Electroanalytical Determination of Escitalopram Oxalate Using Nickel Nanoparticles Modified Carbon Paste Sensor

A sensitive voltammetric method was described for the determination of escitalopram oxalate based on electrocatalytic oxidation at nickel nanoparticles modified chloranil carbon paste sensor in Britton-Robinson buffer (pH range from 2 to 10). The modified electrode was characterized by scanning electron microscopy, electrochemical impedance and cyclic voltammetry. The investigation of electrochemical behavior of escitalopram oxalate was performed using cyclic voltammetry and differential pulse voltammetry. The anodic peak current showed a linear range from 1.0 × 10 to 7.0 × 10 mol L. The detection limit is below 2.0 × 10 mol L. The proposed method is rapid, economical, simple, precise and sensitive voltammetric method for the determination of escitalopram oxalate in bulk, dosage form and urine.


3. Apparatus
AEW2 electrochemical workstation with ECProg3 electrochemistry software (Sycopel, England) was used in this study.A platinum wire (BASi model MW-1032) and an Ag/AgCl/3 mol L -1 NaCl (BASi model MF-2063) were used as a counter electrode and reference electrode, respectively.The pH measurements were carried out utilizing a cyberscan 500 pH meter (EUTECH Instruments, USA).
EIS was performed using IM6e electrochemical workstation (Zahner-electrik GmbH, Germany).All diagrams were recorded by applying 10 mV sinusoidal potential within a frequency range from 100 kHz to 100 mHz.
Scanning electron microscopy (SEM) measurements were achieved by a JSM-6700F scanning electron microscope (Japan Electro Company, Japan).

4. Determination of ESC in Bulk Powder
The working, counter and reference electrodes were submerged in electrolytic cell containing 5 mL of BR buf-fer (pH 7.0).Aliquots of ESC (1.0 × 10 -3 mol L -1 ) were added, and then voltammetric analyses were carried out by using DPV method and the voltammograms were recorded at scan rate of rate of 20 mV s -1 , pulse amplitude of 50 mV and accumulation time of 100 s.

5. Determination of ESC in Tablets
Ten tablets were weighed and crushed to a fine powder using mortar and pestle, and then sufficient amount to prepare 1.0 × 10 -3 mol L -1 ESC was transferred into 100 mL volumetric flask already containing 60 mL of methanol.The flask was sonicated for about 15 min and completed to the volume with methanol.The solution was filtered to remove the insoluble excipients.ESC was determined by standard addition method.

6. Analysis of ESC in Urine
Urine sample obtained from a healthy person (50 mL) was stored in a refrigerator at 8.0 °C for one week.10 mL from urine sample was centrifuged for 10 min at 2000 rpm.The supernatant was filtered using 0.45 μm filter paper, and then diluted ten times with BR buffer of pH 7.0.Successive additions of ESC (1.0 × 10 -3 mol L -1 ) were added to the voltammetric cell containing diluted urine (5.0 mL) and DPV voltammograms were listed.The experiments were performed in compliance with the Helsinki Declaration of 1975, as revised in 2008.The institutional committees (NODCAR, Egypt) have approved these experiments.Informed consent was obtained from all participants.

1. Electrochemical Behavior of ESC
The pharmaceutical and biomedical analysis is among the most important branches of applied analytical chemistry.Analytical measurement procedures should have a critical role in drug analysis as well as in biological samples.DPV method has been developed for determination of ESC in the bulk, tablets and urine using nickel nanoparticles modified chloranil carbon paste sensor.
Fig. 1a presents the cyclic voltammograms of ESC (1.0 × 10 -4 mol L -1 ) in BR buffer of pH 7.0 at different working electrodes: CP, DDQCP, TCNQCP, TCNECP and CACP, exhibiting one well defined anodic peak with no peak on the reverse scan, suggesting the irreversibility of the electrode reaction.This anodic peak may be attributed to the oxidation of tertiary amine group which agree with the reported method 27

(Fig 1c).
Fig. 1b describes the effect of mediator type on the anodic current of ESC in BR buffer of pH 7.0.The anodic peak current values are in the following order: CACP (15.16 μA) < DDQCP (13.02 μA) < TCNECP (10.08 μA) < TCNQCP (9.29 μA) < CP (6.09 μA).CA increases the anodic peak current from 6.09 μA at CP to 15.16 μA at CACP and lowers the oxidation potential from 1.147 V at CP to 1.095 V at CACP, thus CA acts as an electrocatalytic mediator.

2. Effect of pH
Voltammetric behavior of ESC was studied in BR buffer over the pH range from 2 to 10 at CACP as shown in Fig. 2a.The peak current increases as the pH increases up to pH 7.0, after pH 7.0 the peak current decreases as the pH increases (Fig. 2b).Therefore, pH 7.0 was selected as a suitable supporting electrolyte because the peak current reaches its maximum value at this pH value.
Fig. 2c demonstrates that the peak potential varies linearly with pH over the pH values (2-10) with the linear regression equation of E(V) = 1.451-0.054pH, with a correlation coefficient (R) of 0.9937.The slope was found to be -54 mV/pH units, which is close to the theoretical value of -59 mV suggesting that the number of protons and transferred electrons involved in the oxidation mechanism is equal. 56

Effect of Ni(NO 3 ) 2 Concentration
The influence of Ni(NO 3 ) 2 concentration on response of fabricated sensor was investigated using different concentrations of Ni(NO 3 ) 2 (1.0, 2.0 and 3.0 × 10 -3 mol L -1 ) which were deposited at CACP at -1.0 V for different times (120, 180, 240 and 300 s).It was found that 1.0 × 10 -3 mol L -1 Ni(NO 3 ) 2 and 240 s are the optimum concentration and deposition time used to prepare the modified sensor (NiCACP) to give the best results for the determination of ESC.

4. Morphologies of Different Electrodes
Electronic Supplementary Information 1 (ESI 1) displays the significant differences in the surface structure

5. Electrochemical Behavior of ESC at NiCACP
ESI 2A presents the cyclic voltammograms of ESC (1.0 × 10 -4 mol L -1 ) at CP, CACP and NiCACP in BR buffer of pH 7.0.We note that the anodic oxidation peak has the highest current and the lowest potential values (25.03 μA, 0.980 V) in case of NiCACP in comparison with these values in case of CACP (15.16 μA, 1.095 V) and CP (6.09 μA, 1.147 V).NiCACP shows catalytic effect in the anodic oxidation of ESC.Therefore, it was selected to determine ESC in bulk, tablets and urine.
Scan rate (ν) effect on the the peak current (I) of ESC (1.0 × 10 -4 mol L -1 ) was carried out by immersing NiCACP in BR buffer of pH 7.0, and the cyclic voltammograms were recorded over the scan range of 10-250 mV s -1 .ESI 2B shows a linear relationship between log I and log ν as given by the following equation: log I = 0.28 + 0.56 log ν.The slope of 0.56 indicates a diffusion controlled process with some adsorption character. 57ccumulation time (T acc ) effect on the peak current was studied at open circuit condition at NiCACP in BR buffer of pH 7.0 containing 1.0 × 10 -4 mol L -1 ESC.It was found that the peak current increases as the accumulation time increases up to 100 s and then it decreases as T acc increases.100 s was selected as the optimum accumulation time in the determination of ESC (ESI 2C).
The electron transfer coefficient (α) can be calculated using the following equation: α = 47.7/(Ep -E p/2 ) mV, 58 where E p is the peak potential and E p/2 is the potential where the current is at half peak value.α was calculated to be 0.48.

6. Electrochemical Impedance Spectroscopy Study
Nyquist plots of ESC using NiCACP and CACP exhibit the difference in the presence of metallic nickel nanoparticles as shown in Fig. 3a.
A simple equivalent circuit model (Fig. 3b) was used to fit the results.R s is the solution resistance and R p is the polarization resistance.Q represents the constant phase element (CPE) of capacitance for the film, n is its corresponding empirical exponents, C f is the capacitance of the double layer and W is the Warburg impedance due to diffusion (Table 1).The capacitance value for NiCACP is relatively higher than CACP in terms of C f and Q denoting the increase of ionic adsorption at the electrode/electrolyte interface for NiCACP.Moreover, the decrease in the R p is attributed to the selective interaction between nickel nanoparticles and ESC that resulted in the increase of the current in the electro-oxidation process.
NiCACP stability was studied in BR buffer of pH 7.0 containing 1.0 × 10 -4 mol L -1 ESC as a function of immersion time (Fig. 3c).The results show good stability till 12 h, thus NiCACP works well.

7. Determination of ESC in Bulk Powder
DPV method was applied for quantitative analysis of ESC in 5.0 mL of BR buffer (pH 7.0) at NiCACP.Successive additions from ESC solution (1.0 × 10 -3 mol L -1 ) were introduced into the electrolytic cell and the voltammograms were recorded, giving linearity over the concentration range of 1.0 × 10 -6 -7.0 × 10 -5 mol L -1 (0.414 -29.01 μg mL -1 ) as shown in Fig. 4. The validation of the method was performed according to the International Conference on Harmonization (ICH) guideline, 63 through the evaluation of limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, ruggedness and robustness.The LOD and LOQ were found to be 1.98 × 10 -7 mol L -1 and 6.60 × 10 -7 mol L -1 , respectively.The relative standard deviation (RSD) and the percentage recovery values were found in the following ranges: 0.33-0.77%and 99.91-101.35%,respectively.
The robustness of the method was examined by testing the influence of small variations from the optimum conditions: pH (7.00 ± 0.20), scan rate (20 ± 2.00) and accumulation time (100 ± 5.00) on the peak current of ESC (1.00 × 10 -5 mol L -1 ).The RSD values were 0.35%, 0.58% and 0.42% for pH, scan rate and accumulation time, respectively, indicating the robustness of the proposed method.

8. Interference Study
Some interfering species (1.00 × 10 -3 mol L -1 ) such as inorganic cations (Na + , K + , and Ca 2+ ), sugars (glucose and dextrose) and amino acid (valine and alanine) were used to study their interference with ESC (1.00 × 10 -3 mol L -1 ).There is no interference between these species and ESC; NiCACP shows good selectivity for the determination of ESC.

9. Analysis of ESC in Tablets
Standard addition method was successfully applied to determine ESC in Cipralex tablets at NiCACP without any pretreatment or time consuming extraction steps prior to analysis.The mean recovery and mean RSD values for five replicate measurements were 100.58% and 1.18%, respectively.The results listed in Table 2 show there is no interference between ESC and the excipients suggesting the selectivity and the sensitivity of the proposed method in the determination of ESC in dosage forms.

10. Analysis of ESC in Urine
The proposed method was used to determine ESC in urine samples (ESI3) in concentration range of 4.00 ×   10 -6 -6.00 × 10 -5 mol L -1 with correlation coefficient of 0.9998, the LOD and LOQ were 7.57 × 10 -7 mol L -1 and 2.52 × 10 -6 mol L -1 , respectively.Four different concentrations (8.00 × 10 -6 , 2.20 × 10 -5 , 3.60 × 10 -5 , and 4.40 × 10 -5 mol L -1 ) were chosen to be repeated five times to evaluate the accuracy and precision of the method.The RSD and the percentage recovery values were in the following ranges: 0.41-0.89%and 99.38-101.94%,respectively.The proposed method is more sensitive than chromatographic method used to determine ESC in urine (22.80 × 10 -5 mol L -1 ). 64The proposed method is less sensitive than capillary electrophoresis method (1.496 × 10 -9 -1.61 × 10 -6 mol L -1 ) but our method is more simple, cheap and it is used to determine ESC in urine without any extraction steps or pretreatment. 24

Conclusion
It is important to determine drugs at higher sensitivity than in the reported methods, therefore it was our intention to develop a precise and sensitive electroanalytical voltammetric method for the determination of ESC.The use of chloranil as modifier and Ni nanoparticles increases the active sites at the electrode surface which increases the sensitivity toward ESC.The proposed method is more sensitive than some reported methods as mentioned before in the text, thus it is an excellent means for determination of ESC in quality control because of its low cost, accuracy, selectivity and enforcement.The proposed method can be applied in clinical laboratories and pharmacokinetic studies.

Figure 2 :
Figure 2: Cyclic voltammograms of 1.0 × 10 -4 mol L -1 ESC in BR buffer over the pH range of 2-10 at CACP, scan rate of 100 mV s -1 (a).Plots of anodic peak current (b) and peak potential (c) as a function of pH.
.: Electroanalytical Determination of Escitalopram ... of CP, CACP and NiCACP.The surface of CP was predominated by irregular shaped graphite flaks and separate layers (ESI 1A).The SEM of CACP illustrates irregular ice shaped surface (ESI 1B).The SEM of NiCACP shows a tree shaped structure, the nanoparticles appear randomly and space among them produce large surface area (ESI 1C).

Figure 4 :
Figure 4: Calibration curve of ESC at NiCACP, pulse amplitude = 50 mV, T acc = 100 s and scan rate = 20 mV s -1 (a).Plot of anodic current as a function of ESC concentration (b).
DDQ, TCNQ and TCNE were obtained from Merck.CA, nickel nitrate, graphite powder and paraffin oil were supplied by Sigma-Aldrich.

Table 2 :
Determination of ESC in Cipralex tablets by applying standard addition technique.

Table 1 :
Fitting data of electrochemical impedance spectroscopy.