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Electrochemical analysis of amlodipine in some pharmaceutical formulations and biological fluid using disposable pencil graphite electrode
Journal of Electroanalytical Chemistry 788 (2017) 7–13
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Electrochemical analysis of amlodipine in some pharmaceutical formulations and biological fluid using disposable pencil graphite electrode Nimisha Jadon ⁎, Rajeev Jain, Annu Pandey School of Studies in Environmental Chemistry, Jiwaji University, Gwalior 474011, India
a r t i c l e
i n f o
Article history: Received 2 December 2016 Received in revised form 23 January 2017 Accepted 26 January 2017 Available online 30 January 2017 Keywords: Amlodipine Square-wave voltammetry Differential pulse voltammetry Cyclic voltammetry Pencil graphite electrode
a b s t r a c t A reproducible and sensitive voltammetric procedure has been developed for the determination of amlodipine (AML) by using pencil graphite electrode (PGE) as working electrode. The experimental parameters, such as concentration, pH, amplitude, frequency, deposition potential were optimized and the peak potential was found to be 700 mV [vs. Ag/AgCl], by square-wave voltammetry (SWV) and differential pulse voltammetry (DPV). Under optimized conditions in Britton–Robinson buffer (pH 8.5), linear calibration curve was obtained in the range of 0.8 nM–51.2 nM solution. The limit of detection (LOD) and limit of quantification (LOQ) for SWV and DPV were calculated 0.02 pM, 0.04 pM, 0.06 pM and 0.2 pM respectively. The proposed method was used to estimate the amount of drug in different brands of pharmaceutical formulations with almost 99.9% recovery. Developed method was also applied for the detection of AML in spiked human serum with good recovery of almost 99% and limit of detection was found to be 0.21 pM. The proposed methodology is first attempt to develop a method on PGE which represents an effective, cheap, easily available and alternative tool instead of commonly used glassy carbon and other chemically modified electrodes for the electrochemical determination of amlodipine. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Amlodipine (Fig. 1) offers vital economic benefits in people suffering from heart diseases. It acts as calcium channel blocker. This drug dilates the blood vessels, thereby enhancing blood circulation. It is prescribed for patients with diabetes, if they get heart related ailments that require management using calcium channel blocker drugs. This is also using in combination with other drug for treating hypertension, angina, lower cholesterol. Its therapeutic importance and extensive use and benefits for saving the heart patient's life, need a requirement to develop a rapid and simple analytical technique for AML detection in pharmaceutical preparations and in biological fluids [1–5]. On reviewing the literature we found that several conventional techniques GC, LC, HPTLC and some spectrophotometric methods have been developed for the assay of AML in tablets and biological samples. Limitation of spectrophotometric over electrochemical method is that the differential degrades the signal-to-noise ratio. The stray light of UV–Vis spectrophotometer that caused by the faulty equipment design and other factors could influence spectra measurement accuracy of the absorption in substance. However, these methods are also dependent on many parameters, such time of the analysis, character of a compound or mixture of ⁎ Corresponding author. E-mail address: [email protected] (N. Jadon).
http://dx.doi.org/10.1016/j.jelechem.2017.01.055 1572-6657/© 2017 Elsevier B.V. All rights reserved.
compounds (extracts) and show a low sensitivity at low sample concentration over electrochemical methods. This method does not discriminate between the sample of interest and contaminants that absorb at the same wavelength [6–7]. These methods have advantage of high degree of selectivity but need sample clean-up, expensive instrumentation and a highly skilled person. A highly-sensitive, convenient and effective tool for the analysis of important biomolecules including drugs in pharmaceutical formulations and human body fluids are the electrochemical techniques, these techniques have attracted more attention nowadays due to their distinctive nature and owing to their simplicity, low cost and less time consuming as compared to the other reported analytical techniques. Some electrochemical methods have been reported for the determination of AML on different types of electrode such as carbon paste, glassy carbon, diamond electrode and gold electrode. Carbonbased materials such as glassy carbon or carbon nano-tubes or mercury electrode have also been utilized for voltammetric analysis of AML [8– 16]. Demerit of unmodified bare electrodes is sluggish electron transfer and fouling of surface which reduces its sensitivity and selectivity. Different types of electrode modifiers have been applied to overcome these demerits such as a multi-wall carbon nanotubes modified glassy carbon electrode (GCE) [17–23], copper nanoparticles modified carbon paste electrode [14], fullerene-C60-modified GCE [13], polyvinylpyrrolidone modified carbon paste electrode [15,39], ruthenium hexachloroplatinate or hexacyanocobaltate film coated GCE [12] and
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2.3. Preparation of pencil graphite electrode (PGE) The pencil graphite electrode was handmade. It was prepared by using insulin syringe as a holder and bontite for fixing the lead into syringe. HB pencil lead of diameter of 2.0 mm, was used. Prepared PGE was cleaned by rubbing it on butter paper and was pretreated before measurement. 2.4. Analytical procedure Fig. 1. Structure of amlodipine.
MWCNT/iron-doped polypyrrole modified GCE [11] with high sensitivity and selectivity for the quantification amlodipine content in pharmaceutical formulation. In this study, PGE was used for sensitive voltammetric determination of AML without applying any modifier. Earlier it was reported that pencil lead electrodes offer a renewal surface which is simpler and faster than polishing procedures, common with solid electrodes, and result in good reproducibility for individual surface [18]. Pencil graphite electrode (PGE) is a new type of carbon electrode which has been used for the determination of a few varieties of analytes by voltammetric techniques [16,40]. Electro-analysis of AML on edge plane pyrolytic graphite electrode has been reported by Goyal et al. but the detection limit is in micromolar range [40]. PGE has several advantages compared to other carbon-based electrode such as low cost, no need for time-consuming processes like surface polishing and disposability. The surface can be modified easily, has high electrochemical reactivity and surface area. PGE can be also used for stripping voltammetric analysis instead of mercury-based electrodes [24–31]. PGE nowadays could be a better replacement of other costly carbon based electrodes for the detection of organic molecules. This paper is the first systematic report of AML quantification on PGE up to pM level.
Differential pulse voltammetry (DPV), square-wave voltammetry (SWV) and cyclic voltammetry (CV) methods were applied for the investigation of electrochemical behaviour and quantification of drug solution. Voltammograms were obtained by addition of different volumes of prepared working drug solution into the electrochemical cell containing 8.0 mL of BR buffer of desired pH (pH 8.5), 1.0 mL methanol and 1.0 mL 1 M KCl as supporting electrolyte. 2.5. Procedure for assay in pharmaceutical formulation Commercial samples of pharmaceutical formulations with declared AML content were purchased from a local pharmacy. Each tablet contains a dose of 5.0 mg and 2.5 mg AML according to the information. The stock solutions of samples of the tablets were prepared as follows: five tablets were accurately weighed and powdered in mortar. A portion of powdered tablet equivalent to the average weight of one tablet was dissolved into 5.0 mL methanol followed by sonication for 15 min in a 10.0 mL volumetric flask then solution was made up to the mark using methanol to get final concentration. Aliquots of this solution were diluted to final volume of 10.0 mL was completed with buffer and supporting electrolyte and then transferred into a voltammetric cell and voltammograms were recorded. 2.6. Procedure for assay in spiked human serum
2. Materials and methods 2.1. Reagents and solutions All reagents used like boric acid, acetic acid, orthophosphoric acid, sodium hydroxide, and methanol were of analytical grade quality (Merck and Sigma) and were used without further purification. Amlodipine (as besylate, assay: 98.63%) standard was obtained from Cipla Limited, Chandigarh, India. Different pharamaceuticals tablets containing amlodipine (Asomex-2.5, Amlovas-5, Amlodac-5, Amlogard) by different manufacturers were purchased from local market. Ultrapurified water supplied by a Milli-Q system (Millipore) with resistivity greater than 18.0 Ω cm was used for the preparation of solutions. Pencil lead, insulin syringe and bontite for making electrode were also purchased from local market. Stock solution of 1.0 mM concentration was daily prepared in methanol and working solutions were prepared by further dilution of this stock solution. 1.0 M KCl solution was used as supporting electrolyte for all experiments.
2.2. Instrumentation Electrochemical experiments were carried out by Autolab type III (potentiostat-galvanostat with 757VA Computrace software). Pencil graphite electrode (PGE) as working electrode, Ag/AgCl as reference electrode, and a graphite rod as auxiliary electrode were employed. All the measurements were performed under nitrogen purging for 5 min at room temperature. pH measurement of buffers was carried out on a pH meter fitted with a glass electrode.
1.0 mL of human serum was transferred into a centrifugation tube. Aliquot of AML stock solution was added to get the final concentration. It was mixed well using a vortex mixer and then 0.5 mL methanol was added as precipitating agent after that it was centrifuged then contents of the centrifugation tube was transferred quantitatively into the voltammetric cell and voltammograms were recorded for AML. Values of the peak current (ip) vs. the corresponding concentration were plotted to obtain the calibration graph and LOD, LOQ were also calculated. 3. Results and discussion 3.1. Characterization of PGE EIS is a beneficial technique to get information about properties of surface conductivity of electrodes. Conductivity of PGE was taken using the 3.0 mM potassium ferricyanide prepared in phosphate buffer of pH 7.0 (Fig. 2). Electron charge transfer resistance (Rct) was calculated for by using the Randles-Sevcik equation. The Rct value was found for bare PGE as 476.56 Ω, which shows better electrical conductivity and has a faster electron transfer rate on bare PGE. Surface area was also calculated by cyclic voltammograms of 1.0 mM potassium ferricyanide at different scan rate and was found to be 0.165 cm2 using equation given below. SEM images of PGE surface shows regular graphite layer with little rough surface. 1=2 ip ¼ 0:4463 F3 =RT An3=2 DR 1=2 C0 ʋ 1=2
N. Jadon et al. / Journal of Electroanalytical Chemistry 788 (2017) 7–13
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Similarly a linear relationship was obtained in plot of Ep versus ln ʋ (Fig. 3, Inset B). The dependence of Ep can be expressed by equation below: Ep =mV ¼ 0:051
ln ʋ =mV s−1 þ 0:865
ð2Þ
with correlation coefficient of 0.992. Above both result confirmed that oxidation of AML on PGE is irreversible in nature and the overall process is a diffusion controlled process. Peak potential of electrode process is calculated by the equation given below: Ep ¼ E0 þ
ðRTÞ ðRTÞk ðRTÞ þ ln þ logυ αnF αnF αnF
where, R, T, F has its usual meaning and α is transfer coefficient and number of electron transfer in oxidation process was calculated by using slope of this equation and value of α was 0.302 and hence calculated value of n was 1.548, so number of electron transfer is during the oxidation process is (n) 2. Proposed mechanism of electron transfer is shown in Scheme 1.
Fig. 2. EIS spectrum of bare PGE electrode.
3.3. Differential pulse voltammetry and square wave voltammetry 3.2. Cyclic voltammetry Reversibility of drug was observed through cyclic voltammetric technique. Response was recorded for 1.0 μM AML in BR buffer (pH 8.5) at scan rate of 100 mV/s and well-defined anodic peak at 750 mV with high peak current value (20 μA) indicated oxidative nature of AML while absence of reduction peak on reverse sweep indicated that it is irreversibly oxidized at PGE. The scan rate was varied in the range 50 to 700 mV/s at optimized pH on PGE by cyclic voltammetry. On plotting a graph, a linear relationship was found between peak current and scan rate i.e. on increasing scan rate peak current also increases (Fig. 3, Inset A) and can be explained by following equation with correlation coefficient of 0.99 ×. The linear relation between log ʋ and log i can be represented by Eq. (1). Y ip ¼ 0:302 ðmVÞ þ 1:553 ðμAÞ
ð1Þ
Under optimized experimental conditions the electrochemical behaviour of amlodipine was studied by differential pulse voltammetry and square wave voltammetry using bare PGE. Since these techniques are more sensitive than cyclic voltammetry, further analysis was performed utilizing DPV and SWV. Results on PGE and GCE were compared and are shown in Fig. 4 and it can be easily concluded that peak current is much higher on PGE than GCE. 3.4. Optimization of operational parameters The electrochemical behaviour of drug was examined in various buffer systems like Britton Robinson buffer, acetate buffer and phosphate buffer but best results were obtained in BR buffer from pH 2 to 11. Influence of various pH on the peak current and potential of AML was investigated by SWV and DPV techniques. Maximum peak current value was found at pH 8.5. On going towards
Fig. 3. Voltammogram showing effect of increase in scan rate on (A) peak current and (B) peak potential.
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Scheme 1. Mechanism reaction showing transfer of electron during the oxidation process.
more basicity shifting in the peak potential to less positive values (Fig. 5) indicating protonation during electrode process. The slope of the linear Ep and pH equation was found to be 0.002 [41–
42]. The linear relationship between the peak potential (E p ) and pH using PGE can be explained by equation: Ep ðVÞ ¼ −0:002 þ 0:698 pH; R2 0:998
ð3Þ
Effect of deposition potential was examined in the range of 0.0 to 0.9 V. The peak current was highly affected by increasing the deposition potential, best results were observed in the range of 0.6 to 0.9 V. The optimal deposition potential was fixed at 0.9 V. Effect of variation of frequency on peak current was examined over range of 10–70 Hz in BR buffer (pH 8.5). A linear relationship was observed between the peak current and frequency up to 70 Hz, and best frequency was found to be 50 Hz without any distortion in peak of AML with higher sensitivity. Other parameters like deposition time of 0.0 s and pulse amplitude of 50 mV was best selected for further all measurements.
Fig. 4. Differential pulse voltammograms of 22.4 nM AML in BR buffer at pH: 8.5 containing 1.0 M KCl at PGE and GCE at optimized conditions pulse amplitude: 50 mV, pulse time: 0.04 s and sweep rate: 0.0175 V/s.
Fig. 5. Effect of change in pH on peak potential.
N. Jadon et al. / Journal of Electroanalytical Chemistry 788 (2017) 7–13
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Fig. 6. Voltammograms with calibration graph for the dependence of the (a) DPV (b) SWV current for AML at different concentrations range (0.8–51.2 nM) and (2.4–89.6 nM) in BR buffer pH 8.5, Eacc = 0.9 V, frequency (f) = 50 Hz.
4. Validation of the proposed method The method was validated for linearity, LOD, LOQ, accuracy, precision, reproducibility, repeatability and stock stability by examining the peak current value as a function of concentration of drug solution for 3 consecutive days under the optimized parameter. Fig. 6 shows the voltammograms for the oxidation peak of AML at a linear concentration range of 0.8–52.2 nM for SWV and 2.1–89.6 nM for DPV. LOD and LOQ were found to be 0.02 pM and 0.04 pM for SWV and for DPV it was calculated as 0.06 pM and 0.2 pM. The inter-day and intra-day repeatability studies were performed. The repeatability was examined by performing 3 replicate measurements of recovery studies for 44.2 nM drug followed pre-concentration under the same operational conditions. The relative standard deviation was found to 0.7 and 0.3 respectively with good recovery of 99%. Reproducibility was obtained by measuring the peak current response of fresh solution for 5 replicates which were similar after 5 continuous potential scan and compared to original oxidation current value which suggest good reproducibility of about 99% for proposed methodology. Accuracy and precision were examined by calculating recovery % RSD which was indicated in Table 1. Low value of % RSD shows good precision and accuracy of method. Stability of stock solution was examined by recording the voltammogram of a fixed concentration of drug solution daily, over a period of one month and obtained response showed the adequate stability of stock solution.
Voltammograms were recorded for different concentration of drug solution in a linear range of 2.4–89.6 nM in BR buffer pH 8.5. Results of different manufacturers have been compiled in the Table 1 with their % recovery and % RSD. The percentage recovery of AML based on the average of five replicate measurements of different manufacturer of AML was found 99.9% for. The RSD calculated for each brand was nearly 1. The small values for % RSD shows high precision of the method. The good linearity of the calibration graph can be seen by the correlation coefficients. The precision was estimated for 2.4–89.6 nM of the drug using the calibration graph and standard addition method (Fig. 7).
4.1. Pharmaceuticals assay application The applicability of method was verified for determination of AML in dosage forms, from different manufacturer available in market. Table 1 Analysis of pharmaceutical samples containing AML using PGE. Sample
Manufacturer Claimed amount (mg/tablet)
Detecteda amount (mg/tablet)
%R
%RSD
Amlovas-5 Amlodac-5 Amlogard Asomex-2.5
Macleods Zydus Pfizer Emacure
4.99 4.92 4.96 2.48
99.9 99.3 99.4 99.2
0.8 0.8 1.5 0.7
5 5 5 2.5
± ± ± ±
0.08 0.06 0.07 0.06
a Mean of five determinations: Calculated as at the 95% confidence level; theoretical value t = 2.31.
Fig. 7. Voltammograms and calibration graph for AML tablets at different concentrations range (2.4–89.6 nM) in BR buffer pH 8.5, deposition potential = 0.9 V.
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N. Jadon et al. / Journal of Electroanalytical Chemistry 788 (2017) 7–13 Table 3 Comparison of the sensitivity of the developed method with earlier reported voltammetric methods for determination of AML. Electrode Buffer
Ep (V)
Year
Ref
GCE GPE AuE
+0.50 0.017–0.52 μM 0.009 μM −0.39 4.3–170 μM 1.7 μM +0.75 24–34 μM 4.2 μM
2012 2013 2012
[37] [38] [12]
+0.87 8.1–41 μM
12 μM
2002
[34]
0.80 μM 0.001 μM 0.31 μM 0.07 μM 0.003 nM 0.02, 0.04 (pM)
2009 2010 2014 2014 2016 This work
[35] [40] [36] [33] [32]
GCE GCE EPPGE GCE BDDE GPE PGE
BRB, pH 13 BRB, pH 6 PBSB, pH 11 PBSB, pH 5.5 BRB, pH 5 PBS, pH 7.2 BRB, pH 5 BRB, pH 5 BRB, pH 5 BRB, pH 8.5
+0.82 +0.45 +0.95 +0.75 +0.56 +0.70
LCR
4–100 μM 0.005–1 μM 1–35 μM 0.2–6 μM 0.01–1.0 μM 0.8–51.2 (nM)
LOD
Abbreviations: AuE: gold electrode, PGE: pencil graphite electrode, BRB: Britton–Robinson buffer solution, Ep: peak potential, EPPGE: edge plane pyrolytic graphite electrode, GCE: glassy carbon electrode, GPE: graphite paste electrode, LCR: linear concentration range, LOD: limit of detection, PBS: phosphate buffer solution, SWV: square-wave voltammetry.
Fig. 8. Voltammograms for AML in spiked human serum at different concentrations range (2.0–256.0 nM) in BR buffer pH 8.5, deposition potential = 0.9 V.
procedure was applied for the determination of AML in pharmaceutical formulation and human serum with LOD 0.2 pM and LOQ 0.6 pM respectively. Due to the simplicity in procedure, stability and less expensive. Results of this study are more sensitive (up to pM level) and precise in comparison with already reported voltammetric methods (Table.3). The developed technique is an effective electrochemical study of analyte without any need of complex sample preparation and separation as required for the chromatographic and spectrophotometric methods of analysis.
4.2. Human serum assay application
Acknowledgements
Voltammograms were recorded for extracted AML from serum spiked by different concentrations. The peak currents were found to be linearly related to concentration over the range 2.0 nM–256.0 nM (Fig. 8) according to the equation:
This work was supported by Department of Science and Technology, New Delhi, India, by providing INSPIRE fellowship to Annu Pandey [IF150764].
ip ðμAÞ ¼ 0:227 þ 5:486C ðnMÞ; R2 ¼ 0:996:
ð4Þ
The percentage recovery of AML in human serum, based on the average of five replicate measurements, is 99.5% (Table 2). The percentage recovery of AML was determined by comparing the peak currents of known drug concentrations in human serum with their equivalents on the calibration curve. The estimated LOD and LOQ in spiked human serum were found to be 0.2 pM mL−1 and 0.6 pM mL−1 respectively. 5. Conclusion This study is first attempt of development and application of disposable pencil graphite voltammetric sensor for quantification of AML. A good linear response was observed in concentration range from 0.8 nM to 51.2 nM with a low limit of detection (0.02 pM), and quantification (0.06 pM) with a good repeatability. AML in commercial pharmaceutical tablets was also determined. Validated voltammetric
Table 2 Analysis of spiked human serum containing AML using PGE. S. no.
1 2 3 4
Spiked human serum Concentration added (nM)
Concentration found (nM)a
%R
%RSD
2.0 4.0 8.0 16.0
1.99 ± 0.04 3.96 ± 0.05 7.95 ± 0.04 15.87 ± 0.1
99.5 99.2 99.4 99.2
0.2 0.1 0.2 0.1
a Mean of five determinations: Calculated as at the 95% confidence level; theoretical value t = 2.31.
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Electrochemical analysis of amlodipine in some pharmaceutical formulations and biological fluid using disposable pencil graphite electrode Nimisha Jadon ⁎, Rajeev Jain, Annu Pandey School of Studies in Environmental Chemistry, Jiwaji University, Gwalior 474011, India
a r t i c l e
i n f o
Article history: Received 2 December 2016 Received in revised form 23 January 2017 Accepted 26 January 2017 Available online 30 January 2017 Keywords: Amlodipine Square-wave voltammetry Differential pulse voltammetry Cyclic voltammetry Pencil graphite electrode
a b s t r a c t A reproducible and sensitive voltammetric procedure has been developed for the determination of amlodipine (AML) by using pencil graphite electrode (PGE) as working electrode. The experimental parameters, such as concentration, pH, amplitude, frequency, deposition potential were optimized and the peak potential was found to be 700 mV [vs. Ag/AgCl], by square-wave voltammetry (SWV) and differential pulse voltammetry (DPV). Under optimized conditions in Britton–Robinson buffer (pH 8.5), linear calibration curve was obtained in the range of 0.8 nM–51.2 nM solution. The limit of detection (LOD) and limit of quantification (LOQ) for SWV and DPV were calculated 0.02 pM, 0.04 pM, 0.06 pM and 0.2 pM respectively. The proposed method was used to estimate the amount of drug in different brands of pharmaceutical formulations with almost 99.9% recovery. Developed method was also applied for the detection of AML in spiked human serum with good recovery of almost 99% and limit of detection was found to be 0.21 pM. The proposed methodology is first attempt to develop a method on PGE which represents an effective, cheap, easily available and alternative tool instead of commonly used glassy carbon and other chemically modified electrodes for the electrochemical determination of amlodipine. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Amlodipine (Fig. 1) offers vital economic benefits in people suffering from heart diseases. It acts as calcium channel blocker. This drug dilates the blood vessels, thereby enhancing blood circulation. It is prescribed for patients with diabetes, if they get heart related ailments that require management using calcium channel blocker drugs. This is also using in combination with other drug for treating hypertension, angina, lower cholesterol. Its therapeutic importance and extensive use and benefits for saving the heart patient's life, need a requirement to develop a rapid and simple analytical technique for AML detection in pharmaceutical preparations and in biological fluids [1–5]. On reviewing the literature we found that several conventional techniques GC, LC, HPTLC and some spectrophotometric methods have been developed for the assay of AML in tablets and biological samples. Limitation of spectrophotometric over electrochemical method is that the differential degrades the signal-to-noise ratio. The stray light of UV–Vis spectrophotometer that caused by the faulty equipment design and other factors could influence spectra measurement accuracy of the absorption in substance. However, these methods are also dependent on many parameters, such time of the analysis, character of a compound or mixture of ⁎ Corresponding author. E-mail address: [email protected] (N. Jadon).
http://dx.doi.org/10.1016/j.jelechem.2017.01.055 1572-6657/© 2017 Elsevier B.V. All rights reserved.
compounds (extracts) and show a low sensitivity at low sample concentration over electrochemical methods. This method does not discriminate between the sample of interest and contaminants that absorb at the same wavelength [6–7]. These methods have advantage of high degree of selectivity but need sample clean-up, expensive instrumentation and a highly skilled person. A highly-sensitive, convenient and effective tool for the analysis of important biomolecules including drugs in pharmaceutical formulations and human body fluids are the electrochemical techniques, these techniques have attracted more attention nowadays due to their distinctive nature and owing to their simplicity, low cost and less time consuming as compared to the other reported analytical techniques. Some electrochemical methods have been reported for the determination of AML on different types of electrode such as carbon paste, glassy carbon, diamond electrode and gold electrode. Carbonbased materials such as glassy carbon or carbon nano-tubes or mercury electrode have also been utilized for voltammetric analysis of AML [8– 16]. Demerit of unmodified bare electrodes is sluggish electron transfer and fouling of surface which reduces its sensitivity and selectivity. Different types of electrode modifiers have been applied to overcome these demerits such as a multi-wall carbon nanotubes modified glassy carbon electrode (GCE) [17–23], copper nanoparticles modified carbon paste electrode [14], fullerene-C60-modified GCE [13], polyvinylpyrrolidone modified carbon paste electrode [15,39], ruthenium hexachloroplatinate or hexacyanocobaltate film coated GCE [12] and
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2.3. Preparation of pencil graphite electrode (PGE) The pencil graphite electrode was handmade. It was prepared by using insulin syringe as a holder and bontite for fixing the lead into syringe. HB pencil lead of diameter of 2.0 mm, was used. Prepared PGE was cleaned by rubbing it on butter paper and was pretreated before measurement. 2.4. Analytical procedure Fig. 1. Structure of amlodipine.
MWCNT/iron-doped polypyrrole modified GCE [11] with high sensitivity and selectivity for the quantification amlodipine content in pharmaceutical formulation. In this study, PGE was used for sensitive voltammetric determination of AML without applying any modifier. Earlier it was reported that pencil lead electrodes offer a renewal surface which is simpler and faster than polishing procedures, common with solid electrodes, and result in good reproducibility for individual surface [18]. Pencil graphite electrode (PGE) is a new type of carbon electrode which has been used for the determination of a few varieties of analytes by voltammetric techniques [16,40]. Electro-analysis of AML on edge plane pyrolytic graphite electrode has been reported by Goyal et al. but the detection limit is in micromolar range [40]. PGE has several advantages compared to other carbon-based electrode such as low cost, no need for time-consuming processes like surface polishing and disposability. The surface can be modified easily, has high electrochemical reactivity and surface area. PGE can be also used for stripping voltammetric analysis instead of mercury-based electrodes [24–31]. PGE nowadays could be a better replacement of other costly carbon based electrodes for the detection of organic molecules. This paper is the first systematic report of AML quantification on PGE up to pM level.
Differential pulse voltammetry (DPV), square-wave voltammetry (SWV) and cyclic voltammetry (CV) methods were applied for the investigation of electrochemical behaviour and quantification of drug solution. Voltammograms were obtained by addition of different volumes of prepared working drug solution into the electrochemical cell containing 8.0 mL of BR buffer of desired pH (pH 8.5), 1.0 mL methanol and 1.0 mL 1 M KCl as supporting electrolyte. 2.5. Procedure for assay in pharmaceutical formulation Commercial samples of pharmaceutical formulations with declared AML content were purchased from a local pharmacy. Each tablet contains a dose of 5.0 mg and 2.5 mg AML according to the information. The stock solutions of samples of the tablets were prepared as follows: five tablets were accurately weighed and powdered in mortar. A portion of powdered tablet equivalent to the average weight of one tablet was dissolved into 5.0 mL methanol followed by sonication for 15 min in a 10.0 mL volumetric flask then solution was made up to the mark using methanol to get final concentration. Aliquots of this solution were diluted to final volume of 10.0 mL was completed with buffer and supporting electrolyte and then transferred into a voltammetric cell and voltammograms were recorded. 2.6. Procedure for assay in spiked human serum
2. Materials and methods 2.1. Reagents and solutions All reagents used like boric acid, acetic acid, orthophosphoric acid, sodium hydroxide, and methanol were of analytical grade quality (Merck and Sigma) and were used without further purification. Amlodipine (as besylate, assay: 98.63%) standard was obtained from Cipla Limited, Chandigarh, India. Different pharamaceuticals tablets containing amlodipine (Asomex-2.5, Amlovas-5, Amlodac-5, Amlogard) by different manufacturers were purchased from local market. Ultrapurified water supplied by a Milli-Q system (Millipore) with resistivity greater than 18.0 Ω cm was used for the preparation of solutions. Pencil lead, insulin syringe and bontite for making electrode were also purchased from local market. Stock solution of 1.0 mM concentration was daily prepared in methanol and working solutions were prepared by further dilution of this stock solution. 1.0 M KCl solution was used as supporting electrolyte for all experiments.
2.2. Instrumentation Electrochemical experiments were carried out by Autolab type III (potentiostat-galvanostat with 757VA Computrace software). Pencil graphite electrode (PGE) as working electrode, Ag/AgCl as reference electrode, and a graphite rod as auxiliary electrode were employed. All the measurements were performed under nitrogen purging for 5 min at room temperature. pH measurement of buffers was carried out on a pH meter fitted with a glass electrode.
1.0 mL of human serum was transferred into a centrifugation tube. Aliquot of AML stock solution was added to get the final concentration. It was mixed well using a vortex mixer and then 0.5 mL methanol was added as precipitating agent after that it was centrifuged then contents of the centrifugation tube was transferred quantitatively into the voltammetric cell and voltammograms were recorded for AML. Values of the peak current (ip) vs. the corresponding concentration were plotted to obtain the calibration graph and LOD, LOQ were also calculated. 3. Results and discussion 3.1. Characterization of PGE EIS is a beneficial technique to get information about properties of surface conductivity of electrodes. Conductivity of PGE was taken using the 3.0 mM potassium ferricyanide prepared in phosphate buffer of pH 7.0 (Fig. 2). Electron charge transfer resistance (Rct) was calculated for by using the Randles-Sevcik equation. The Rct value was found for bare PGE as 476.56 Ω, which shows better electrical conductivity and has a faster electron transfer rate on bare PGE. Surface area was also calculated by cyclic voltammograms of 1.0 mM potassium ferricyanide at different scan rate and was found to be 0.165 cm2 using equation given below. SEM images of PGE surface shows regular graphite layer with little rough surface. 1=2 ip ¼ 0:4463 F3 =RT An3=2 DR 1=2 C0 ʋ 1=2
N. Jadon et al. / Journal of Electroanalytical Chemistry 788 (2017) 7–13
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Similarly a linear relationship was obtained in plot of Ep versus ln ʋ (Fig. 3, Inset B). The dependence of Ep can be expressed by equation below: Ep =mV ¼ 0:051
ln ʋ =mV s−1 þ 0:865
ð2Þ
with correlation coefficient of 0.992. Above both result confirmed that oxidation of AML on PGE is irreversible in nature and the overall process is a diffusion controlled process. Peak potential of electrode process is calculated by the equation given below: Ep ¼ E0 þ
ðRTÞ ðRTÞk ðRTÞ þ ln þ logυ αnF αnF αnF
where, R, T, F has its usual meaning and α is transfer coefficient and number of electron transfer in oxidation process was calculated by using slope of this equation and value of α was 0.302 and hence calculated value of n was 1.548, so number of electron transfer is during the oxidation process is (n) 2. Proposed mechanism of electron transfer is shown in Scheme 1.
Fig. 2. EIS spectrum of bare PGE electrode.
3.3. Differential pulse voltammetry and square wave voltammetry 3.2. Cyclic voltammetry Reversibility of drug was observed through cyclic voltammetric technique. Response was recorded for 1.0 μM AML in BR buffer (pH 8.5) at scan rate of 100 mV/s and well-defined anodic peak at 750 mV with high peak current value (20 μA) indicated oxidative nature of AML while absence of reduction peak on reverse sweep indicated that it is irreversibly oxidized at PGE. The scan rate was varied in the range 50 to 700 mV/s at optimized pH on PGE by cyclic voltammetry. On plotting a graph, a linear relationship was found between peak current and scan rate i.e. on increasing scan rate peak current also increases (Fig. 3, Inset A) and can be explained by following equation with correlation coefficient of 0.99 ×. The linear relation between log ʋ and log i can be represented by Eq. (1). Y ip ¼ 0:302 ðmVÞ þ 1:553 ðμAÞ
ð1Þ
Under optimized experimental conditions the electrochemical behaviour of amlodipine was studied by differential pulse voltammetry and square wave voltammetry using bare PGE. Since these techniques are more sensitive than cyclic voltammetry, further analysis was performed utilizing DPV and SWV. Results on PGE and GCE were compared and are shown in Fig. 4 and it can be easily concluded that peak current is much higher on PGE than GCE. 3.4. Optimization of operational parameters The electrochemical behaviour of drug was examined in various buffer systems like Britton Robinson buffer, acetate buffer and phosphate buffer but best results were obtained in BR buffer from pH 2 to 11. Influence of various pH on the peak current and potential of AML was investigated by SWV and DPV techniques. Maximum peak current value was found at pH 8.5. On going towards
Fig. 3. Voltammogram showing effect of increase in scan rate on (A) peak current and (B) peak potential.
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Scheme 1. Mechanism reaction showing transfer of electron during the oxidation process.
more basicity shifting in the peak potential to less positive values (Fig. 5) indicating protonation during electrode process. The slope of the linear Ep and pH equation was found to be 0.002 [41–
42]. The linear relationship between the peak potential (E p ) and pH using PGE can be explained by equation: Ep ðVÞ ¼ −0:002 þ 0:698 pH; R2 0:998
ð3Þ
Effect of deposition potential was examined in the range of 0.0 to 0.9 V. The peak current was highly affected by increasing the deposition potential, best results were observed in the range of 0.6 to 0.9 V. The optimal deposition potential was fixed at 0.9 V. Effect of variation of frequency on peak current was examined over range of 10–70 Hz in BR buffer (pH 8.5). A linear relationship was observed between the peak current and frequency up to 70 Hz, and best frequency was found to be 50 Hz without any distortion in peak of AML with higher sensitivity. Other parameters like deposition time of 0.0 s and pulse amplitude of 50 mV was best selected for further all measurements.
Fig. 4. Differential pulse voltammograms of 22.4 nM AML in BR buffer at pH: 8.5 containing 1.0 M KCl at PGE and GCE at optimized conditions pulse amplitude: 50 mV, pulse time: 0.04 s and sweep rate: 0.0175 V/s.
Fig. 5. Effect of change in pH on peak potential.
N. Jadon et al. / Journal of Electroanalytical Chemistry 788 (2017) 7–13
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Fig. 6. Voltammograms with calibration graph for the dependence of the (a) DPV (b) SWV current for AML at different concentrations range (0.8–51.2 nM) and (2.4–89.6 nM) in BR buffer pH 8.5, Eacc = 0.9 V, frequency (f) = 50 Hz.
4. Validation of the proposed method The method was validated for linearity, LOD, LOQ, accuracy, precision, reproducibility, repeatability and stock stability by examining the peak current value as a function of concentration of drug solution for 3 consecutive days under the optimized parameter. Fig. 6 shows the voltammograms for the oxidation peak of AML at a linear concentration range of 0.8–52.2 nM for SWV and 2.1–89.6 nM for DPV. LOD and LOQ were found to be 0.02 pM and 0.04 pM for SWV and for DPV it was calculated as 0.06 pM and 0.2 pM. The inter-day and intra-day repeatability studies were performed. The repeatability was examined by performing 3 replicate measurements of recovery studies for 44.2 nM drug followed pre-concentration under the same operational conditions. The relative standard deviation was found to 0.7 and 0.3 respectively with good recovery of 99%. Reproducibility was obtained by measuring the peak current response of fresh solution for 5 replicates which were similar after 5 continuous potential scan and compared to original oxidation current value which suggest good reproducibility of about 99% for proposed methodology. Accuracy and precision were examined by calculating recovery % RSD which was indicated in Table 1. Low value of % RSD shows good precision and accuracy of method. Stability of stock solution was examined by recording the voltammogram of a fixed concentration of drug solution daily, over a period of one month and obtained response showed the adequate stability of stock solution.
Voltammograms were recorded for different concentration of drug solution in a linear range of 2.4–89.6 nM in BR buffer pH 8.5. Results of different manufacturers have been compiled in the Table 1 with their % recovery and % RSD. The percentage recovery of AML based on the average of five replicate measurements of different manufacturer of AML was found 99.9% for. The RSD calculated for each brand was nearly 1. The small values for % RSD shows high precision of the method. The good linearity of the calibration graph can be seen by the correlation coefficients. The precision was estimated for 2.4–89.6 nM of the drug using the calibration graph and standard addition method (Fig. 7).
4.1. Pharmaceuticals assay application The applicability of method was verified for determination of AML in dosage forms, from different manufacturer available in market. Table 1 Analysis of pharmaceutical samples containing AML using PGE. Sample
Manufacturer Claimed amount (mg/tablet)
Detecteda amount (mg/tablet)
%R
%RSD
Amlovas-5 Amlodac-5 Amlogard Asomex-2.5
Macleods Zydus Pfizer Emacure
4.99 4.92 4.96 2.48
99.9 99.3 99.4 99.2
0.8 0.8 1.5 0.7
5 5 5 2.5
± ± ± ±
0.08 0.06 0.07 0.06
a Mean of five determinations: Calculated as at the 95% confidence level; theoretical value t = 2.31.
Fig. 7. Voltammograms and calibration graph for AML tablets at different concentrations range (2.4–89.6 nM) in BR buffer pH 8.5, deposition potential = 0.9 V.
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N. Jadon et al. / Journal of Electroanalytical Chemistry 788 (2017) 7–13 Table 3 Comparison of the sensitivity of the developed method with earlier reported voltammetric methods for determination of AML. Electrode Buffer
Ep (V)
Year
Ref
GCE GPE AuE
+0.50 0.017–0.52 μM 0.009 μM −0.39 4.3–170 μM 1.7 μM +0.75 24–34 μM 4.2 μM
2012 2013 2012
[37] [38] [12]
+0.87 8.1–41 μM
12 μM
2002
[34]
0.80 μM 0.001 μM 0.31 μM 0.07 μM 0.003 nM 0.02, 0.04 (pM)
2009 2010 2014 2014 2016 This work
[35] [40] [36] [33] [32]
GCE GCE EPPGE GCE BDDE GPE PGE
BRB, pH 13 BRB, pH 6 PBSB, pH 11 PBSB, pH 5.5 BRB, pH 5 PBS, pH 7.2 BRB, pH 5 BRB, pH 5 BRB, pH 5 BRB, pH 8.5
+0.82 +0.45 +0.95 +0.75 +0.56 +0.70
LCR
4–100 μM 0.005–1 μM 1–35 μM 0.2–6 μM 0.01–1.0 μM 0.8–51.2 (nM)
LOD
Abbreviations: AuE: gold electrode, PGE: pencil graphite electrode, BRB: Britton–Robinson buffer solution, Ep: peak potential, EPPGE: edge plane pyrolytic graphite electrode, GCE: glassy carbon electrode, GPE: graphite paste electrode, LCR: linear concentration range, LOD: limit of detection, PBS: phosphate buffer solution, SWV: square-wave voltammetry.
Fig. 8. Voltammograms for AML in spiked human serum at different concentrations range (2.0–256.0 nM) in BR buffer pH 8.5, deposition potential = 0.9 V.
procedure was applied for the determination of AML in pharmaceutical formulation and human serum with LOD 0.2 pM and LOQ 0.6 pM respectively. Due to the simplicity in procedure, stability and less expensive. Results of this study are more sensitive (up to pM level) and precise in comparison with already reported voltammetric methods (Table.3). The developed technique is an effective electrochemical study of analyte without any need of complex sample preparation and separation as required for the chromatographic and spectrophotometric methods of analysis.
4.2. Human serum assay application
Acknowledgements
Voltammograms were recorded for extracted AML from serum spiked by different concentrations. The peak currents were found to be linearly related to concentration over the range 2.0 nM–256.0 nM (Fig. 8) according to the equation:
This work was supported by Department of Science and Technology, New Delhi, India, by providing INSPIRE fellowship to Annu Pandey [IF150764].
ip ðμAÞ ¼ 0:227 þ 5:486C ðnMÞ; R2 ¼ 0:996:
ð4Þ
The percentage recovery of AML in human serum, based on the average of five replicate measurements, is 99.5% (Table 2). The percentage recovery of AML was determined by comparing the peak currents of known drug concentrations in human serum with their equivalents on the calibration curve. The estimated LOD and LOQ in spiked human serum were found to be 0.2 pM mL−1 and 0.6 pM mL−1 respectively. 5. Conclusion This study is first attempt of development and application of disposable pencil graphite voltammetric sensor for quantification of AML. A good linear response was observed in concentration range from 0.8 nM to 51.2 nM with a low limit of detection (0.02 pM), and quantification (0.06 pM) with a good repeatability. AML in commercial pharmaceutical tablets was also determined. Validated voltammetric
Table 2 Analysis of spiked human serum containing AML using PGE. S. no.
1 2 3 4
Spiked human serum Concentration added (nM)
Concentration found (nM)a
%R
%RSD
2.0 4.0 8.0 16.0
1.99 ± 0.04 3.96 ± 0.05 7.95 ± 0.04 15.87 ± 0.1
99.5 99.2 99.4 99.2
0.2 0.1 0.2 0.1
a Mean of five determinations: Calculated as at the 95% confidence level; theoretical value t = 2.31.
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