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Anal. Chem. 1989, 6 1 , 170-173
Hydrophobic Stripping Voltammetry Using a Lipid-Modified Glassy Carbon Electrode Olivier Chastel, Jean-Michel Kauffmann, a n d Gaston J. Patriarche*
Institut de Pharmacie, Universitt?Libre d e Bruxelles, Campus Plaine, CP 20516 Boulevard du Triomphe, 1050 Bruxelles, Belgium Gary D. Christian
Department of Chemistry, BG-IO, University of Washington, Seattle, Washington 98195-9550
A glassy carbon electrode Is modified by depositlon of a hydrophoMc coating of the llpld asolectln. I n neutral aqueous media, this negatively charged ilpid-modlfled electrode will selectlvely accumulate organk molecules, dependlng on thelr charge and their hydrophilic-hydrophobk balance. A study of the antitumor drug, marcellomycln, is made to determine the effects of operatlng parameters. The accumulation step Is accomplished in the absence of an applied potential. The molecule can then be quantlfled by voltammetric oxidatlon. The oxldation current is enhanced 4-fold with 11.6 pg of ilpld/mm2. Optimal accumulatlon time Is 3 mln; at longer times ilpid Is lost. The oxidation current Is maximum at pH 6, and the oxidation potential decreases linearly with increasing pH from 4 to 10 (slope = 80 mV/pH). To point out the charge effect in the accumulation process, three purlfied lipids were investlgated: cardtdlpln, L-a-phosphatldylserlne, and L-ar-phosphatldylethanolambre. The more phosphate Sites the llpld contalns, the more marcellomycln Is accumulated. Other negatlveiy charged compounds do not accumulate (ferrocyanide, uric acld, ascorbic acid) while posltiveiy charged and hydrophobic molecules give hlgher responses than marcellomycln (tetracain, chlorpromazine).
INTRODUCTION Progress has been achieved in electrochemistry due to the development of modified electrodes. Intense activity is under way to produce new types of electrodes that could lead to the production of highly specific types of materials designed for the analysis of pharmaceutical compounds (I).The principal route is to cover the electrode with an appropriate polymer, thus selecting the components arriving at the electrode surface. Cellulose acetate (2,3), poly(4-vinylpyridine) (4),and Ndion (5-7) are commonly used to modify electrochemical detectors (8-10). In the present study, we report the coating of the surface of a glassy carbon electrode (GCE) with lipids for the selective accumulation of the antitumor compound marcellomycin (Figure l) which can then be electrochemically oxidized. This anthracyclin glycoside antitumor agent is used in association with other cytostatic agents to reduce their required therapeutic concentrations because of their cytotoxicity (11-13). The anthracyclin glycoside family is one of the most effective against several types of cancer (leukemia and solid tumors). The quinone function is responsible for its pharmacological activity (14,15).Its mechanism of action is considered to involve an intercalation in the DNA structure and subsequent inhibition of DNA and RNA synthesis (16). Marcellomycin was chosen for this study because of the high affinity between the anthracyclin group and phospholipids (17). The anthracyclin binding to phospholipids has been extensively studied by Ruysschaert and co-workers (17,18).
The reaction proceeds electrostatically between the protonated amino groups of the sugar residues and the ionized phosphate residues of the phospholipids. Moreover, an interaction between adjacent anthraquinone chromophores has been suggested (18).Considering some phospholipids present in the membrane of heart hypochondria, the anthracyclin drugs show the highest affinity in the order cardiolipin > L-a-phosphatidylserine > L-a-phosphatidylethanolamine(18).The strong anthracyclin-cardiolipin complex (association constant = 2 x lo6 M-I) has been shown to be responsible for the anthracyclin cardiotoxicity (18).This places a limit on the total dose of marcellomycin that may be administered (19). In the present studies, a mixture of phospholipids was deposited onto the electrode surface of a glassy carbon eiectrode by using asolectin, as the source. The electroactive character of marcellomycinallows its voltammetric oxidation measurement a t solid electrodes (20,21).The molecule is irreversibly oxidized a t the glassy carbon electrode in a single two-electron, two-proton step. Cyclic voltammetric measurements as a function of pH have shown absorption phenomena in alkaline media. Quantitative measurements in acidic media at the glassy carbon electrode were possible down to 5 X lo4 M (21). A similar compound, adriamycin, has been adsorbed on a carbon paste electrode prior to measurement (22). In this paper the electrochemicaloxidation of marcellomycin is investigated a t the glassy carbon electrode in the absence and presence of adsorbed lipids. The comparison of voltammetric curves demonstrates the utility of this lipid-modified electrode for the sensitive and selective analysis of marcellomycin. The compound is measured directly in diluted urine. EXPERIMENTAL SECTION Apparatus. Voltammetric measurements were made with a BAS CV27 voltammograph and a Hewlett-Packard 7090A recorder. The scan rate was 10 mV/s for all measurements. The working electrode was a glassy carbon electrode (0.40 cm2geometric area, Metrohm AG). A saturated calomel reference and platinum wire counter electrode were employed. Before each experiment,the glassy carbon electrode was polished mechanically with alumina on a smooth cloth. The three-electrodecell was kept at 37 f 1 " C with a Haake FJ thermostat bath. The solution pH was measured with a Tacussel Mini 80 pH meter. When necessary, removal of oxygen was accomplished by passing purified nitrogen through the solution for 15 min and then passing over the solution during the experiment. Accumulation of the marcellomycin on the electrodes was accomplished by stirring the analyte solution at a constant rate with a magnetic stirring bar (approximately 200 rpm) with an open circuit. Current-voltage curves were run with the electrode in the same solution. Reagents and Solutions. All reagents were of analyticalgrade. Drugs were of pure grade or pharmocoepia quality and were used without further purification. Triple distilled water used to prepare solutions was stored in polyethylene bottles. Marcellomycin, or 2-ethyl-1,2,3,4,6,1 l-hexahydro-2,5,7,10-tetrahydroxy-6,1 l-dioxo-
0003-2700/89/0361-0170$01.50/00 1989 American Chemical Society
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ZH5 OH
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Figure 2. Relative electrode response as a function of the deposited lipid amount: Ia, peak current intensity at the llpld modifled electrode; Ib, peak current intensity at the glassy carbon electrode.
44 [2,3,6-trideoxy-4-0-(2,6-dideoxy-4-( 0-2,6-dideoxy-a-~-lyxola hexop~anosyl)-cy-~-lyxo-hexopyranosyl)-3-(dimethylamino)-cy-~I lyxohexopyranosyl]oxy)-1-naphthacenecarboxylicacid methyl E ester, was obtained from Bristol Meyers (Brussels) and was used as received. Buffer solutions were prepared from Na2HP0,.12H20 (Merck P.A.), with the pH adjusted with hydrochloric acid or sodium hydroxide. The lipid mixture used to coat the electrode was asolectin (L-cy-phosphatidylcholine, type 11-S) extracted from soybean, containing 18% of the compound based on choline determination, along with other lipids to mimic a membrane (Sigma P5638); the most important are phosphatidylinositol,phosphatidylserine (PS), phosphatidylethanolamine (PE), and cardiolipin (CL) (20). Purified PS, PE, and CL were from Sigma. Lipid solutions in chloroform were prepared daily. Electrode Modification. A reproducible lipid coating was adsorbed on the electrode surface by pipetting 2 X 12.5 pL of lipid solution on the electrode surface and allowing the solvent to evaporate at room temperature. As the modified electrode is not reusable after each measurement, the lipid coating is removed by washing with acetone, and the electrode is polished. Flgure 3. Voltammetrlc curves as a functlon of accumulation time: (a) glassy carbon electrode, (b) lipid modified electrode. RESULTS AND DISCUSSION Different parameters were studied for optimization, inpH 7, using a lipid layer of 1.6 pg/mm2, was varied from 1to cluding the quantity of lipid, the accumulation time, and the 15 min. The results show a maximum in the oxidation current for an accumulationtime of 3 min (Figure 3). In the presence solution pH. of lipid there is a slight positive potential shift in the voltEffect of Quantity of Lipid. Accumulation from a 5 X ammetric wave, with decreased electron transfer rate ( E pM aqueous solution of marcellomycin at pH 7 was performed with a stirring time of 3 min. The quantity of asoledin E p / 2 increases from 50 to 70 mV), probably due to complexation or association with the lipid. In the absence of on the electrode varied between 0.1 and 3.5 pg/mm2. The results show an increase in the marcellomycin oxidation accumulation, there is a decrease in the current, due to decurrent with increased lipid deposition up to 1.6 I.lg/mm2, at creased diffusion rate in the lipid layer. which point the current becomes constant (Figure 2). The As the stirring time exceeds 3 min, the current decreases, current is increased by as much as 4-fold over that in the apparently due to a loss of the lipid layer due to aqueous solubility. When stirring was performed for 10 min in the absence of lipid. The quantity of lipid calculated to represent absence of analyte followed by 3 min of stirring in the presence a bilayer (averagemolecular weight, 750, one molecule, 60 A2) of added analyte, the current was 30% of that shown in Figure is indicated by the arrow in the figure (0.15 Mg/mm2). The molecule accumulates in the lipid layer and the di3 for 3 min of accumulation time, demonstrating the loss of hydroxy functions of this group are then oxidized upon anodic lipid layer. The loss of lipid layer due to aqueous solubility may act as a solution-based sink for the analyte. To check scanning. This accumulation and orientation are responsible this, small amounts of asolectin were added in the bottom of for the increase in the measured signal. There is no change the cell. After solvent evaporation the analyte solution (20 in the oxidation current using an unmodified glassy carbon electrode when the solution is stirred in contact with the mL) was added. After the mixture was stored for periods as electrode. As the lipid layer is increased, more of the marlong as 10 min, no detectable depletion of marcellomycin signal was observed a t the conventional GCE. Despite a rather cellomycin is extracted and contacted with the electrode, but unstable lipid layer, the modified GCE allows (1)a rapid study eventually the electrode surface becomes saturated with reof the marcellomycin-lipid interaction and (2) the quantitative spect to the analyte. This is further supported by a leveling estimation of this process in a reproducible manner (see off of the current at high marcellomycin concentrations (beanalytical performances). low). Effect of Solution pH. Optimum accumulation conditions Effect of Accumulation Time. The stirring time for (3 min, 1.6 wg/mm2) were employed to study the effect of pH accumulation from a 5 X M marcellomycin solution at
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B C Flgure 4. Schematic structure of phospholipids: (A) cardiolipin, (B) phosphatidylserine, and (C) phosphatidylethanolamine. on the current for a 5 X M marcellomycin solution. The pH was varied from 4 to 10. In the absence of lipid, the oxidation current is independent of pH (20).In the presence of lipid at pH 6, the oxidation current reaches a maximum and is more than 5 times higher than the value in the absence of lipid. Also, the peak potential E, decreases as a linear function of increased pH (with a correlation coefficient of 0.9992) in the presence of lipid (80 mV/pH) but is constant from pH 4 to 5 in the absence of lipid. The pH for maximum current may be related to the pH of maximum ionic interaction between the marcellomycin and the negatively charged lipid (17, 18). A t pH less than 6, marcellomycin is positively charged but the amount of lipids in their anionic form is low. At pH greater than 6, more phospholipids become negatively charged, but the amount of positively charged marcellomycin is low. A t high pH, accu50 100 0 10 Cone. ( ~ x i 0 3 mulation is nearly zero. In the absence of lipid at lower pH Figure 5. Peak intensity as a function of marcellomycin Concentration: the potential of oxidation is independent of pH and the ox(a) lipid modified electrode, (b) glassy carbon electrode. idation is easier than in the presence of lipid, perhaps due to the association of the analyte with the lipid. with accumulation in the presence of lipid it is linear between Glassy Carbon Electrode Modified with Pure Lipids. 2 X lo4 and 5 x lod M,with a correlation coefficient of 0.9999 In order to demonstrate the importance of electrostatic in(Figure 5). At concentrations below 2 x lo4 M,the slope teractions in the accumulation process and to attempt to is higher for the latter, suggesting that at low concentrations, increase the adherence of the lipid coating, studies were adsorption may occur, and at higher concentrations diffusion performed by coating the surface of the GCE with purified control is observed. The reproducibility of the response for lipids. The lipids used are structurally closely related. 5X M marcellomycin was determined for seven trials. Cardiolipin (CL) contains two phosphate sites, while phosThe average voltammetric peak height was 9.6 pA with a phatidylserine (PS) and phosphatidylethanolamine (PE) coefficient of variation of 2.7%. With accumulation, the contain one phosphate (Figure 4). At physiological pH, CL sensitivity of measurement is significantly enhanced, the peak bears two negative charges and PS one negative charge and current being about 4-fold greater in the linear region than PE is a neutral lipid. Accumulation was performed at pH 7 with the unmodified electrode. The minimum detectable with a lipid coating of 1.6 bg/mm2. The three purified lipids concentration is 2 X lo* M at the glassy carbon electrode and show similar stability. A maximum response is obtained for 5 min accumulation, then the response decreases. Thus a 5x M at the modified electrode. slight amelioration in stability is observed in comparison with Selectivity. Under the same analytical conditions, several asolectin. In terms of magnitude of the electrode response, pharmaceutically interesting compounds were investigated. This accumulation process is specific for molecules positively under optimal conditions ( 5 min accumulation for CL, PS, charged and which present a hydrophobic character. Adriaand PE and 3 min for asolectin), the CL-modified GCE gives mycin shows the same response intensity as marcellomycin. the highest response. With respect to CL, the asolectin Tetracain and chlorpromazine, positively charged on their electrode shows a 10% decrease, PS a 12% decrease, and PE tertiary nitrogen and which present a high solubility in lipids, a 40% decrease. These observations suggest a chargecharge accumulate to a greater extent: gain factor, respectively, of complex; the more the lipid is negatively charged the more &fold and &fold. marcellomycin is accumulated. Negatively charged molecules react in an entirely different Analytical Performance. Further studies were performed way. The voltammetricoxidation waves for both ferrocyanide to more closely model physiological pH conditions. The above ( 5 X low4M)and ascorbic acid (1 X lo4 M) (Figure 6) are studies indicate optimum conditions for the measurement of marcellomycin to be an asolectin deposition of 1.6 pg/mm2, decreased and shifted to more positive potentials in the with accumulation for 3 min at pH 6. When calibration curves presence of lipid layer (1.6 pg/mm2), but accumulation does not occur. The same is observed for uric acid (5 X lo4 M) are prepared at pH 7 (5 X to 1 X M), the response at the unmodified glassy carbon electrode is nonlinear, but and NADH ( 5 x M). A
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ANALYTICAL CHEMISTRY, VOL. 61,
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Figure 6. Voltammetric oxidation curves of ascorbic acid (1 X lo-' M): (a) glassy carbon; (b) lipid-modified electrode (asolectin = 1.6 pg/mml), no accumulation: (c) IIpM modified electrode (asolectln = 1.6 pg/mm2), 3-min accumulation: (d) supporting electrolyte.
Determination of Marcellomycin in Urine. It is not possible to measure the compound adriamycin directly in urine at the carbon paste electrode (23),but instead the electrode was changed to a separate solution following accumulation to allow the sensitive determination. In the present study an interfering oxidation current was observed in diluted (150) urine which precludes determination of marcellomycin at the unmodified glassy carbon electrode (24). This interference is suspected to be due to uric acid as indicated by adding known amounts of uric acid to the sample. Quantitative determination of relatively high concentrations of marcellomycin is, however, possible with the modified electrode. By measurement of the peak in both the absence and the presence of lipid accumulation, the increase in current is due only to the marcellomycin. The increase varies linearly with concentrations between 5 X lo4 and 5 X M in urine diluted 150, with a correlation coefficient of 0.9997. CONCLUSION This voltammetric study shows that a phospholipid-modified glassy carbon electrode may give valuable qualitative and quantitative information with respect to drug analysis and drug-membrane interaction. Indeed, the response at a phospholipid electrode can be inhibited, unchanged, or enhanced, depending on the charge of the molecule and its hydrophobic-hydrophilic balance and on the nature of the phospholipid. Further work is progress in order to improve
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the lipid adherence by using porous carbon electrodes. Other structurally related anthracyclin compounds are being investigated to determine the relative affinities toward phospholipids. The ease and rapidity of electrode preparation constitute interesting advantages over more sophisticated techniques used to study the drug-phospholipid interaction (25). With respect to hydrophobic stripping performances, the use of differential pulse voltammetry should allow improvement in the sensitivity (23),and the use of more water insoluble lipids should improve the electrode stability.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18)
Patriarche. G. J. J . Pharm. B M . Anal. 1988, 4 , 789-797. Sittampalam, G.; Wilson, G. S. Anel. Chem. 1983, 55, 1608-1611. Wangand, J.; Hutchins, D. Anal. Chem. 1985. 5 7 , 1536-1541. Wang, J.: Golden, T.; Turhi, P. Anel. Chem. 1987, 59. 740-744. Ji, H.; Wang, E. J . Chromotogr. 1987, 70, 111-120. Whitely, L. D.; Martin, 0. R. Anal. Chem. 1987, 5 9 , 1746-1751. Hoyer, B.; Florence, T. M.; Batiey. G. E. Anal. Chem. 1987. 5 9 , 1608- 1614. Murray, R. W. I n Electroanalyticel Chemistry; Bard, A. J.. Ed.; Marcel Dekker: New York, 1984; Voi. 13, pp 191-368. Anson, F. C. Acc. Chem. Res. 1975, 8 , 400-407. Faulkner. L. 0. Chem. Eng. News 1984, 62, 28-40. Carter, S. K. Cancer Chemother. Phamcol. 1980, 4 . 5-10. Bruchner, H. W.; Cohen, C. J.; Goldberg, J. D.; Kabakow, R.; Wallach. R. C.; Deppe, G.; Greenspan, E. M.; Gusberg, S. 6.; Holland, J. F. Cancer 1981, 47, 2288. Tatika, H.; Edgerton, F.; Marabella, P.; Conway, D.; Harguindem, S. Cancer 1981, 48, 1528. Crooke, S. T.; Bustoyko, S. Cancer Chemotherapy; Crooke, S. T., Ed.; Academic: New York, 1981; Voi. 13, Chapter 8. Yong, R. C.; Ozois, R. F.; Meyers, C. E. New England J . Med. 1981, 305, 139. Colendi, E.;DiMarco, A.; Regiani, M.; Scarpinato, B.; Valentini, L. B b chim. Siophys. Acta 1985, 103. 25-49. Goormaghtigh, E.; Chatehin, P.; Caspers, J.; Ruysschaert, J. M. B b chlm. Blophys. Acta 1980, 597, 1-14. Goormaghtigh, E.; Ruysschaert, J. M. Colblds Surf. 1984, 10, 239-247. -.- . . .
(19) Minow, R. A.; Banjamin, R. S.; Gottlleb, J. A. Cancer Chemother. Res. 1975. 6. 195-202. (20) Leyters,'R. Biochem. J. 1964, 9 3 , 313-316. (21) Mebsout, F.; Kauffmann, J. M.; Patriarche, G. J. Analusis 1987, 75, 243-247. (22) Mebsout, F.; Vire, J.C.; Patriarche, G. J. Anal. Lett. 1985, 78, 1437- 1440. (23) Chaney, E. N.; Baldwin, R. P. Anal. Chem. 1982, 5 4 , 2556-2560. (24) Moutet, M.; Vailot, R.; Buret, R. Bioeiectrochem. Bloenerg. 1987, 78, 137- 146. (25) Goormaghtigh, E. Ph.D. Thesis, Universite Libre de Bruxelles, Belgium, 1983.
RECEIVED for review June 6,1988. Accepted October 27,1988. Thanks are expressed to the Fonds National de la Recherche Scientifique (FNRS Belgium) for help to G.J.P. and to the SPPS (Belgium Politic Research, A.R.C.) Contract No. 86/ 91-89.
