carbon Electrode Anodized in Triethylene Glycol and Its Application to Electrochemical HPLC Analysis of Protein-Containing Samples Hatsuo MAEDA, Kazunori KATAYAMA, Rie MATSUI, Yuji YAMAUCHI, and Hidenobu OHMORI Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-Oka, Suita, Osaka 565 0871, Japan In order to improve electrochemical performance of a glassy carbon (GC) electrode anodized in triethylene glycol (TEG), a GC electrode was anodized in H 2O prior to TEG anodization. The effect of this pretreatment was evaluated by comparing electrochemical responses of acetaminophen on HPLC using the following electrodes: a GC electrode anodized in both H 2O and TEG (double modified GC electrode); an unmodified GC electrode; GC electrodes anodized in either H 2O or TEG. HPLC was carried out for samples containing uric acid and acetaminophen with or without various proteins. The results reveal that the double modified GC electrode has satisfactory sensitivity, reproducibility, and durability in electrochemical detection of acetaminophen by HPLC, compared with other electrodes. In order to demonstrate the advantage of the double modified GC electrode, analysis of acetaminophen from a urine sample was performed by HPLC without carrying out any tedious procedures such as the removal of proteins. In addition, electrochemical analyses of various compounds were performed using the double modified GC electrode in a near-flow injection mode, suggesting that the electrode has potential applications for HPLC analysis of biologically important cationic and neutral compounds. (Received November 5, 1999; Accepted January 17, 2000) Although several biologically important organic compounds are electrochemically detectable without troublesome derivatization, electrochemical analysis of biological samples is hampered by electrode fouling due to non-specific protein adsorption. Therefore, efforts have been made to develop modification methods to increase the surface hydrophilicity, which decreases protein adsorption. Polymeric coating has proved to be one feasible technique for this purpose. 1 8 However, polymer-coated electrodes appear to have a limited number of applications, 2,7 with the exception of stripping analysis of trace metals. 3 6,8 This may be due to slow mass transport of organic analytes in the membrane. Thus, electrodes having specific desired surface conditions, that may be prepared without relying on polymer chemistry, hold great promise for electrochemical analysis of biological samples. In particular, the development of such electrodes will serve to extend the applicability of reversed-phase HPLC using an electrochemical detector as a useful tool for the routine analysis of clinical samples. This is because columns packed with stationary phases that allows proteins to pass through have recently become commercially available for reversed-phase HPLC, and the removal of proteins from samples is no longer required for HPLC analysis of biological fluids. However, only two studies on such modified electrodes have been reported. 9,10 Recently, anodization of a glassy carbon (GC) electrode in triethylene glycol (TEG) was found to be useful for preventing adsorption of bovine serum albumin (BSA) on the electrode surface. 11 In this treatment, TEG molecules are fixed on the surface of GC via ether-linkage with one of two terminal hydroxy groups (depicted in Fig. 1 as electrode 1). The covalently formed hydrophilic surface not only resists protein adsorption, but also has pinhole defects, allowing analytes to show electrochemical responses at the electrode. Preliminary examination indicates that electrode 1 has potential application in electrochemical HPLC analysis of samples containing BSA. 11 However, the electrochemical performance of electrode 1 was found to be poorer than that of the unmodified GC electrode, resulting in the requirement of a high applied potential for HPLC. Therefore, in the present study, anodic treatment of a GC electrode in H 2O prior to that in TEG is examined, since anodization in H 2O introduces oxygen functionalities on the surface of a GC electrode (cf. electrode 2 in Fig. 1), leading to enhanced electrochemical performance. 9,12 16 The present paper describes the resisting ability against adsorption of various Fig. 1 Schematic drawing of surface structures of modified electrodes used in the present study.
294 ANALYTICAL SCIENCES MARCH 2000, VOL. 16 Table 1 Relative electrochemical responses of acetaminophen (1 µm) on HPLC using an unmodified GC electrode or electrodes 1 3, respectively Electrode Electricity passed in anodization a Relative responses b in triethylene in H 2 O 0.4 V glycol c 0.6 V c Unmodified 1.00 1.98 1 100 mc 0.19 1.95 3 100 mc 100 mc 0.86 2.16 3 1 C 100 mc 1.27 2.16 2 100 mc 1.24 2.14 a. Carried out at 2.0 V vs. Ag wire in H 2 O or triethylene glycol containing LiClO 4 (0.1 M). b. Against a peak area obtained using an unmodified GC electrode at 0.4 V. c. Applied potential (vs. Ag/AgCl). Fig. 2 Chromatograms of a sample containing uric acid and acetaminophen (1 µm each) in a phosphate buffer at 0.4 V (vs. Ag/AgCl) using an unmodified GC electrode (A) and electrode 3 (B) as a detector. Peaks: a, uric acid; b, acetaminophen. proteins as well as oxidized products of analytes, and the electrochemical performance of a GC electrode anodized successively in H 2O and TEG (cf. electrode 3 in Fig. 1) as a working electrode for electrochemical analysis by HPLC. Experimental Materials Deionized and distilled water was used throughout the present study. Human serum albumin (HSA), BSA, ribonuclease A, lysozyme, pyruvate kinase fibrinogen (Type III), and human serum were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Epinephrine, norepinephrin, 3-methoxytyramine hydrochloride (all from Sigma), dopamine hydrochloride, ascorbic acid, uric acid, catechol (all from Wako Pure Chemical Industries Ltd., Osaka, Japan), 3,4-dihydroxyphenylacetic acid (DOPAC)(Aldrich), homovanillic acid (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan), acetaminophen, and vanillomandelic acid (all from Nacalai Tesque Inc., Kyoto, Japan) were used as supplied. All other chemicals were of reagent grade and were used without further purification. GC plates (GC 20, 30 15 3 mm) used as a working electrode for an electrochemical cell were obtained from Shimadzu (Kyoto, Japan). Sample preparation All solutions were prepared in phosphate buffer (0.1 M, ph 7.0; Na 2HPO 4+NaH 2PO 4). A urine sample was collected from one of the researchers two hours after a dose of three 100 mg acetaminophen tablets. The urine sample was then diluted 100 times with phosphate buffer. Prior to HPLC analysis, all samples and the urine sample were passed through a 0.45 µm filter. Apparatus A GC plate was polished using a polishing system (Model ML-150P, Maruto, Tokyo, Japan). Electrochemical modification of a GC electrode was performed using a potentiostat/galvanostat (Model HA 301, Hokuto Denko, Tokyo, Japan) connected to a coulomb/amperehour meter (Model HF201, Hokuto Denko). The HPLC system used for the analysis of samples containing uric acid and acetaminophen with or without a protein consisted of a pump (LC-10AD, Shimadzu), an on-line degasser (Model KT-17, Showa Denko, Osaka, Japan), an injector with a 20-µl sample loop (Model 7125, Rheodyne, Cotati, CA, USA), a separation column (Wakopak Wakosil-GP-N6, 250 4.6 mm i.d., Wako Pure Chemical Industries Ltd.), an electrochemical detector (L-ECD- 6A, Shimadzu), a column oven (CTO-10A, Shimadzu) and a recorder (C-R2A, Shimadzu). The same system was used for the comparison of electrochemical responses of various compounds on hydrodynamic voltammetry, except that a Cosmosil 5C18-MS guard column (10 4.6 mm i.d., Nacalai Tesque, Inc.) was used in place of the above-mentioned separation column. Electrode modification and measurement Electrode 1 was prepared using a revised method: 17 a GC plate was subjected to controlled-potential electrolysis in TEG containing LiClO 4 (0.1 M) at 2.0 V vs. Ag wire, in which 100 mc of electricity was consumed; after treatment, the anodized electrode was washed using MeOH and water, and was subjected to cyclic voltammetry five times in phosphate buffer between 0 and 0.5 V vs. SCE. Electrode 2 was obtained as follows: a GC plate was anodized in H 2O containing LiClO 4 (0.1 M) at 2.0 V vs. SCE (100 mc or 1 C) and was treated five times using cyclic voltammetry in the same solution between 0 and 0.5 V vs. SCE. The anodic treatment in TEG to prepare electrode 1 was applied to electrode 2 to produce electrode 3. HPLC analysis was carried out under the following conditions: mobile phase, phosphate buffer (0.1 M, ph 7.0); column temperature, 35 C; injection volume, 20 µl; flow rate, 1.0 ml/min. Results and Discussion Sensitivity in detection of acetaminophen by HPLC using electrode 3 As reported previously, 11 anodization of a GC electrode for an HPLC electrochemical detector at 2.0 V vs. Ag wire in TEG containing LiClO 4 (0.1 M) is an effective tool for the prevention
ANALYTICAL SCIENCES MARCH 2000, VOL. 16 295 Table 2 Effects of various proteins and a human serum on electrochemical responses of acetaminophen (1 µm) by HPLC using an unmodified GC electrode or electrode 3 a Unmodified GC electrode Electrode 3 Protein 0.4 V b 0.6 V b 0.4 V b 0.6 V b PA 12 /PA 1 PA 12 /PA 1 RSD c PA 12 /PA 1 RSD c PA 12 /PA 1 RSD c BSA 0.44 1.05 4.2% 1.00 5.0% 1.05 3.9% HSA 0.46 0.91 3.3% 0.97 4.8% 1.04 2.6% Ribonuclease A 0.45 0.99 3.3% 1.07 8.3% 1.02 1.9% Lysozyme 0.33 0.94 3.3% 0.95 5.3% 0.96 1.9% Pyruvate kinase 0.26 0.96 3.5% 0.98 5.7% 1.03 3.1% Fibrinogen 0.37 0.87 e 0.93 4.8% 1.04 4.1% Human serum d 0.37 0.81 e 0.94 e 0.96 e a. Evaluated by comparison of peak areas for acetaminophen obtained by HPLC analysis for a sample containing acetaminophen and uric acid before (PA 1 ) and after (PA 12 ) ten repetitions of HPLC analysis of a sample containing acetaminophen, uric acid, and a protein (0.1%w/v). b. Applied potential (vs. Ag/AgCl). c. Determined using PA 1, PA 12, and peak areas observed by ten injections of a protein-containing sample: n=12. d. A sample containing only a human serum was injected ten times instead. e. Not determined. of BSA absorption to the electrode surface, when more than 100 mc of electricity was passed during the anodic treatment. Accordingly, electrodes 1 and 3 were prepared by essentially the same method, in which 100 mc of electricity was passed. Electrode 3 was obtained by successive anodization in H 2O and TEG, in this order. Preparation of electrode 3 by successive anodic treatment in the reverse order was not attempted because 1-alkanol molecules anodically fixed to a GC surface are detached by applying a potential more than 1.5 V vs. SCE in an aqueous solution. 18 Anodization in H 2O for the preparation of electrodes 2 and 3 was carried out at 2.0 V vs. SCE using LiClO 4 as a supporting electrolyte. In order to evaluate the effects of anodization in H 2O prior to that in TEG, that is, in order to evaluate whether the use of electrode 3 rather than electrode 1 improves sensitivity in electrochemical analysis, HPLC with an electrochemical detector using the modified electrodes was carried out. The system using electrode 3 was applied to the analysis of urine samples by using a buffer solution of uric acid and acetaminophen as a model sample. Detection potential was set at 0.4 or 0.6 V vs. Ag/AgCl. The electrochemical performance of electrode 3 was evaluated by comparing the peak area due to acetaminophen on a chromatogram obtained using electrode 3 with those obtained using an unmodified GC electrode, electrode 1, and electrode 2. Figure 2 shows typical chromatograms obtained using an unmodified GC electrode and electrode 3 at 0.4 V. The results are summarized in Table 1, in which peak areas obtained using each of the electrodes are expressed in values relative to the peak area obtained using an unmodified GC electrode at 0.4 V. Responses at electrode 3 increased remarkably compared to those at electrode 1. The increment in peak areas of acetaminophen obtained using electrode 3 at 0.4 V became larger as the double modified electrode was prepared by passing more electricity during anodization in H 2O. Even using the electrode 3 produced by pretreatment in H 2O at the expense of only 100 mc, peak area increased more than 4.5 times that observed at electrode 1. With a detection potential at 0.6 V, at which electrode 1 had an electrochemical performance similar to that of an unmodified GC electrode, sensitivity in the detection of acetaminophen was improved by about 10% by using electrode 3. In addition, as shown in Table 1, electrode 2 exhibited a higher electrochemical performance toward acetaminophen than did the unmodified GC electrode. Accordingly, the observed results for electrode 3 were ascribed to the formation of oxygen functionalities introduced on the GC surface by anodic treatment in H 2O prior to anodization in triethylene glycol. Since anodization in H 2O passing more than 100 mc resulted in an unstable background upon HPLC using electrode 3, electrode 3 was prepared by passing 100 mc of electricity in each anodization process for further examination. Resisting ability of electrode 3 against electrode fouling The effects of various proteins on the detection of acetaminophen by HPLC using electrode 3 were evaluated as follows: (1) a sample containing uric acid and acetaminophen (1 µm each) was subjected to HPLC, and a peak area (PA 1) for the latter was obtained; (2) ten chromatograms for a sample containing uric acid (1 µm), acetaminophen (1 µm), and a protein (0.1%w/v) were obtained successively; (3) HPLC was performed for a sample containing uric acid and acetaminophen (1 µm each), and a peak area (PA 12) for the latter was measured again; (4) the resisting ability of electrode 3 against surface passivation was estimated by the ratio of PA 12 to PA 1. The following proteins were examined: BSA, HSA, ribonuclease A, lysozyme, pyruvate kinase, and fibrinogen. The effect of human serum was also evaluated. Human serum exhibited an unknown peak overlapping to that of acetaminophen on a chromatogram obtained at an unmodified GC electrode as well as electrode 3 under the conditions. Accordingly, in order to estimate the fouling of electrode 3 by human serum, a sample containing only human serum (0.1%w/v) was successively injected ten times before measurement of PA 12. The results are summarized in Table 2, which also contains measurements obtained for the unmodified GC electrode. For HPLC using an unmodified GC electrode at 0.4 V, ten injections of a sample containing each of proteins or human serum remarkably affected the responses to acetaminophen. Compared to PA 1, the peak areas of acetaminophen on chromatograms for a protein-containing sample gradually became smaller as the number of analyses for the sample increased, and the values of PA 12/PA 1 were less than 0.5. The results indicate that the surface of an unmodified GC electrode was apparently fouled by repetitive analyses of a proteincontaining sample. When a mixture of uric acid and acetaminophen, rather than a protein-containing sample, was analyzed successively ten times, PA 12/PA 1 was 0.73. Thus, the observed loss in detection sensitivity for acetaminophen is attributable to adsorption not only of each protein, but also of oxidized products from uric acid and acetaminophen to the
296 ANALYTICAL SCIENCES MARCH 2000, VOL. 16 Fig. 3 Chromatograms of a sample containing uric acid (1 µm), and acetaminophen (1 µm), and BSA (A) or fibrinogen (B) (0.1%w/v) in a phosphate buffer at 0.6 V (vs. Ag/AgCl) using an unmodified GC electrode as a detector. Peaks: a, uric acid; b, acetaminophen. electrode surface. When HPLC using an unmodified GC electrode was carried out at 0.6 V, the results were considerably different from those at 0.4 V. As shown in Fig. 3, a new peak, in addition to those for uric acid and acetaminophen, emerged on each chromatogram obtained by injections of protein-containing samples. The largest peak was recognized upon performing HPLC for a sample containing fibrinogen (Fig. 3B). HPLC performed for a sample containing only one of the examined proteins without uric acid and acetaminophen showed essentially the same peak, demonstrating that the proteins themselves exhibit electrochemical responses as peaks. Repeated analysis of a sample containing proteins other than fibrinogen and human serum caused no significant effects on the detection of acetaminophen, and PA 12/PA 1 was close to unity. After twelve repetitions of analysis of a sample, acetaminophen was reproducibly detected, as shown by an RSD of less than 5%. Even after ten measurements of a sample containing fibrinogen or human serum, a peak area for acetaminophen was maintained in more than 80% of PA 1. These results appeared to suggest the absence or reduction of electrode fouling by adsorption of each protein and anodically generated products on the surface of a bare GC electrode induced at 0.6 V. However, the following results clearly demonstrate that adsorption of these compounds also occurs at 0.6 V similar to or greater than that observed at 0.4 V, whereas the higher applied potential enables acetaminophen to show similar HPLC responses, even for an unmodified GC electrode, the surface of which is covered with proteins and other chemicals, resulting in a decrease in active surface area. HPLC using an unmodified GC electrode was performed by changing the applied potential to 0.4, 0.6, and 0.4 V. With HPLC at 0.4 V, a sample containing only acetaminophen was analyzed, while the system at 0.6 V was applied to analysis for a sample containing both acetaminophen and BSA. The peak area due to acetaminophen at 0.4 V decreased more than 10% by the intervention of the analysis at 0.6 V, clearly indicating that when the analysis is carried out at 0.6 V, protein adsorption takes place, but there is no fouling effect. At present, the origins of the peaks due to the proteins observed at an unmodified GC electrode remain unclear. Since none of the proteins exhibited anodic peaks on cyclic voltammetry in phosphate buffer below 1.0 V vs. SCE, the proteins are assumed to disturb the structure of the double layer on an unmodified GC electrode more significantly at 0.6 V than 0.4 V, leading to a large increase in charging currents observed as HPLC responses at 0.6 V. In contrast with the HPLC results for an unmodified GC electrode, ten injections of a sample containing a protein had almost no effect on the analysis of acetaminophen by HPLC using electrode 3, regardless of the applied potential. Although lysozyme, pyruvate kinase, and fibrinogen were shown to significantly foul the surface of an unmodified GC electrode when samples containing these proteins were repeatedly analyzed by HPLC at 0.4 V, acetaminophen from samples including these proteins could be detected quite reproducibly by HPLC using electrode 3 at the potential, RSD being less than 6%. However, 0.6 V appears to be the potential of choice, which is similar to the case using an unmodified GC electrode: when the detection potential was set at 0.6 V, inhibition by repeated analysis of a sample containing each protein was much smaller, and the RSD for peak areas of acetaminophen were improved. When HPLC using electrode 1 at 0.6 V was performed successively ten times for a sample containing uric acid (1 µm), acetaminophen (1 µm), and BSA (0.1%w/v), almost no changes were observed in the responses to acetaminophen. The ratio of peak areas on the first and tenth chromatograms, and the RSD were 1.04 and 1.82%, respectively. By HPLC using electrode 2 at 0.4 V, detection of acetaminophen from a sample containing uric acid (1 µm), acetaminophen (1 µm), and BSA (0.1%w/v) was hindered by the fact that the peak area of acetaminophen decreased as the analysis of the sample was repeated and the sensitivity was decreased by 30% after ten analyses of the sample. The results described thus far demonstrate that electrode 3 has electrochemical performance combining advantages exhibited by either electrodes 1 or 2: the surface of electrode 3 effectively resists adsorption of various proteins and oxidized products from uric acid and acetaminophen and allows acetaminophen to smoothly enter an electrochemical reaction. Calibration curves for acetaminophen and analysis of urine sample By HPLC using electrode 3, calibration curves for acetaminophen were obtained. Figure 4 shows a log log plot of the HPLC responses versus the acetaminophen concentration. At 0.4 V, the detection limit was 100 nm (S/N=3) and a linear relationship was observed between the peak areas and the concentrations of acetaminophen over 100 nm 10 µm, the slope and correlation coefficient (r) being 205.7 mc/m and 1.000, respectively. Utilizing 0.6 V as applied potential increased the sensitivity of HPLC detection for acetaminophen. The detection limit was 20 nm (S/N=3), and the linear relationship defined by a slope of 580.7 mc/m and a correlation coefficient of 0.999 was exhibited over the concentration range of 60 nm 10 µm. A calibration curve was also obtained by HPLC using electrode 1. As a detection potential, only 0.6 V was examined, since almost no response to acetaminophen, even at 10 µm, was observed for HPLC at 0.4 V. The detection limit was 60 nm (S/N=3), and a linear relationship was observed over 60 nm 10 µm, over which slope and r were 593.7 mc/m and 1.000, respectively. For HPLC performed at 0.6 V, using
ANALYTICAL SCIENCES MARCH 2000, VOL. 16 297 Fig. 4 A log log plot of the acetaminophen concentration versus the responses on HPLC using electrode 3 at 0.4 V (squares) and 0.6 V (circles) (vs. Ag/AgCl). Fig. 5 Chromatogram of a urine sample in a phosphate buffer at 0.6 V (vs. Ag/AgCl) using electrode 3 as a detector. Peak: a, acetaminophen. electrode 3 rather than electrode 1 slightly improved the detection limit. Electrode 3, however, has been proven to enable HPLC analysis at a low potential. A urine sample, which was collected from one of the researchers two hours after taking a dose of 300 mg of acetaminophen, was analyzed by HPLC using electrode 3 at 0.6 V. The sample was prepared merely by dilution of the urine. Figure 5 shows a typical chromatogram of the sample. Using the above calibration curve, the mean value for ten repetitions of analysis of the sample was 1.94 µm, and RSD was 2.9%. Although the result was not compared to those of other methods, HPLC using electrode 3 has been demonstrated to yield satisfactory sensitivity and reproducibility, and is useful for performing urinalysis without pretreatment to remove proteins. Electrochemical performance of electrode 3 with respect to other compounds Applicability of HPLC using electrode 3 with respect to the analysis of various compounds was evaluated by hydrodynamic voltammetry (HV). HV was performed between 0 and 0.9 V vs. Ag/AgCl in a near-flow injection mode, for which a flow system including an ODS short column was used to clearly discriminate a peak due to each compound from a base-line distortion induced by the injection of a sample. In addition to uric acid and acetaminophen, epinephrine, norepinephrin, dopamine, 3- methoxythyramine, catechol, DOPAC, homovanillic acid, vanillomandelic acid, and ascorbic acid were subjected to HV. HV responses for electrode 3 and an unmodified GC electrode were compared at a given potential, at which the peak area of each analyte obtained using the unmodified GC electrode reached an almost constant value. The results of this comparison are summarized in Table 3. The following findings were observed: the responses of cationic and neutral compounds for HV using electrode 3 were maintained at greater than 80% of those using an unmodified GC electrode; for anionic compounds, a loss in responses of over 40% was observed using electrode 3 compared with those obtained using an unmodified GC electrode. Thus, HPLC using electrode 3 has been shown to be a useful analytical method for biologically important cationic and neutral compounds, and appears to have a similar sensitivity, better durability and reproducibility without electrode fouling, compared to HPLC using an unmodified GC electrode. Table 3 Comparison of responses of various electroactive compounds on hydrodynamic voltammetry using an unmodified GC electrode or electrode 3 Compound Potential (V vs. Ag/AgCl) a Relative response b Epinephrine 0.3 1.17 Norepinephrine 0.3 0.84 Dopamine 0.2 1.01 3-Methoxytyramine 0.5 0.91 Catechol 0.3 1.06 Acetaminophen 0.5 0.86 DOPAC 0.5 0.66 Homovanillic acid 0.6 0.55 Vanilomandelic acidc 0.9 0.61 Aascorbic acid 0.3 0.35 Uric acid 0.4 0.63 a. Applied potential at which a response reached an almost constant value on hydrodynamic voltammetry using an unmodified GC electrode. b. Ratio of peak area obtained using electrode 3 to that obtained using an unmodified GC electrode at the specified potential. c. No plateau was observed upon hydrodynamic voltammetry. The observed lower sensitivity of electrode 3 against anionic compounds can be explained as follows. Recently, anodic peaks of DOPAC and ascorbic acid on cyclic voltammetry at a GC electrode anodized in TEG were found to be suppressed, whereas epinephrine exhibits an anodic response quite similar to that of an unmodified GC electrode. 19 The degree of retardation of anodic peaks for these anionic compounds varies depending on identities of the supporting electrolytes used in anodic treatment in TEG: for example, the degree of the observed depression increases in the following order: LiClO 4 < H 2SO 4 < HOCH 2CH 2SO 3Na. These results are ascribed to the oxidation of a portion of the terminal hydroxyl groups to carboxylates. Thus, a portion of the terminal hydroxy groups on electrode 3 (cf. Fig. 1) seem to be transformed during anodization in TEG into the anionic functional groups, which prevents anionic compounds from approaching the vicinity of the electrode surface due to electrostatic repulsion. However, the distribution ratio of carboxylates to hydroxyl groups on electrode 3 is small, since LiClO 4 was used as a supporting electrolyte for anodic treatment.
298 ANALYTICAL SCIENCES MARCH 2000, VOL. 16 In conclusion, we have demonstrated that successive anodization in H 2O and TEG not only confers on a GC electrode a surface that is free from fouling due to adsorption of proteins and anodized products of uric acid and acetaminophen, but also enables the modified electrode to retain the original electrochemical performance of the unmodified GC electrode toward acetaminophen. Thus, anodization in H 2O prior to that in TEG has been shown to improve the sensitivity of HPLC using a GC electrode anodized only in TEG. A GC electrode anodized successively in H 2O and TEG was used to perform HPLC analysis of acetaminophen from a urine sample without the need for tedious pretreatment, such as the removal of proteins. In addition, the modified electrode is shown to have good electrochemical performance for cationic and neutral compounds. The obtained results indicate that the modified electrode has useful applications in HPLC analysis of biologically important compounds. Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research (09470494) from the Ministry of Education, Sciences, Sports, and Culture, Japan. References 1. G. Sittampalam and G. S. Wilson, Anal. Chem., 1983, 55, 1608. 2. J. Wang and L. D. Hutchins, Anal. Chem., 1985, 57, 1536. 3. J. Wang and L. D. Hutchins-Kumar, Anal. Chem., 1986, 58, 402. 4. B. Hoyer, T. M. Florence, and G. E. Batley, Anal. Chem., 1987, 59, 1608. 5. B. Hoyer and T. M. Florence, Anal. Chem., 1987, 59, 2839. 6. J. Wang and Z. Lu, J. Electroanal. Chem., 1989, 266, 287. 7. J. Wang and T. Golden, Anal. Chem., 1989, 61, 1397. 8. J. Wang and Z. Taba, Electroanalysis, 1990, 2, 383. 9. A. J. Downard and A. D. Roddick, Electroanalysis, 1994, 6, 409. 10. A. J. Downard and A. D. Roddick, Electroanalysis, 1995, 7, 376. 11. H. Maeda, M. Itami, K. Katayama, Y. Yamauchi, and H. Ohmori, Anal. Sci., 1997, 13, 721. 12. R. C. Engstrom, Anal. Chem., 1982, 54, 2310. 13. R. C. Engstrom and V. A. Strasser, Anal. Chem., 1984, 56, 136. 14. G. E. Cabaniss, A. A. Diamantis, W. Rorer Murphy Jr., R. W. Linton, and T. J. Meyer, J. Am. Chem. Soc., 1985, 107, 1845. 15. T. Nagaoka and T. Yoshino, Anal. Chem., 1986, 58, 1037. 16. L. J. Kepley and A. J. Bard, Anal. Chem., 1988, 60, 1459. 17. H. Maeda, M. Itami, Y. Yamauchi, and H. Ohmori, Chem. Pharm. Bull., 1996, 44, 2294. 18. H. Maeda, Y. Yamauchi, M. Hosoe, T.-X. Li, E. Yamaguchi, M. Kasamatsu, and H. Ohmori, Chem. Pharm. Bull., 1994, 42, 1870. 19. H. Maeda, T. Kitano, C.-Z. Huang, K. Katayama, Y. Yamauchi, and H. Ohmori, Anal. Sci., 1999, 15, 531.