A study of the bielectronic electro-reduction of cercosporin phytotoxin in highly acidic non-aqueous medium

Journal of Electroanalytical Chemistry 465 (1999) 225 – 233

A study of the bielectronic electro-reduction of cercosporin phytotoxin in highly acidic non-aqueous medium Marı´a Alicia Zo´n, Nancy Cristina Marchiando, He´ctor Ferna´ndez * Departamento de Quı´mica y Fı´sica, Uni6ersidad Nacional de Rı´o Cuarto, Agencia Postal No 3 (5800) -Rı´o Cuarto, Co´rdoba, Argentina Received 14 July 1998; received in revised form 5 November 1998

Abstract The kinetics of the electro-reduction of the cercosporin phytotoxin in 1 M HClO4 + ACN is analyzed on the basis of the theory presented for the nine-member square scheme when protonations are assumed to be at equilibrium. Experimental results obtained fit fairly well the theoretical model proposed by Laviron for 2e − , 2H + reactions. The formal heterogeneous rate constant, the voltammetric half wave potential and the cathodic transfer coefficient for the overall electrode process were determined from a fitting procedure of experimental square wave voltammograms by employing the COOL algorithm. Average values of 0.184 V, 0.45 and 0.019 cm s − 1 were calculated for the half wave potential, the cathodic transfer coefficient and the formal heterogeneous rate constant, respectively. The convolution analysis of cyclic voltammograms has been used to obtain the individual heterogeneous rate constants of the separate one-electron processes. Average values calculated were 0.013 and 0.008 cm s − 1, respectively. However, a complete description of the redox behaviour of cercosporin through the nine-member square scheme could not be achieved due to the lack of some thermodynamic parameters. An average value of 8 × 10 − 6 cm2 s − 1 was obtained for the diffusion coefficient of cercosporin from both chronocoulometry and convolution voltammetry measurements. Square wave voltammetry was also used to generate Ip versus c*cer calibration curves for this fungal metabolite. Detection limits of 5.8 ×10 − 7 and 2.8×10 − 7 M could be determined theoretically from calibration curves performed at 40 and 100 Hz, respectively, while minimal concentrations in the range of 1.9 to 3.8 ×10 − 6 M could be measured experimentally by the same technique at those frequencies. © 1999 Elsevier Science S.A. All rights reserved. Keywords: Electroreduction; Cercosporin phytotoxin

1. Introduction Cercosporin is a toxin produced by members of the genus Cercospora, a large group of fungal pathogens which cause damaging leaf spot diseases on a wide range of economically important crops. Cercosporin was first isolated in 1957 by Kuyama and Tamura [1] from Cercospora kikuchii, a soybean pathogen. Its chemical structure was elucidated independently by Lousberg et al. [2] and Yamazaki et al. [3]. Cercosporin is a perylenequinone derivative (see Fig. 1). * Corresponding author. Fax: +54-358-4676224. E-mail address: [email protected] (H. Ferna´ndez)

Cercosporin is a nonspecific toxin, which also kills cells of plants that are not a host of pathogenic Cercospora species. Moreover, cercosporin is also toxic to mammalian cells and to bacteria. Its phytotoxicity was widely studied and reviewed by Daub et al. [4–7]. The killing capability of cercosporin is highly light dependent [6,8], acting as a photosensitizing agent in host plants [4]. Processes involved in the killing of cells by photodynamic agents are complex and actual mechanisms remain to be elucidated in several cases [9]. Some hypotheses have been discussed which state that resistance to cercosporin may result from a reducing environment at the cell surface [10,11]. The toxicity of cercosporin appears to be dependent on its redox-state.

0022-0728/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PII: S 0 0 2 2 - 0 7 2 8 ( 9 9 ) 0 0 1 0 1 - 1

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It has been shown that reduced derivatives of cercosporin are less toxic than oxidized cercosporin [11]. As far as we know, a few reports related to the electro-reduction of cercosporin have been reported recently in the literature [12,13]. Clark et al. [13] have proposed the following overall reaction scheme for the electro-reduction of cercosporin in acetic acid+ acetate buffers containing 50% acetone on a glassy carbon electrode by using cyclic voltammetry in the pH range 4.7–7.1: Q +2H + +2e − UQH2 where Q and QH2 represent the quinone and hydroquinone forms of cercosporin, respectively. Therefore, the electrochemical behaviour of cercosporin has been proposed to be similar to other quinoid-type systems under the experimental conditions reported. We are interested in the study of the kinetics and heterogeneous reaction mechanisms of some mycotoxins produced by fungi in the last few years, particularly Alternaria alternata and Cercospora genus mycotoxins. Also, the use of electroanalytical techniques to detect and quantify these metabolites in real samples belong to the area of our interest. In this work, we report results obtained in our laboratory on the bielectronic reduction of cercosporin in 1 M HClO4 +acetonitrile (ACN) on a glassy carbon disk electrode by using cyclic (CV) and square wave voltammetry (SWV) as well as double potential step chronocoulometry. The highly acidic medium was chosen to ensure that the bielectronic charge transfer is accomplished in just one cathodic/one anodic quasi-reversible peak system. We have found that in a neutral (0.2 NaClO4 +ACN) or slightly acidic non-aqueous medium the bielectronic transfer is split into a two cathodic/two anodic quasi-reversible peak system. The study of the effect of the medium acidity on the electrochemical behaviour is being carried out and will be published separately [14].

Fig. 1. Chemical structure of cercosporin (1,12-bis (2-hydroxypropyl)-2,11-dimethoxy-6,7-methylenedioxy-4,9-dihydroxyperylene-3, 10-quinone).

2. Experimental

2.1. Reagents Cercosporin was obtained from Sigma and it was used as received. ACN and acetone (Ac) were Sintor˚ molecugan, HPLC grade. They were dried over 3 A lar sieves for 48 h prior to use and then used without further purification. Perchloric acid (Merck p.a.) was used as received. Stock solutions of cercosporin were prepared in acetone. They were stored at 5°C in the dark. Working solutions were prepared daily by adding aliquots of stock solutions to 1 M HClO4 + ACN medium and were protected from light. Manipulation of all laboratory material was done using thin plastic gloves for safety reasons. Experiments were conducted at a temperature of 25°C with light excluded from the cell.

2.2. Apparatus and experimental measurements The measuring system for the electrochemical techniques was built from a EG&G PARC Model 273 potentiostat/galvanostat equipped with model PAR270 electrochemical analysis software. Voltammograms at low metabolite concentrations were subjected to a 5-point moving average smoothing after acquisition by using the filtering facility in the electrochemical software. Cyclic voltammograms were obtained at scan rates (6) in the range 0.050 to 1 V s − 1. They were convoluted, after background current subtraction, by using the method proposed by Oldham [15]. Characteristic parameters used for obtaining square wave voltammograms were: pulse half-peak-to-peak (DESW)= 25 mV; staircase step height (DEs)=5 mV and the square wave frequency ( f ) was in the range of 20–100 Hz. The ratio of peak current (Ip) to peak width (W1/2) exhibited a maximum at nDESW =50 mV in good agreement with the theoretical behaviour predicted for a reversible electron transfer [16]. Square wave voltammograms were fitted by using the well-known COOL algorithm [17]. Background currents were subtracted from experimental square wave voltammograms by using a home made programme. Chronocoulometric measurements were performed for potential steps where the charge transfer rate was controlled by semi-infinite diffusion to the electrode surface [18]. The initial and final potentials were chosen as 0.4 and 0 V versus SCE, respectively. The pulse time was 10 s. Electrochemical measurements were performed in a two-compartment Pyrex cell [19]. The working electrode was a glassy carbon disk. It was polished successively with wet alumina powder (0.3 and 0.05 mm, from Fischer), rinsed copiously with distilled water

M.A. Zo´n et al. / Journal of Electroanalytical Chemistry 465 (1999) 225–233

Fig. 2. Nine-member square scheme.

and sonicated in a water bath for 2 min. The polished electrode was further activated electrochemically in 1 M KOH (Merck p.a.) aqueous solution by a potential step of 1.2 V over 5 min according to a procedure described previously by Anjo et al. [20]. Its electrochemical area (A = 0.089 cm2) was calculated from the well known I versus t 1/2 Cottrell plots [18] by studying the ferrocene (Fc)/ferrocinium (Fc’ + ) redox couple in 0.1 M NaClO4 + ACN at 293 K since the Fc diffusion coefficient in this reaction medium has been reported already [21]. Voltammetric responses show a simple electrode reaction on glassy carbon electrodes while those obtained on platinum electrodes appear to be complicated by adsorption of reactant and/or product species upon the electrode surface. The counter electrode was a platinum foil of large area (approx 2 cm2). The reference electrode was an aqueous saturated calomel electrode (SCE) fitted with a fine glass Luggin capillary containing a bridge solution identical to that of the sample being measured or a silver wire as pseudo-reference. Positive feedback technique was employed to compensate for solution resistance. Solutions were deaerated by bubbling pure nitrogen. The blank standard deviation was determined from 30 replications through square wave voltammograms obtained by following a methodology reported already [22,23]. This standard deviation (2.8× 10 − 8 A) was then used to calculate the detection limits from calibration curves obtained at 40 – 100 Hz [22,23].

3. Theory Several classes of biologically important organic redox systems [24,25] show quasi-reversible behaviour in protic solvents with an apparent direct 2e − , 2H + exchange. Laviron [26,27] has developed a theoretical treatment for 2e − , 2H + reaction, which can be described using the nine-member square scheme [28] (Fig. 2). It has been shown that when the protonation reactions are assumed to be much faster than the electron

227

exchanges, i.e. when they are assumed to be at equilibrium, the symmetry factors of all the individual electrochemical reactions are equal to 0.5 and when no dimerization or disproportionation of the radical intermediate occurs, the complex nine-member scheme behaves as a simple electrochemical reaction with two successive one-electron exchanges, with apparent rate constants k1,app and k2,app and apparent standard potentials Er1 and Er2 (see Fig. 2). Er1 and Er2 are the equilibrium potentials for each of the six-member ladder schemes which compose the nine-member square scheme. k1,app and k2,app are complex functions of the heterogeneous rate constants for the individual electrochemical steps (ki ), the dissociation constants (Kai ) and the proton concentration. The rate constants which can be determined experimentally are [26]: k*1 = r 1/2 k1,app

(1)

and k*2 = r 1/2 k2,app

(2)

with r 1/2 = exp[(F/4RT)(Er1 − Er2)]

(3)

The kinetics of the overall reaction are controlled by the kinetic of the ladder scheme on the left when EBEt or by the kinetics of the ladder scheme on the right when E\Et, with: (RT/F) ln(k*2 /k*1 ) Et = E °+ f

(4)

where E °f is the equilibrium potential for the 2e − , 2H + reaction. E °f is defined by: E °= (1/2)(Er1 + Er2) f

(5)

If the oxidized form is the only species initially present in the bulk solution, the current is given by (anodic currents taken as positive) [26]: − 1/4 I= − 2FAk*1 c*Q[exp(2F/RT)(E − E °)] f

(6)

or − 3/4 I= −2FAk*2 c*Q[exp(2F/RT)(E − E °)] f

(7)

where c*Q is the bulk concentration of the oxidized form of the quinoid compound, A is the electrode area and F the Faraday constant. Eqns. 6 and 7 are applied in different potential regions depending on the kinetics of the overall process being controlled by reaction 1 or reaction 2, respectively (see Fig. 2). The kinetics of the benzoquinone/hydroquinone couple on a platinum electrode in aqueous medium [27] as well as the electrooxidation of three substituted catechols on a carbon paste electrode in buffered media [29] have been analyzed successfully and discussed on the basis of the theory proposed for the nine-member square scheme.

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3.1. Square wa6e 6oltammetry

4. Results and discussion

The voltammetric half wave potential (E1/2 :E °), the f formal heterogeneous rate constant (k °) and the caf thodic transfer coefficient (a) for an overall two-electron process can be obtained from square wave voltammograms using non-linear least squares analysis through the COOL algorithm [17]. For reactions controlled by semi-infinite linear diffusion conditions, the slope (a) of the linear regression between experimental and dimensionless currents is given by [30]:

A representative cyclic voltammogram of cercosporin in 1 M HClO4 + ACN after subtraction of background currents is shown in Fig. 3a. A separation between the cathodic and anodic peak potentials (Epc − Epa) of 36 mV was found at 0.050 V s − 1, this value being close to the theoretical one predicted for a bielectronic nernstian redox couple, i.e. 30 mV at 25°C [18]. Moreover, the ratio between anodic and cathodic peak currents (Ipa/ Ipc) determined by using the method proposed by Nicholson [33] was 1.01, which is indicative of nernstian waves with a stable reaction product. The Ipc versus 6 1/2 plot was linear (correlation coefficient, r= 0.9992). From this plot, a value of 6.6× 10 − 6 cm2 s − 1 was calculated for the diffusion coefficient of cercosporin (DCer) through the well-known Ipc versus 6 1/2 expression deduced for a reversible redox couple [18] by using n= 2. We have also found by using double potential step chronocoulometry that cercosporin does not adsorb significantly on a glassy carbon electrode from 0.37 mM Cer+ 1 M HClO4 + ACN solutions, at least within the potential range studied. Plots of Q (tBt)

a =nFAD 1/2c*Q/(ptp)1/2

(8)

where n is the number of electrons transferred, D is the diffusion coefficient of the reactant and tp is the characteristic time, i.e. the pulse width of the excitation waveform. When a quasi-reversible electrochemical reaction is chosen as the mechanistic model for performing the fit, the COOL algorithm gives the best values of E1/2, a and the dimensionless function log(kt 1/2 p ), which is related to the heterogeneous charge transfer rate constant through: 1/2 k= k °/D f

(9)

by assuming that the diffusion coefficients for the oxidized and reduced species are taken as equal [17].

3.2. Con6olution potential sweep 6oltammetry The advantages that the convolution technique offers in the treatment of experimental data from linear sweep voltammograms are well known [18,31]. In the Butler– Volmer formalism the rate constant for a quasi-reversible cathodic reaction may be expressed as follows [18,31]: k(E) = D 1/2{I/IL,c − I(E){1 +exp[(nF/RT)(E − E °)]}} f (10) where IL,c is the cathodic limiting convoluted current and I(E) is the convoluted current at a given potential (E). k(E) is the potential-dependent rate constant of the forward reaction. Under purely diffusion controlled conditions, IL,c is defined by [18,31]: IL,c =nFAD 1/2c*Q

(11)

On the return scan of the cyclic voltammograms the current, I, passes through zero. At this point, the formal potential (E °) f for a quasi-reversible system may be determined from [32]: E °= EI = 0 − (RT/nF) ln[(IL,c −II = 0)/II = 0] f

(12)

where EI = 0 is the potential corresponding to the intersection of the backward cyclic voltammogram curve with the horizontal axis (I = 0) and II = 0 is the convoluted current value at that potential.

Fig. 3. Cyclic voltammogram after background subtraction (a) and the convoluted current of the cyclic voltammogram (b) for a solution of c*Cer =0.37 mM in 1 M HClO4 +ACN on a glassy carbon disk (A= 0.089 cm2). Reference electrode: SCE. 6 =0.050 V s − 1.

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229

on indium–tin oxide (ITO) electrodes. In addition, these authors also reported that a minor component (B 10%) of the faradaic current results from strongly adsorbed cercosporin on glassy carbon electrodes in aqueous media [13].

4.1. Analysis of square wa6e 6oltammograms

Fig. 4. Square wave voltammograms for reduction of 0.37 mM of cercosporin in 1 M HClO4 + ACN at 80 Hz. Forward (If), reverse (Ir) and net (In) experimental currents are shown by circles while the corresponding currents from the best fitting are shown by solid lines. Working electrode: glassy carbon disk (A =0.089 cm2). Reference electrode: SCE.

versus t 1/2 and Q(t Bt B2t) versus [t 1/2 +(t −t)1/2 − t 1/2] gave equal (and of opposite sign) slopes within experimental error (slopes =(2.02 90.19) ×10 − 5 C s − 1/2) and intercepts corresponding to cercosporin-free background solutions. From the slope of the Q(tB t) versus t 1/2 plot indicated above, a value of DCer = 7.8× 10 − 6 cm2 s − 1 was calculated, which can be compared reasonably with the value determined previously from cyclic voltammetry. These results show that reactant (and/or product) adsorption is negligible [18] at least under the experimental conditions mentioned. Besides, the chemical stability of the electrode reaction product was also checked by employing the Q(2t)/Q(t) charge ratio, for which an experimental value of 0.44 was calculated. This value can be also taken as evidence that the redox couple under study is not complicated by coupled homogeneous chemical reactions [18]. Daub et al. [12] found no evidence for adsorption of cercosporin

The forward, reverse and net experimental currents obtained from a typical square wave experiment after background current subtraction are shown in Fig. 4 (circles). The best fitting curves for both net and forward and reverse currents (solid lines in Fig. 4) were achieved when the quasi-reversible dimensionless current function was chosen to perform the fitting. After the most probable mechanism was identified, calculations were carried out with a 95% confidence level. The minor differences found for forward currents (experimental and calculated) at potentials smaller than about  0.12 V might be explained by considering that the reduction peak for cercosporin in 1 M HClO4 + ACN is displayed at potentials close to the lower potential limit given by the background solution. This behaviour made a complete background currents suppression difficult, particularly in that potential region. A small difference was also observed for net current voltammograms in that potential region (see Fig. 4). On the other hand, the net current responses showed a constant peak width in the frequency range studied. Peak widths were about 64 mV, close to the value expected theoretically for a bielectronic reversible reaction, i.e. 63 mV at 25°C [17,34]. Characteristic values for cercosporin in 1 M HClO4 + ACN obtained from the best fitting curves for individual forward and reverse currents, which are more sensitive to the kinetic effects than net currents [35,36], are reported in Table 1. Besides, results of fitting performed on net currents did not differ significantly from those obtained when the fitting was carried out on forward and reverse currents, as expected for

Table 1 Results of the best fitting obtained from the individual forward and reverse square wave voltammograms from data corresponding to a solution of c*Cer = 0.37 mM in 1 M HClO4+ACN on a glassy carbon disk (A = 0.089 cm2)d f/Hz

E1/2/V

a

log(kt 1/2 p )

Slopea/mA

k

104 (1−r)b

1013x 2c/mA2

20 40 80 80 100 100

0.183 0.186 0.185 0.185 0.184 0.184

0.23 0.41 0.47 0.46 0.45 0.47

0.0771 −0.1000 −0.2442 −0.2109 −0.2408 −0.2257

54.05 82.82 115.3 113.8 128.7 127.2

7.55 7.10 7.21 7.78 8.12 8.41

8.45 2.40 6.28 4.08 6.76 3.03

3.2 1.02 8.53 6.15 11.4 56.4

a

Ratio of experimental to dimensionless currents. Complement of correlation coefficient of experimental versus dimensionless currents linear regression. c 2 x : Chi2 function. d Reference electrode: SCE. Temperature: 25°C. b

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230

Table 2 Data corresponding to convoluted voltammograms. c*Cer = 0.37 mM in 1 M HClO4+ACNg 6/V s−1

105IL,c/C s−1/2

#E/#log[IL,c−I(E))/I(E)]a

E °f b/V

Slopes of (I) linesc/V−1

Slopes of (II) linesd/V−1

k*1 e/cm s−1

k *2 f/cm s−1

0.050 0.075 0.100 0.500 1

1.83 1.84 1.83 1.84 1.85

0.034 0.035 0.035 0.034 0.035

0.182 0.181 0.181 0.182 0.181

−(17.590.5) −(189 1) −(17.890.6) −(189 1) −(189 2)

−(5992) −(5793) −(6192) −(5892) −(6092)

0.011 0.013 0.014 0.013 0.014

0.007 0.010 0.007 0.008 0.007

a

Slopes of logarithmic analysis of convoluted forward current curves. Formal potential for the overall bielectronic process, determined by applying Eq. (12). c Slopes of (I) lines from ln k(E) versus E plots with its corresponding standard deviation (see Fig. 5). d Slopes of (II) lines from ln k(E) versus E plots with its corresponding standard deviation (see Fig. 5). e Individual rate constants determined from ln k(E) versus E plots as indicated in the text. f Individual rate constants determined from ln k(E) versus E plots as indicated in the text. g Working electrode: glassy carbon disk (A= 0.089 cm2). Reference electrode: SCE. Temperature: 25°C. b

systems not complicated by complex reaction mechanisms [36]. The fits were very good, as can be inferred from the complement of the correlation coefficient (1− r) of the linear regression between experimental and dimensionless currents and the chi2 (x 2) function values (see Table 1), two parameters which measure the quality of fitness. From these results, an average value for k 1/2 of 7.7090.50 was calculated. A plot of a versus t − p (Eq. (8)) was linear (r=0.9991). From the slope of this plot, a value of DCer =6.13 ×10 − 6 cm2 s − 1 was determined. This value, which does not differ significantly from values obtained from both chronocoulometry and cyclic voltammetry, was then used to obtain the overall formal rate constant for the bielectronic process by using Eq. (9), i.e. k °= 0.019 cm s − 1. Calculated averf age values for E1/2 and a of the overall electrode process with the corresponding standard deviations were: (0.18490.001) V and (0.4590.02), respectively. The statistical Q-test (at 95% confidence level) performed on the a values showed that the value of a =0.23 obtained at 20 Hz had to be rejected. However, k and E1/2 values at the same frequency were included to determine average values on the basis of the results of the application of the same statistical test. The apparently odd result obtained for a at a frequency of 20 Hz may be explained by considering that the experimental system would be moving from quasi-reversible to nearly reversible behaviour for that long measurement time [36], although a definitive reason is not known yet.

4.2. Determination of indi6idual heterogeneous rate constants. An application of con6olution 6oltammetry Convoluted cyclic voltammograms for cercosporin in 1 M HClO4 + ACN after background current subtraction showed a very small hysteresis between the forward and backward convoluted currents Fig. 3b, at least at the sweep rates studied. This behaviour is

characteristic of nearly reversible electrochemical reactions [18,31,37]. On the other hand, the convoluted current returns closely to zero during the reverse scan, which is a characteristic of an electrode reaction with no significant loss of the electroactive species through follow-up homogeneous chemical reactions [38]. Logarithmic analysis of convoluted forward I− E curves was another test to check the nearly reversible behaviour for this redox couple in the medium studied. E versus log [(IL,c − I(E))/I(E)] plots showed slopes close to the 0.030 V dec − 1 value predicted for a reversible two-electron process [18,31] (see Table 2). The potential dependent forward rate constant (k(E)) was obtained from Eq. (10) at different 6. Values of DCer and E °f required for applying Eq. (10) were determined from Eq. (11) and Eq. (12), respectively. A value of DCer = 8.20× 10 − 6 cm2 s − 1 was calculated from the average of IL,c values shown in Table 2. This value agrees well with that previously determined by chronocoulometry and it is slightly higher than those obtained by cyclic and square wave voltammetries. From the techniques performed in this work, chronocoulometry as well as convolution voltammetry are perhaps the most reliable techniques for the determination of diffusion coefficients [18]. Therefore, an average value of DCer = (8.09 0.2)× 10 − 6 cm2 s − 1 was obtained by using these two methods. This value is higher than that reported previously in the literature by other authors for a different reaction medium using cyclic voltammetry (i.e. DCer = 1×10 − 6 cm2 s − 1 in acetic acid +acetate buffers containing 50% acetone) [13]. On the other hand, an average value of (0.181 9 0.001) V was found for E °, f which agrees well with the E1/2 value determined previously by square wave voltammetry (i.e. E1/2 = 0.184 V) (see Tables 1 and 2). Then, plots of ln k(E) versus E showed the same characteristic shape at different sweep rates analyzed, as can be observed in Fig. 5. They consisted of two straight lines, I and II, with slopes close to (− F/2RT) and (− 3F/2RT), respec-

M.A. Zo´n et al. / Journal of Electroanalytical Chemistry 465 (1999) 225–233

tively (see Table 2). The observed variations in ln k(E) with potential as well as experimental slope values obtained from both linear portions fit fairly well the theoretical model proposed by Laviron for 2e − , 2H + reactions [26,27] (see Eqs. (6) and (7)). The change in slope for these logarithmic plots occurs when k1,app = k2,app. Extension of (I) and (II) lines in ln k(E) versus E plots up to the overall formal potential can be used to determine k*1 and k*2 , respectively (see Fig. 5). Experimental values obtained for these parameters at different 6 are gathered in Table 2. Average values of (0.0139 0.001) cm s − 1 and (0.008 90.001) cm s − 1 were calculated for k*1 and k*2 , respectively. Nevertheless, it is assumed that a complete description of the redox behaviour for cercosporin should require a knowledge of eight independent variables, i.e. the six pKai s of the acid/base pairs involved in the nine-member square scheme and two formal potentials (see Fig. 2). Unfortunately, these thermodynamic parameters for cercosporin are not known yet and it is not possible, at present, to make a complete study about kinetic behaviour for this redox couple. While the theoretical treatment developed for reactions which show an apparent direct exchange of 2e − , 2H + was in most cases applied to the study of redox couples in buffered aqueous media [27,29], experimental results obtained in this work show that the rates of protonation are also apparently larger than the rates of electron exchange in 1 M HClO4 +ACN medium. The protonation rate constants may be of the same order of magnitude as, or a little larger than, the electron-exchange rate constants when the protonation site is an oxygen or a nitrogen atom [39]. On the other hand, acidity of the medium plays also an important role in the reaction mechanism. Thus, the electro-reduction of p-benzoquinone, dimethoxy-2,6-p-benzoquinone

231

(DMQ) and tetramethoxy-3,3%,5,5% p-diphenylquinone (TMBQ) in 0.1 M Et4NClO4 + ACN showed two successive reversible monoelectronic waves corresponding to the formation of Q’ − and Q2 − [40]. However, the addition of perchloric acid to quinone solutions in ACN caused the appearance of a new redox couple at potentials more positive than those of the first wave observed in the neutral medium and corresponding to the exchange of two electrons [40]. On the basis of the results discussed above, we can infer that the electrochemical behaviour of cercosporin appears to be less complicated than that previously found for ATX-I, an A. alternata genus mycotoxin related structurally to cercosporin, which has also been studied by us [41]. Cyclic as well as square wave voltammograms obtained for ATX-I in 1 M HClO4 + ACN medium on glassy carbon electrodes showed a quasi-reversible adsorption peak system at potentials lower than those corresponding to the diffusion controlled electro-oxidation peak. A considerable increase in peak currents was found for different pre-concentration times of ATX-I on the glassy carbon surface [42]. This characteristic behaviour, which might be ascribed to a specific interaction between ATX-I and the glassy carbon surface, was not observed for cercosporin under the same experimental conditions [43].

4.3. Analytical application Square wave voltammetry is an effective and rapid electroanalytical technique with well-established advantages, including good discrimination against background currents and low concentration detection limits [34,44]. The net current-potential curve is the most useful analytical signal. In the absence of side reactions, it is symmetrical about the E1/2 and its peak height is proportional to concentration. Square wave voltammograms of cercosporin in 1 M HClO4 + ACN were obtained at 40–100 Hz in the 1.9×10 − 6 to 1.5×10 − 4 M range. The relationship between peak currents (Ip) and c*Cer is shown in Fig. 6. As it can be observed, good linear calibration curves were obtained. The linear regressions can be expressed by least-square procedures as: Ip/A=(0.1459 0.001)c*Cer/M−(1.149 0.06)× 10 − 7

r

= 0.9976 and Ip/A=(0.3019 0.003) c*Cer/M−(1.990.2)× 10 − 6 r = 0.9985

Fig. 5. Variation of heterogeneous rate constant with potential. Experimental conditions are the same as in Fig. 3 except 6 = 0.100 V s − 1.

at 40 and 100 Hz, respectively (lines a and b in Fig. 6). Forty five and thirty two experimental points were taken into account to obtain the calibration curves at 40 and 100 Hz, respectively.

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de la Provincia de Co´rdoba (CONICOR) and Secretarı´a de Ciencia y Te´cnica from the Universidad Nacional de Rı´o Cuarto are gratefully acknowledged. N.C. Marchiando thanks FOMEC for a research fellowship. We thank the Referees for valuable suggestions. We are indebted to Lilia Ferna´ndez for language assistance.

References

Fig. 6. Peak current versus cercosporin concentration in 1 M HClO4 + ACN. Working electrode: glassy carbon disk (A= 0.089 cm2). A silver wire was used as pseudo-reference electrode. (a) f= 40 Hz; (b) f =100 Hz.

The sensitivity found at 100 Hz, higher than at 40 Hz, agrees with the theoretically predicted dependence between square wave peak current and frequency [35,44]. Theoretical detection limits (dl) were calculated by using the following equation [22,23]: dl = 3s/m

(13)

where s is the blank standard deviation (see Experimental) and m is the slope of the calibration curve. Detection limits determined theoretically were found to be 5.8 ×10 − 7 and 2.8 ×10 − 7 M at 40 – 100 Hz, respectively. In addition, the lowest concentration values measured experimentally for a signal to noise ratio of 2:1 were about 1.9×10 − 6 M for both frequencies. However, for the 100 Hz measurements a departure from linearity was observed at concentrations lower than about 4×10 − 6 M. Recently, the HPLC method was used to quantify Cercospora beticola toxins in crude mycelial extracts, the amount of cercosporin detected being 270 ng for a 50 ml culture medium (about 1× 10 − 5 M) [45], although concentrations as low as 0.1 mM have been determined by spectrophotometric measurements [46]. Our preliminary analytical results encourage us to continue studying the possibility of performing the quantitative determination of this fungal secondary metabolite in crude samples by pulse electrochemical methods.

Acknowledgements Financial support from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Consejo de Investigaciones Cientı´ficas y Tecnolo´gicas

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