Journal of Pharmaceutical and Biomedical Analysis 26 (2001) 873– 881 www.elsevier.com/locate/jpba Electroanalysis of dapsone, an anti-leprotic drug P. Manisankar *, A. Sarpudeen, S. Viswanathan Department of Industrial Chemistry, Alagappa Uni6ersity, Karaikudi-630 003, India Received 24 November 2000; received in revised form 4 April 2001; accepted 15 April 2001 Abstract The electrochemical oxidation and adsorption of dapsone, an anti-leprotic drug were studied in aqueous alcohol medium at a stationary glassy carbon electrode. Cyclic voltammetry studies showed one well-defined oxidation peak in the potential range 1.2– 1.9 V at pH conditions 1.0, 4.0, 7.0, 9.2 and 13.0. The oxidation was irreversible and exhibited diffusion controlled adsorption. Controlled potential coulometry revealed one electron oxidation of the amino group in the molecule. A systematic study of the experimental parameters that affect the squarewave stripping response was carried out and the optimized experimental conditions were arrived at. A calibration plot was derived for the determination of the compound in solution. This method was used for the determination of dapsone in tablets and urine. The limits of determination was 0.0036 and 3.56 mg/ml and the relative standard deviation (n= 10) was 4 ppt (0.4%) at a concentration level 0.100 mg/ml. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Dapsone; Cyclic voltammetry; Squarewave voltammetry; Tablets; Urine 1. Introduction Dapsone, 4,4-diamino-biphenyl-sulphone (DDS) is one of the most important and main drug available for the treatment of leprosy [1]. It is also used in the treatment of malarial diseases and as an anti-inflammatory [2] in acute ileitis. Dapsone with clofazimine prevents the inhibitory effect on neutrophil motility [3]. With ethambutol and rifampin it is used for the treatment of human eye disease [4]. The structural formula of dapsone is: * Corresponding author. Fax: + 91-4565425-202. E-mail address: pms11@rediffmail.com (P. Manisankar). Perusal of literature reveals the availability of methods like HPLC [5,6], LC [7], spectrophotometry and polarography for the determination of DDS in tablets, blood and urine [1– 11]. The cleavage of DDS using a mercury cathode and tetraalkyl ammonium salts in both protic and aprotic media has also been reported [12]. A number of procedures were available for the determination of dapsone using HPLC technique. HPLC, at its current stage of development, is clearly not a method for analytical problems with a high repetition rate because the receptive condition of the system requires 24– 36 h. On the other hand, electroanalysis is a manageable method, which is suitable for various problems [13]. Hence the development of electrochemical determination 0731-7085/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 7 3 1 - 7 0 8 5 ( 0 1 ) 0 0 4 8 0 - 0 874 P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 assumes importance. No electroanalytical data based on stripping voltammetry are available for the determination of this compound. Stripping voltammetry is a simple, highly sensitive and selective technique for the determination of electroactive compounds at ppm and ppb levels. Determination of drugs in pharmaceutical formulations and urine samples using differential pulse polarography was already reported [8]. Mercury is unjustly marked as one of the most insidious hazards within the laboratory. Solid electrode is better than the liquid electrode. Hence we selected glassy carbon as a working electrode. The present paper reports the cyclic voltammetric behaviour of DDS and square wave stripping voltammetric procedures for the determination of this drug. The analytical procedure proposed for the determination of DDS is easy to adopt and the lowest concentration level possible is better than the existing LC and polarographic methods. This method can be adopted for the determination of DDS in pharmaceutical formulations and urine samples. 2. Experimental 2.1. Apparatus and reagents The voltammetric experiments and stripping analysis were performed using a Bioanalytical Systems (BAS 100A) electrochemical analyser. A glassy carbon working electrode (BAS Model MF 2012; geometric area=0.0774 cm2), a silver– silver chloride reference electrode (BAS Model MF2021), a platinum counter electrode and a standard one-compartment three-electrode cell of 15 ml capacity were used in all experiments. The compound DDS was prepared [14] by the action of thionyl chloride on acetanilide followed by the oxidation with CrO3 to its sulphone. Then it was hydrolysed to 4,4%-diamino-biphenyl-sulphone (DDS). DDS was recrystallized from ethanol to white crystals (m.p. 178–179 °C). The stock solution was prepared in alcohol. The supporting electrolytes solutions, 10% H2SO4 in 50% aqueous alcohol (pH 1.0), B.R. buffer in 50% aqueous alcohol (pH 4.0 and 9.2), 0.1 M KCl in 50% aqueous alcohol (pH 7.0) and 0.1 M NaOH in 50% aqueous alcohol (pH 13.0) were prepared and used for the voltammetric studies. 2.2. Procedure Under the experimental conditions, a solution of DDS was placed in the electrochemical cell and purified nitrogen gas was passed for 20 min to remove the dissolved oxygen under stirred conditions. The glassy carbon electrode was pretreated in two ways to get reproducible results: (1) mechanical polishing over a velvet micro-cloth with alumina suspension, and (2) electrochemical treatment by applying a potential of + 1.5 V for 4 s in the same solution in which the measurements was carried out. The electrode cleaning procedures were carried out for each and every experiment and this pretreatment requires only 5 min. The suitability of glassy carbon electrode was checked after every 100 stripping analysis by running cyclic voltammogram of potassium ferricyanide and measuring the potential difference between reversible peaks. If the potential difference was greater than 60 mV, then the electrode was boiled in 10% potassium hydroxide solution for 10 min, washed with water and the usual treatments mentioned above were done. The electrode was fairly stable for long periods. The Ag/AgCl reference electrode and the platinum counter electrode were used. The analyte solution was prepared in the ratio of 1:9 using the analyte and appropriate amount of the buffer solution and the total volume was kept constant (10 ml). Measurements were made using cyclic voltammetry, squarewave voltammetry, chronocoulometry and controlled potential coulometry for the reaction mechanism study. Squarewave stripping voltammetry was employed for the analytical study at pH 1.0. Exactly 9 ml 10% sulphuric acid in 50% aqueous ethanol and 1 ml of the ethanolic solution of DDS were taken in a 15 ml undivided cell for normal studies. There was no change observed in the stripping response when the volume of the pH solution was reduced to 8 ml and the substrate volume was increased to 2 ml. The total analyte volume was always kept at 10 ml. All the three electrodes were inserted, deaerated and the experiment was car- P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 875 Fig. 1. Cyclic voltammogram of 7.7 mM DDS at various scan rates in 0.1 M H2SO4 in 50% aqueous ethanol. ried out depending upon the necessity of the medium volume. Background values were recorded and subtracted. 3. Results and discussion 3.1. Cyclic 6oltammetric beha6iour Cyclic voltammograms of DDS at all the five pH conditions exhibited one distinct and welldefined anodic peak in the potential range 1.2– 1.9 V at concentrations ranging from 3.2 to 29.4 mM and sweep rate from 0.1 to 1.0 V/s (Fig. 1). Apart from this, a cathodic peak with low peak current is observed at pH 7.0, 9.2 and 13.0. However, it does not satisfy the criteria for reversibility [15]. The cyclic voltammetric study was not considered beyond the concentration 29.4 mM of DDS in this medium due to the precipitation of substrate in the solution. As the sweep rate is increased from 0.10 to 1.00 V/s at a fixed concentration of DDS: (i) the peak potential shifts anodically, (ii) the peak current increases steadily, (iii) the peak current function, ip/ACw 1/2 exhibits almost constancy. If the polishing was not done between two sweep rate changes then a decrease in the peak current is observed. This indicates the possibility of adsorption. A straight line is obtained when ip is plotted against w 1/2 (Fig. 2). This reveals that the anodic oxidation is diffusion controlled. A straight line (Ep = 0.088 log w+ 0.0017) is observed when Ep is plotted against log w at a particular concentration in pH 1.0 medium. From the slope of the straight line (DE/Dlog w), the hna value is calculated by using the formula, DE/Dlog w = −30/hna. The hna value is found to be 0.34 and is taken for further calculation for the number of electron transferred. Fig. 2. Dependance of the peak current on the square root of scan rate. 876 P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 Fig. 3. Dependence of the peak current on the various concentrations of DDS. Similar correlations were made for other pH conditions. With the increase of concentration from 7.7 to 29.4 mM: (i) the peak potential shifts anodically, (ii) the peak current increases, (iii) the peak current function decreases steadily. As the compound has the character of adsorption during the cycle itself on electrode surface, the polishing was given for every concentration changes. As a result of linear variation between ip and concentration C, a straight line is obtained when ip is plotted against C (Fig. 3). However, the peak current function exhibits a decrease with increase of concentration. This reveals the blocking effect due to adsorption of the substrate on the electrode surface. The peak current, ip and the peak current function, Ip values almost decrease gradually from pH 1.0 to 13.0 except in basic medium (pH 13.0) where an increase in the peak current is observed. The peak potential value decreases from pH 1.0 to 4.2 and then slight increase is observed at pH 7.0. Then it is decreased up to alkaline pH conditions (Fig. 4). This shows that the mechanism is different at acid, neutral and basic conditions. Since, no cathodic peak is observed when the potential scan direction is reversed the anodic oxidation is irreversible reaction. The fractional hna value confirms the irreversible oxidation of DDS. After the first cycle, the peak current decreases tremendously and comes closer to the background current as the number of cyclic voltammetric cycles increased from 2 to 10 for 20 mM and sweep 0.300 V/s (Fig. 5). This indicates and confirms the adsorption behaviour of substrate. 3.2. Controlled potential coulometric beha6iour By using controlled potential coulometry, the number of electrons transferred, n values were found out from the charge consumed by 1×10 − 9 M concentration of the compound DDS. The charge consumed for every electrolysis was found as 774×10 − 7 C in acid medium. The coulometric n was calculated by using the equation, Q= nFN, where Q is charge in coulombs, F is Faraday’s constant and N is number of moles of the substrate. The n value is found to be one (rounded Fig. 4. (a) Plot of peak versus pH; (b) plot of peak potentials versus pH. P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 877 Fig. 6. Plot of log ip versus log Hz. Fig. 5. Ten continuous cycles voltammagram of 7.7 mM DDS in pH 1.0 at scan rate 0.3 V/s. value) for anodic peak of the compound DDS in all mediums (Table 1). 3.3. Chronocoulometric beha6iour In chronocoulometry, a plot of charge versus square root of time, Q versus t 1/2 transforms the data into a linear relationship whose slope was 1 given as DQ/Dt 1/2 =2 nFACD 1/2/y2. The number of electrons transferred, n is one, taken from the controlled potential coulometric studies. By using forward slope and above equations, diffusion coefficient of DDS was calculated. It exhibits almost constancy except in basic medium (Table 2). 3.4. Square wa6e 6oltammetric beha6iour To confirm the results found in the cyclic voltammetry studies, the same experiments were carried out in pH 1.0 with the squarewave technique, maintaining the pulse amplitude at 25 mV and modifying the frequency used. The logarithm of the peak current was directly proportional to the logarithm of the frequency (Fig. 6). Table 1 Controlled potential coulometry pH Electrolytic potential (mV) Current (A) Charge (C) Number of electrons transferred 1.0 4.0 7.0 9.2 13.0 1800 1725 1750 1600 1200 2023×10−8 1994×10−8 1983×10−8 2017×10−8 2239×10−8 774×10−7 781×10−7 789×10−7 799×10−7 895×10−7 0.80 0.81 0.82 0.83 0.93 Table 2 Chronocoulometry pH Concentration (mol/cm3) Forward slope (mC/s) Intercept Correlation coefficient Diffusion coefficient (cm2/s) 1.0 4.0 7.0 9.2 13.0 3.2×10−6 9.1×10−6 3.2×10−6 9.1×10−6 9.1×10−6 8.5456 8.5290 2.6976 8.0956 15.1921 37.7749 −39.7049 4.9286 −80.8418 11.6859 0.9986 0.9975 0.9994 0.9999 0.995 1.238×10−5 1.230×10−5 1.001×10−5 1.113×10−5 3.918×10−5 878 P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 All the above facts reveal that the anodic oxidation of DDS at a glassy carbon electrode in acid medium is an irreversible one-electron transfer, the overall reaction is diffusion-controlled and the compound is having adsorptive behaviour. At acid medium, the NH2 group is protonated and hence the loss of electron becomes difficult. This is understood from higher Ep values observed in acid medium. In alkaline medium the oxidation becomes easier because of the easy availability of electrons from the NH2 group and removal of H+ from NH2 as H2O. Thus hydroxide present in the medium facilitates the oxidation. Hence lowest Ep values are observed here. In neutral, slightly acidic and basic medium, intermediate values are possible because of the maximum possible oxidation of neutral substrate. The formed intermediates, radical cations or radical may undergo some coupled chemical reactions. Further deelectronation is not realized because of the absence of second anodic peak. On the basis above discussion the probable oxidation mechanism is depicted as follows: To carry out the square wave stripping voltammetry the optimum conditions of various instrumental parameters which lead to well-defined peaks with maximum peak current were fixed by varying them over a range (Table 3). A lower concentration of 1.42 mM was chosen to establish optimum conditions (deposition time 10 s, a.c. amplitude 50 mV, frequency 15 Hz, quiet time 5 s, step potential 15 mV and deposition potential 50 mV). Under optimum conditions the square wave stripping voltammograms were recorded for various concentrations varying from 0.0142 to 14.2 mM. (0.0036– 3.56 mg/ml). As an illustration, the squarewave stripping voltammogram recorded for DDS under optimum conditions is presented in Fig. 7. The peak currents obtained for various concentrations were plotted against concentration, which give a straight line, ip(mA) =60.534 C where ip is the peak current in mA and C is the concentration in mg/ml with good correlation (r 2 =0.9923).The relative standard deviation for 10 measurements (n =10) was 4 ppt (parts per 3.5. Squarewa6e stripping 6oltammetric beha6iour Since highest peak current with no complication from other peaks was observed in pH 1.0, the acid medium was chosen for stripping. The test solution was purged with nitrogen for about 20 min and an accumulation potential of 0.0 V was applied to the electrode. The stirring was stopped after accumulation time of 15 s. After a 10 s rest period, Osteryoung squarewave stripping pulse sweep varying a.c. amplitude and frequency was carried out. The set conditions given in the instrument were square wave amplitude= 50 mV, frequency= 15 Hz, quiet time=10 s, sampling point=256, step potential =4 mV and deposit potential=0.0 V. thousand; 0.4%) at a concentration level 0.100 mg/ml. 4. Determination of DDS in pharmaceutical sample One tablet of dapsone is dissolved in 100 ml of 50% aqueous alcohol and 1.5 ml of dapsone solution was pipetted out into a cell containing 5.5 ml of buffer solution (pH 1.0). By using optimum conditions, the squarewave stripping voltammogram was recorded (Fig. 7) and the peak current value was noted. The corresponding amount of DDS present in 1 ml of the tablet solution was P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 found out from the calibration equation. From this the amount of DDS present in the whole of the 100 ml of solution was calculated. The experimentally calculated value of DDS present in one tablet was 109.1 mg. The theoretical calculated value is 110.0 mg. Deviation in percentage is 0.82. This experiment was repeated 5 times to get concordant value. Similarly by pipetting out 0.5, 1.0 and 2.0 ml of tablet solution and keeping the buffer volume at 7.0 ml, the stripping voltammograms were recorded. By employing the same 879 procedure the amount of DDS present was calculated. The results are presented in Table 4. A minimum of 3.6 ppm (0.0036 mg/ml) concentration of DDS was determined by this procedure. 5. Determination of DDS assay in urine Measurement of DDS in an urine sample collected after 8 h from administration was demonstrated and 0.5 ml of the urine sample was mixed Table 3 Square wave stripping voltammetry Constant parameters Varied parameter C 1.42 ACA 50 FR 15 QT 10 SP 04 DP 0.0 C 1.42 DT 10 FR 15 QT 10 SP 04 DP 0.0 Deposition Time (DT, s) C 1.42 DT 10 ACA 50 QT 10 SP 04 DP 0.0 C 1.42 DT 10 ACA 50 FR 15 SP 04 DP 0.0 C 1.42 DT 10 ACA 50 FR 15 QT 05, DP 0.0 C 1.42 DT 10 ACA 50 FR 15 QT 05, SP 15 Frequency (FR, Hz) C= concentration (mM). a.c. Amplitude (ACA, mV) Quiet time (QT, s) Step potential (SP, mV) Deposition potential (DP, mV) 10 20 30 40 50 60 25 50 75 100 125 150 175 200 5 7 9 11 13 15 5 10 15 20 25 30 5 10 15 20 25 0 50 300 500 600 Ep (mV) ip (mA) 1724 1624 1628 1620 1636 1632 1572 1568 1532 1504 1460 1452 1400 1344 1508 1512 1532 1508 1508 1516 1516 1524 1508 1544 1512 1544 1530 1560 1495 1500 1525 1465 1495 1525 1495 1525 36.23 29.73 26.53 31.01 27.90 28.54 29.98 44.43 34.73 28.12 36.93 40.53 38.42 33.70 16.01 19.73 17.23 25.89 27.84 28.21 30.74 30.62 28.88 28.82 29.37 26.35 31.53 34.00 60.17 54.22 30.04 63.61 116.05 44.28 42.63 32.17 P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 880 Fig. 7. Square wave voltammogram of DDS under optimum conditions for: (a) 11.8 mM; (b) tablet; (c) urine sample; (d) urine sample before DDS dosage. Table 4 Determination of DDS in dapsone tablet Volume (ml) Ep (mV) ip (mA) Experimental weight per tablet (mg) Theoretical weight per tablet (mg) Deviation (%) 0.5 1.0 1.5 2.0 1568 1555 1554 1574 35.40 66.61 96.84 127.39 109.0 109.1 109.1 109.1 110 110 110 110 0.91 0.82 0.82 0.82 with 6.5 ml of the supporting electrolyte solution and the pH was brought to 1.0. The squarewave stripping voltammogram was recorded under the optimum experimental conditions. The squarewave voltammogram was presented in Fig. 7. This experiment was repeated 5 times to get concordant value. There is no appreciable interference due to the presence of a small amount urine present in the electrolyte hence the same calibration plot can be used. By employing the calibration plot the amount of DDS present in 1.0 ml of the urine sample was calculated. Similar experiments were carried out with vary- ing amount of urine sample, 1.0, 1.5 and 2.0 ml. In all the cases the suitability of the procedure was verified. The results are presented in Table 5. There is no degradation of the analyte in solution during experiment. Only electrooxidation of the amino group of DDS is taking place. The other matters present in tablets and urine samples are not interfering with the study. This method is a stable method. Repetition rate is found to be high. Hence the proposed method can be used as a stable method like spectrophotometric or chromotographic methods. P. Manisankar et al. / J. Pharm. Biomed. Anal. 26 (2001) 873–881 881 Table 5 Determination of DDS in urine sample Volume (ml) Ep (mV) ip (mA) Concentration (mg/ml) Experimental weight per ml of urine (mg) 0.5 1.0 1.5 2.0 1551 1556 1560 1564 9.26 23.51 27.64 37.01 0.1531 0.3067 0.4569 0.6117 0.3061 0.3067 0.3046 0.3059 6. Conclusion References DDS is anodically oxidized irreversibly on glassy carbon electrode in the potential range of 0.10–0.18 V at all pH conditions. The oxidation is diffusion-controlled adsorption and loss of one electron is observed. Effect of pH leads to the conclusion that pH 1.0 is suitable for analytical studies. The adsorptive stripping voltammetric studies are carried out by employing Osteryoung squarewave stripping voltammetry. Optimum conditions are arrived at. The concentration effect was studied and a calibration plot was obtained. This is taken as a standard and used to find out the unknown concentration of DDS present in the pharmaceutical tablet and urine samples. Dapsone is determined over the range of 0.5–20.0 mg/ml by HPLC [6]. Compared to this a better limit of determination of DDS between 0.0036 and 3.56 mg/ml is obtained in this stripping voltammetry method. This technique is simple and easy to carry out. Once the instrument is set, just by changing the analyte and polishing the electrode, within a few minutes, the amount of DDS can be determined. 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