Simultaneous Voltammetric Determination of

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Journal of The Electrochemical Society, 164 (9) B482-B487 (2017) 0013-4651/2017/164(9)/B482/6/$37.00 © The Electrochemical Society

Simultaneous Voltammetric Determination of Acetaminophen and Its Fatal Counterpart Nimesulide by Gold Nano/L-Cysteine Modified Gold Electrode Shalini Menon and K. Girish Kumarz Department of Applied Chemistry, Cochin University of Science and Technology, Kochi-22, India A gold electrode (GE) modified with L-cysteine and gold nanoparticles (AuNPs) has been generated for computing the individual and simultaneous determination of nimesulide (NM) and acetaminophen (APAP). The electrochemical behavior of the pair of molecules has been inspected utilizing square wave voltammetry (SWV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Studies indicated that the oxidation of APAP and NM is expedited at AuNPs/L-cysteine GE giving well defined peaks for APAP and NM at 0.596 V and 1.072 V respectively in Britton-Robinson (B-R) buffer (pH 2). The assay enabled simultaneous determination of these drugs in the linear working range of 3.0 × 10−4 M–1.0 × 10−5 M with detection limits of 4.06 × 10−7 M and 6.49 × 10−6 M for APAP and NM respectively. The high selectivity and sensitivity of the proposed assay were illustrated by its active application in the ascertainment of both APAP and NM in pharmaceutical preparations. © 2017 The Electrochemical Society. [DOI: 10.1149/2.0181712jes] All rights reserved. Manuscript submitted April 11, 2017; revised manuscript received July 5, 2017. Published August 1, 2017.

Acetaminophen or N-acetyl-p-aminophenol is a common antipyretic and analgesic whereas nimesulide, (N-(4-nitro-2phenoxyphenyl)methanesulfonamide) is a non-steroidal antiinflammatory drug with anti-pyretic and analgesic properties.1–3 It is claimed that the combination of NM with APAP in pharmaceutical formulations has rapid onset and longer term of analgesic and antipyretic impacts than either medication individually.4 Therefore, combined tablets of NM and APAP were introduced by some companies at a ratio of 1:3 or 1:5.4 Even though these drugs have been projected as potent drugs for pain and fever; serious hepatic, renal and other adverse effects have been reported following their administration and chronic use.5 NM was never allowed to be used in US, Britain, Canada etc., but is available in more than 50 countries around the globe, including Greece, France, Belgium, Portugal, Switzerland, Thailand, Russia and Brazil.6 Therefore, it is quite essential to generate simple, sensitive, and precise techniques for the determination of APAP and NM in pharmaceutical formulations, as drug monitoring plays a significant role in drug quality control, which has a great impact on public health.7 Different techniques have been used for the individual as well as simultaneous determination of NM and APAP which include titrimetry, thin layer and liquid chromatography, optical methods etc.3,8 However, most of these techniques for simultaneous determination suffer from drawbacks like long analysis time, high costs, and the need for sample pre-treatment, making the assay unsuitable for routine analysis.3 There are reports1,2,7–9 for the individual voltammetric determination of NM and APAP and also for their simultaneous determination using amperometric technique4 but to our knowledge there are no reports to date about the use of voltammetric technique for the simultaneous determination of these analytes. The aim of this work was to develop a simple yet sensitive voltammetric sensor capable of determining NM and APAP individually as well as simultaneously. This was achieved through AuNPs/L-cysteine modified GE that showed improved electrochemical oxidation properties. The potential utilization of the developed sensor in the quantitative determination of NM and APAP in pharmaceutical formulations has also been demonstrated. Experimental Materials and measurements.—The reagents and solvents were of analytical grade and used without further purification. Solutions were made in Millipore water. NM and APAP were obtained as gift samples. Ascorbic acid (AA), uric acid (UA), citric acid (CA), sodium chloride (NaCl), potassium chloride (KCl), dextrose, potassium sulfate (K2 SO4 ), phosphoric acid, boric acid, acetic acid and sodium z

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hydroxide (NaOH) were purchased from s.d.fine Chemicals, India. Application studies were performed on commercially available pharmaceutical formulations. Electrochemical and impedance measurements were made on a CHI600C electrochemical analyzer (CH Instruments Inc. USA) integrated to a desktop computer. A three electrode set up with Ag/AgCl reference electrode, platinum wire auxiliary electrode and the GE (3 mm diameter) or AuNPs/L-cysteine GE as the working electrode was employed for the measurements. pH measurements were made using a Metrohm pH meter. Scanning electron microscopic (SEM) images were obtained on a JEOL 6390LV. Preparation of AuNPs/L-cysteine GE.—The GE was polished with 0.3 μm alumina slurry on a polishing pad, rinsed thoroughly with doubly distilled water, sonicated alternatively in ethanol and doubly distilled water for 5 minutes each, cycled between 0.0 and 1.5 V in 0.5 M H2 SO4 solution at a scan rate (v) of 0.1 Vs−1 until a stable cyclic voltammogram was obtained. GE was then immersed in 1 mg mL–1 L-cysteine solution for 24 hours and washed with doubly distilled water.10 AuNPs were deposited at a voltage of –0.2 V for 30 s on the L-cysteine modified GE by immersing in 2 mg mL–1 HAuCl4 solution in 0.05 M H2 SO4 , and washing with doubly distilled water.10 Thus the AuNPs/L-cysteine composite modified GE was obtained. Before voltammetric measurements, the modified electrode was cycled between –0.6 and 0.6 V (scan rate 0.1 Vs–1 ) in 0.04 M B-R buffer solution several times until reproducible response was achieved. Results and Discussion Characterization of AuNPs/L-cysteine composite modified GE.—The active surface area of the modified electrode was calculated using cyclic voltammetric studies with 2 mM K3 [Fe(CN)6 ] in 0.1 M KNO3 as probe at different scan rates. According to Randles Sevcik equation,11 the peak current (ip ) for a reversible electrochemical reaction for a species of concentration C involving n electrons, with diffusion coefficient D, is given by I p = 2.69 × 105 An 3/2 D 1/2 Cν1/2 Here n = 1, D = 7.60 × 10−6 cm2 s−1 and C is the concentration of K3 Fe(CN)6 which is 2 mM. From the slope of ip vs square root of scan rate (v1/2 ) relation, the effective surface area of the AuNPs/L-cysteine composite modified GE was calculated to be 0.08 cm2 which is about 1.6 times larger than the bare GE (0.05 cm2 ) (Fig. 1). SEM was used to study the surface morphology of bare and modified electrodes. SEM images of bare and modified electrodes are shown in Fig. 2. From the SEM images it is clear that the surface character of GE has changed upon modification with L-cysteine and AuNPs and these distribute almost homogeneously at the electrode

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Journal of The Electrochemical Society, 164 (9) B482-B487 (2017)

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Figure 1. Surface area study of a) bare GE and b) AuNPs/L-cysteine modified GE in 2 mM K3 Fe(CN)6 solution in 0.1 M KNO3 . Plot of oxidation peak current vs square root of scan rate for c) bare GE and d) AuNPs/L-cysteine modified GE.

surface. The particle diameter of AuNPs obtained from SEM images is of the order of 89–93 nanometers. AuNPs/L-cysteine modification provides increased number of catalytic sites for the oxidation of APAP and NM. Electrochemical impedance spectroscopic measurements were engaged to analyze the charge transfer resistance of bare and modified electrodes. The semicircle portion of the Nyquist plot corresponds to the parallel combination of charge transfer resistance and double layer capacitance resulting from electrode impedance.12 The results revealed semicircles of different diameters by both the electrodes, modified and unmodified. Furthermore, the revelation goes on to show that the area of the semicircle decreases upon immobilization of AuNPs on L-cysteine modified GE surface, which is indicative of the decrease in the charge transfer resistance upon modification

Figure 2. SEM images of a) bare GE b) L-cysteine modified GE c) AuNPs/Lcysteine composite GE.

with AuNPs/L-cysteine composite (Fig. 3). This decrease in charge transfer resistance suggests that the modified electrode can effectively increase the electron exchange rate between the electrode surface and the electrolyte solution,12 thereby offering a higher sensitivity for the determination of APAP and NM using AuNPs/L-cysteine modified GE. Electrochemical behavior of NM and APAP.—Square wave voltammetry (SWV) was employed to explore the electrochemical behavior of the two drugs on the bare and modified electrodes in a 0.04 M B-R buffer solution containing 1 × 10−4 M each of APAP and NM (Fig. 4). The electro oxidation of APAP and NM was found to be catalysed by AuNPs/L-cysteine composite on GE. In the bare GE, oxidation peaks for APAP and NM appear at 0.612 V and 1.168 V respectively against Ag/AgCl electrode in B-R buffer (pH 2) with peak currents 3.53 × 10−6 A and 6.64 × 10−6 A respectively whereas on AuNPs/L-cysteine modified GE the same appears at 0.596 V and 1.072 V with peak currents 6.95 × 10−6 A and 1.40 × 10−5 A

Figure 3. Nyquist plots for a) bare GE b) L-cysteine modified GE c) AuNPs/Lcysteine composite GE.


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Journal of The Electrochemical Society, 164 (9) B482-B487 (2017) of protons taking part in the oxidation of NM was calculated to be approximately 1.1,12 Mechanistic study by varying scan rate.—Linear sweep voltammograms (LSV) of APAP and NM (1.00 × 10−4 M) were noted at different scan rates to study the mechanism of electro-oxidation (Fig. 5). The oxidation peak currents for APAP and NM were found to vary linearly with v1/2 in the range 0.01–0.20 V s−1 and 0.01– 0.50 mV s−1 respectively which is indicative of a typical diffusion controlled process.14 For acetaminophen: I p (A) = −3.07 × 10−7 + 1.00 × 10−5 v 1/2 (V 1/2 s −1/2 ) (R 2 = 0.993) For nimesulide:

Figure 4. SWV of a) bare GE b) AuNPs/L-cysteine modified GE in a solution of 0.04 M B-R buffer solution containing 1.0 × 10−4 M each of APAP and NM.

respectively. Well characterized peaks with a peak potential difference (Ep ) of 0.476 V was obtained with SWV which is large enough to enable simultaneous determination of the two drugs without mutual interference. These results indicate that the AuNPs/L-cysteine composite on GE promotes the electrochemical oxidation of APAP and NM by significantly increasing the rate of electron transfer. Optimization of experimental conditions.—Electro-oxidation processes can be greatly influenced by the choice of supporting electrolyte. The electrochemical oxidation of APAP and NM was studied in 0.1 M concentration of various supporting electrolytes such as sulphuric acid, phosphoric acid, phosphate buffer, B-R buffer, acetate buffer, citrate buffer and NaOH. The lowest oxidation potential with maximum peak current was obtained with a 0.04 M B-R buffer solution and hence it was selected as the supporting electrolyte. Effect of pH of solution on the electrochemical response of APAP and NM were investigated in the pH range 2 to 9 in 0.04 M BR buffer solution. Simultaneous response of the analytes at lower oxidation potentials giving well defined peaks with maximum current was obtained only at lower pH and hence a 0.04 M B-R buffer solution with a pH of 2 was chosen. For individual determination of these drugs, B-R buffer solution with pH 2 for NM and pH 7 for APAP were found to be suitable. The peak potential (Ep ) of APAP showed a linear negative shift on increasing the pH of the solution. A slope of −0.0548 is obtained, which is approximately close to the theoretical value of −0.0576, suggesting that the electron transfer is associated with an equal number of electrons and protons during the electrochemical oxidation of APAP.12,13 From the equation dEp /dpH = 2.303 RTm/αFn (m is the number of protons taking part in the electrochemical reaction; n is the number of electrons, α is the charge transfer coefficient, and the other letters represent the accepted scientific terms); number

I p (A) = −1.07 × 10−5 + 9.52 × 10−5 v 1/2 (V 1/2 s −1/2 ) (R 2 = 0.987) Further, the plot of log Ip vs. log v was found to have slopes of 0.55 and 0.68 for APAP and NM respectively which are closer to the theoretical value of 0.50 for a diffusion controlled process.15 These results imply that the oxidation of APAP and NM at AuNPs/L-cysteine modified GE is controlled by the mass transport of these drugs from the bulk of the solution to the surface of the electrode. Peak potentials of APAP and NM shift to more positive values as the scan rate increases pointing to the chemical irreversibility of electrocatalytic oxidation process.16 In addition, it was observed that peak potential varied linearly with natural logarithm of scan rate and according to Laviron’s theory,17 the linear dependency between Ep and ln v confirms the irreversible nature of the processes. Using the slope obtained from the plot of Ep vs. ln v, the number of electrons involved in the oxidation process was calculated to be 2 each for APAP and NM. The plausible reaction ways for the electrooxidation of these two drugs in accordance with this data are shown in Scheme 1.

Scheme 1. Plausible reaction ways for electro-oxidation of a) APAP b) NM.

Figure 5. Overlay of LSV of a solution of 0.04 M B-R buffer containing 2.0 × 10−4 M of a) APAP b) NM on AuNPs/L-cysteine modified GE. Inset of each overlay shows the plot of peak current vs. square root of scan rate. Downloaded on 2017-08-02 to IP 128.122.230.148 address. Redistribution subject to ECS terms of use (see ecsdl.org/site/terms_use) unless CC License in place (see abstract).

Journal of The Electrochemical Society, 164 (9) B482-B487 (2017)

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Figure 6. Overlay of SWV obtained for the oxidation of a) 2.5 × 10−4 M to 1.0 × 10−6 M of APAP in 0.04 M B-R buffer (pH 7) b) 4.5 × 10−4 M to 1.0 × 10−5 M of NM in 0.04 M B-R buffer (pH 2). Inset shows calibration graph in the corresponding linear range.

Individual and simultaneous determination of APAP and NM.— Determination of APAP and NM individually.—On the basis of the above results, an analytical technique has been suggested for the determination of APAP and NM employing AuNPs/L-cysteine modified GE (Fig. 6). The optimized conditions were applied for finding the linear working range (LWR), limit of detection (LOD), linear regression equation (LRE), and regression coefficient (R2 ) (Table I). Determination of APAP and NM simultaneously.—APAP and NM coexist in various pharmaceutical formulations. The proposed strategy has therefore been utilized for the simultaneous determination of APAP and NM by employing the modified electrode. In this regard, two cases were considered. Firstly, the concentration of APAP was increased linearly keeping the concentration NM constant (Fig. 7a) and vice versa (Fig. 7b). Secondly, the simultaneous determinations of these analytes were carried out by increasing their concentrations concurrently (Fig. 8).

Factual outcomes for both the cases have been outlined in Table I. LOD and LWR attained in both of the above-mentioned cases are comparable with the instance when both the analytes have been studied independently. Therefore, it could be presumed that, by using the developed strategy, simultaneous determination of both APAP and NM is quite effective as their individual determinations. Selectivity of the assay.—In order to assess the selectivity of the technique toward simultaneous determination of APAP and NM, influence of foreign species in the analyses of these analytes was studied in a 0.04 M B-R buffer solution. The maximum concentration of foreign species that gave a relative error less than 5% in presence of both APAP and NM at a concentration of 2.0 × 10−5 M was considered as its tolerance limit. Glucose, UA, AA and CA are the most common compounds usually present with these drugs in biological fluids. The effect of certain salts viz., K2 SO4 , NaCl, KCl on the peak current of APAP and NM was also studied (Fig. 9). Results obtained indicate

Table I. Statistical parameters for the electrochemical determination of APAP and NM. Analyte

LWR (M)

LRE

a)Analytical data for individual analytes I p (A) = 1.84 × 10−7 + 0.03C(M) APAP 2.5 × 10−4 –1.0 × 10−6 NM 4.5 × 10−4 –1.0 × 10−5 I p (A) = 1.11 × 10−5 + 0.03C(M) b)Analytical data for APAP when the concentration of NM is kept constant (1 × 10−4 M) I p (A) = 7.80 × 10−7 + 0.93C(M) APAP 4.0 × 10−4 –2.0 × 10−7 c)Analytical data for NM when the concentration of APAP is kept constant (1 × 10−5 M) I p (A) = 9.36 × 10−6 + 0.04C(M) NM 4.5 × 10−4 –1.0 × 10−5 d)Analytical data for APAP and NM simultaneously I p (A) = 1.03 × 10−5 + 0.03C(M) APAP 3.0 × 10−4 –1.0 × 10−5 NM 3.0 × 10−4 –1.0 × 10−5 I p (A) = 7.13 × 10−8 + 0.08C(M)

R2

LOD (M)

0.9923 0.9948

4.90 × 10−7 4.52 × 10−7

0.9926

7.09 × 10−9

0.9861

1.98 × 10−6

0.9779 0.9903

4.06 × 10−7 6.49 × 10−6

Figure 7. Variation of peak current with concentration for the electro-oxidation of a) 4.0 × 10−4 - 2.0 × 10−7 M concentration of APAP in presence of 1 × 10−4 M of NM b) 4.5 × 10−4 - 1.0 × 10−5 M concentration of NM in presence of 1 × 10−5 M of APAP in 0.04 M B-R buffer (pH 2). Inset shows calibration graph in the corresponding linear range. Downloaded on 2017-08-02 to IP 128.122.230.148 address. Redistribution subject to ECS terms of use (see ecsdl.org/site/terms_use) unless CC License in place (see abstract).

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Journal of The Electrochemical Society, 164 (9) B482-B487 (2017) investigated for 2.0 × 10−5 M APAP and NM. The peak current response of APAP and NM was determined with 5 electrodes which were produced under the same conditions. The response peak intensity showed a relative standard deviation of 1.9% for APAP and 1.4% for NM confirming that the results are reproducible. To study the repeatability of the developed sensor, the experiments were executed in five replicates using the same sensor.

Figure 8. a) Overlay of SWV of electro-oxidation of a solution containing 3.0 × 10−4 M to 1.0 × 10−5 M each of APAP and NM in 0.04 M B-R buffer (pH 2) b) Inset shows the calibration graph.

Analytical application.—APAP and NM analyses in all pharmaceutical formulations have been carried out using standard addition method. The measure of APAP and NM acquired in these tablets using the proposed technique agree well with the label values. These molecules were simultaneously determined in tablets in the same mode as for the individual samples (Table II). The peaks appear at 0.588 V and 1.064 V corresponding to the oxidation of APAP and NM respectively. Hence individual as well as simultaneous determination of APAP and NM can be done in various matrices using the developed technique.

Conclusions that the signal change produced by species like AA, glucose, KCl, NaCl, K2 SO4 and CA was within the tolerance limit when present in the same concentration as that of the target analytes. A tenfold excess of glucose and KCl produced interference in the determination of APAP and NM respectively. UA interfered in the determination of NM even when present at same concentration whereas it did not produce any significant interference in the signal for APAP even at tenfold excess concentration. This shows that the determination of both these analytes in pharmaceutical preparations and biological samples at AuNPs/L-cysteine modified GE is not influenced greatly by the commonly interfering compounds found along with these drugs of concern. Reproducibility and repeatability of AuNPs/L-cysteine modified GE.—The reproducibility of the AuNPs/L-cysteine modified GE was

Employing AuNPs/L-cysteine as modifiers on GE, a unique sensor has been fabricated for the simultaneous determination of APAP and NM using square wave voltammetry. Well separated and well defined irreversible peaks were obtained for the electro-oxidation of APAP and NM, which enable their simultaneous determination. The sensitivity was improved significantly upon modification making the proposed method highly sensitive giving submicromolar detection limits for both the drugs. It also provides a barrier to interferences from the compounds usually found in biological fluids and pharmaceutical formulations. The sensor has been employed for the simultaneous as well as individual determination of both APAP and NM in pharmaceutical formulations and the results obtained are satisfactory, which suggests that gold nanoparticles/L-cysteine composite modified gold electrode can act as a promising sensor for the voltammetric determination of APAP and NM.

Figure 9. Effect of foreign species on the electcro-oxidation of APAP and NM (concentration of APAP and NM being 2.0 × 10−5 M). Table II. Determination of APAP and NM in pharmaceutical formulations. Declared (mg/tablet)

Found (mg/tablet)

S.D.

Sample

APAP

NM

APAP

NM

APAP

NM

WELSET-500 (Ranbaxy Laboratories Limited) NICIP (CIPLA Limited) Nimisul-Forte (Manish Pharma Lab)

500.00 500.00

100.00 100.00

502.45 497.16

99.75 97.97

2.51 1.54

0.96 0.93

S.D.- Standard deviation.


Journal of The Electrochemical Society, 164 (9) B482-B487 (2017) Acknowledgments Author Shalini Menon hereby acknowledges KSCSTE- Kerala State Council for Science Technology and Environment for financial aid in the form of Research Fellowship. This article does not contain any studies with human participants or animals performed by any of the authors. The authors declare that there is no conflict of interests regarding the publication of this paper. References 1. 2. 3. 4.

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