Electrooxidation and determination of perphenazine on a graphene oxide nanosheet-modified electrode

Abstract

The electrochemical behavior of perphenazine was investigated on a graphene oxide nanosheet-modified glassy carbon electrode in a phosphate buffer solution at pH 7.4. Cyclic voltammetry was employed to study the drug electrooxidation process. Perphenazine was electrooxidized on the modified electrode surface at lower potentials with a higher rate through an irreversible process. The kinetic parameters of the electrooxidation reaction, including the standard rate constant as well as diffusion and charge transfer coefficients were obtained. Amperometric and differential pulse voltammetric procedures were developed for the determination of perphenazine. Linear dynamic ranges of 0.8–8.0 and 0.7–7.0 mmol L⁻¹ with calibration sensitivities of 35.23 and 36.54 mA mol⁻¹ L cm⁻² and detection limits of 46.6 and 38.4 μmol L⁻¹ were obtained using amperometry and differential pulse voltammetry, respectively. The amperometric method was applied in the analysis of perphenazine tablets, and the applicability of the method for the direct assays of spiked human serum, urine and breast milk fluids was investigated.

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Introduction

The study of the redox properties of drugs and biologically active compounds can provide insight into their in vivo processes, pharmacological activity, molecular mechanisms, and metabolic fate; moreover, drug analysis may lead to the development of new drugs.^1–3 For such purposes, electrochemical techniques were employed and a wide range of drugs have been electroanalyzed. Electroanalytical methods present the great advantage of permitting a direct, simple and rapid determination route, which requires a minimal volume of the sample, in most instances, with no need for derivatization and no interference of matrix effects.^1–5

Perphenazine, (2-[4-[3-(2-chloro-10H-phenothiazin-10-yl) propyl]piperazin-1-yl]ethanol), is a piperazinyl phenothiazine and a typical antipsychotic drug. It acts on all the levels of the central nervous system, particularly the hypothalamus. Perphenazine blocks postsynaptic mesolimbic dopaminergic receptors in the brain, binds to the α-andrenergic receptor, and exhibits an α-adrenergic blocking effect. This receptor's action is mediated by association with G proteins that activate a phosphatidylinositol–calcium second messenger system. Perphenazine depresses the release of hypothalamic and hypophyseal hormones and also binds to the dopamine D1 and dopamine D2 neurotransmitter receptors and inhibits their activity. The mechanism of the anti-emetic effect is predominantly due to blockage of the dopamine D2 receptors in the chemoreceptor trigger zone and vomiting centre. It is used to treat the manic phases of bipolar disorder, psychosis (such as schizophrenia and schizoaffective psychoses), agitated depression, and Parkinson's disease.^6,7

Due to these functions and applications, up to now, some detection methods have been reported for the determination of perphenazine. These methods include high-performance liquid chromatography,^8,9 gas chromatography,^10–12 chemiluminescence,¹³ spectrophotometry^14–16 and titrimetry.^17–19 However, few studies on the electrochemical behavior of this drug have been reported.^20,21 Moreover, chromatographic methods are elaborate, costly and need large amounts of organic solvents. They are also time-consuming, rather complicated, and cannot be used for field determination. In addition, sensitivity for the reported spectrophotometric and titrimetric methods was low and complex procedures are needed. Therefore, there is a need for the development of new methods for the determination of perphenazine in physiological pH conditions.

The electroanalysis of drugs and biologically active compounds has been developed in recent years using the design of novel transducers and sensing elements based on nanostructured materials. Nanostructured materials represent excellent electronic and ionic conductivity, catalytic and electrocatalytic activity, and redox behavior, which cause the acceleration of direct or mediated electron transfer rate between the electrode surface and redox species.^{3–5,22–25} Voltammetric and amperometric sensors and biosensors fabricated by these materials have many applications in the development of drug analysis techniques in various pharmaceutical forms and biological fluids.^4,5,25–28 Among the nanomaterials, carbon nanostructure-based sensors and biosensors exhibit unique sensing utility and figures of merit.^29–31 In this regard, graphene has attracted attention for the construction of electrochemical devices,^22,32–34 and has been applied in the electroanalysis of dopamine, ascorbic acid and uric acid,^35,36 cadmium and lead,^37,38 glucose,³⁹ some drugs,^22,40,41 and cancer markers.^42,43

In the present study, the electrochemical behavior of perphenazine was first investigated on a graphene oxide nanosheet-modified electrode in phosphate buffer solution at pH 7.4. Then, both amperometric and differential pulse voltammetric methods were developed for the analysis of perphenazine in bulk and pharmaceutical forms and also in biological fluids.

Experimental

Reagents and chemicals

All chemicals used in this study were of analytical grade from Merck and used without further purifications. Graphite fine powder with an average size of <50 μm was received from Merck (Darmstadt, Germany). Perphenazine was received from the Center of Quality Control of Drug, Tehran, Iran. The perphenazine tablets were obtained from a local drugstore. All solutions were prepared using redistilled water.

Apparatus

Electrochemical measurements were carried out in a conventional three-electrode cell containing 100 mmol L⁻¹ Na-phosphate buffer solution with pH 7.4 (PBS) powered by an μ-Autolab potentiostat/galvanostat (Utrecht, The Netherlands). An Ag/AgCl 3 mol L⁻¹ KCl, platinum disk, and modified glassy carbon disk (GC) electrode were used as the reference, counter and working electrodes, respectively. The system was run on a PC using a GPES 4.9 software. In order to obtain information about the morphology and size of the graphene oxide nanosheets, scanning electron microscopy (SEM) was performed using an X-30 Philips instrument (Amsterdam, The Netherlands) and transmission electron microscopy (TEM) was performed using a CEM 902A ZEISS instrument (Goettingen, Germany) with an accelerating voltage of 80 kV. For TEM, the samples were prepared by placing a drop of the particles, dispersed in acetone, on a carbon covered nickel grid (400 mesh) and evaporating the solvent.

Procedures

Graphene oxide nanosheets were synthesized using the previously reported method^22,32,34 based on a modified Hummers method.⁴⁴ In a typical procedure, graphite powder (2.0 g) was dispersed in concentrated sulfuric acid (140 mL), and sodium nitrate (1.0 g) was added to the reaction mixture in an ice bath. Then, potassium permanganate (6.0 g) was slowly added to this mixture and stirred for 2 h to obtain graphite oxide. Afterwards, the mixture was diluted (3 times) with redistilled water. Then, H₂O₂ (5% v/v) was added to the mixture until the color of the mixture turned light yellow. The resultant suspension was filtered, and the obtained graphene oxide was thoroughly washed with redistilled water. The graphene oxide was then re-dispersed in redistilled water (∼30% w/v) and exfoliated to graphene oxide nanosheets by an ultrasonic bath for 3 h. The suspension gradually evolved into a brown solution during the ultrasonication, and the graphene oxide was transformed into graphene oxide nanosheets. The final product was filtered, washed with redistilled water and ethanol and dried in an oven at 80 °C.

In order to prepare the graphene oxide nanosheet-modified glassy carbon (GC/GO) electrode, graphene oxide nanosheets (3.0 mg) were first suspended into acetone (1 mL) by ultrasonication for 2 h. The GC electrode with a geometric surface area of 0.071 cm² was polished with 0.05 μm alumina powder on a polishing micro-cloth and ultrasonicated for 3 min in an ultrasonic bath in acetone–water (1 : 1 v/v) to remove the residual adsorbed alumina powder and then washed using redistilled water. Then, graphene oxide suspension (10 μL) was dropped onto the GC electrode surface and dried at room temperature.

The real surface areas of the GC and GC/GO electrodes were determined by cyclic voltammetry using K₄[Fe(CN)₆] (1 mmol L⁻¹) as a redox probe. For a reversible redox process, the peak current is as follows:⁴⁵

Ip = (2.69 × 10⁵)n^3/2AC*D^1/2ν^1/2 (1)

where I_p is the peak current. For the redox transition of [Fe(CN)₆]⁴⁻, n = 1 and D = 7.60 × 10⁻⁶ cm s⁻¹.⁴⁶ Therefore, from the slope of the I_p versus ν^1/2 plots, the real surface areas could be obtained. Evidently, the modified electrode had a surface area of ∼2.6 times of the unmodified electrode surface. In order to make the comparison, currents in the cyclic voltammograms were represented as current densities.

The standard solutions of perphenazine were prepared by dissolving the drug in PBS (which was also used as the supporting electrolyte) and stored in the dark at 4 °C. Additional dilutions were performed daily just before use. The drug solutions were stable and their concentrations did not change with time. For pH adjustment at different values, appropriate volumes of hydrochloric acid or sodium hydroxide solutions (100 mmol L⁻¹) were added to PBS.

The calibration curve for the drug in PBS was measured with amperometric and differential pulse voltammetric techniques. For amperometry, a working potential of 650 mV was applied; moreover, the transient currents were allowed to decay to steady-state values. For differential-pulse voltammetry (DPV), a pulse width of 25 mV, a pulse time of 50 ms, and a scan rate of 10 mV s⁻¹ were employed.

For the analysis of the drug tablets, the average weight of ten tablets from each sample was determined. Then, the tablets were finely powdered and homogenized in a mortar. Appropriately weighed amounts of the homogenized powder were transferred into calibrated flasks (25 mL) containing 100 mmol L⁻¹ HCl solution (4.0 mL). The contents of the flasks were ultrasonicated for 45 min, and then the undissolved excipients were removed by filtration and diluted to volume with the same supporting electrolyte. Appropriate solutions were prepared by taking suitable aliquots of the clear filtrate and diluting them with PBS. The drug tablets were analyzed by amperometric technique.

The drug-free blood samples were obtained from healthy male volunteers. The fresh samples were stored at 4 °C until two phases formed. The supernatant phase was separated by centrifuge as the serum blood. The serum samples were stored frozen until the assay. They were diluted with PBS. Various portions of the stock perphenazine solutions were transferred into volumetric flasks (10 mL) containing the serum sample (5 mL). These solutions were then diluted to the mark with PBS for the preparation of spiked samples (final dilution of 3 : 7 with PBS). The spiked serum solutions were directly analyzed by the calibration method, based on the amperometric procedure.

Urine samples taken from a healthy person were diluted with PBS (3 : 7) after adding an appropriate amount of perphenazine standard solution. The resulting solutions were then directly analyzed according to the proposed procedure without any pre-treatment or extraction steps. The calibration curves for the drug in urine samples were measured with amperometric technique.

The breast milk samples were diluted (3 : 10) with the supporting electrolyte after adding an appropriate amount of the drug standard solution. Then, the resulting solutions were directly analyzed by amperometry. All measurements were carried out at room temperature.

Results and discussion

Fig. 1A and B show the SEM and TEM images of the graphene oxide nanosheets. In the SEM image, a folded paper-like structure is observed. The graphene oxide sheets tended to wrinkle and curl together. In the TEM image, entangled transparent sheets with a few square micrometer dimensions were observed on the edges of the graphene oxide agglomerates.

Fig. 1 SEM (A) and TEM (B) images of the graphene oxide nanosheets.

The typical cyclic voltammograms of GC and GC/GO electrodes recorded in PBS in the absence and presence of 8.0 mmol L⁻¹ perphenazine are shown in Fig. 2. Perphenazine presented one anodic peak using both the GC and GC/GO electrodes. In all the voltammograms, no reduction peak appeared in the backward sweep, indicating the irreversible nature of the electrooxidation of perphenazine on both the electrode surfaces. The anodic peak potentials using the GC and GC/GO electrodes are 675 and 640 mV, respectively. Comparison of the peak potentials indicates that perphenazine was oxidized on the GC/GO electrode surface at lower potentials; therefore, graphene oxide enhanced the oxidation process from the thermodynamic point of view. On the other hand, the anodic peak current densities for perphenazine electrooxidation using the electrodes were the same. Therefore, it is revealed that the modification of the electrode surface by graphene oxide leads to an increase in the rate of the oxidation process by increasing the real surface area of the modified electrode. Therefore, the modification of the GC electrode with graphene oxide nanosheets leads to the electrooxidation of perphenazine, occurring at lower potentials and also increases the sensitivity of the electrode, when the GC/GO electrode is employed for the determination of perphenazine.

Fig. 2 Typical cyclic voltammograms of GC and GC/GO electrodes recorded in PBS in the absence and presence of 8.0 mmol L⁻¹ perphenazine. The potential sweep rate was 50 mV s⁻¹.

The linear sweep voltammograms of the electrooxidation of perphenazine on the GC/GO electrode surface at different pH values are shown in Fig. 3, and the dependencies of the anodic peak potential and current of perphenazine electrooxidation on the solution pH are shown in the inset. Upon increasing the solution pH, the peak current increased up to pH ∼ 6.5 and then decreased. The change in the peak currents can be related to the protonation of amine groups or deprotonation of the alcohol group of perphenazine (vide infra). However, because it aimed for the determination of the drug at physiological conditions, pH = 7.40 was chosen as the working pH throughout the study. Moreover, the peak potential shifted to less positive values upon increasing the solution pH. Because the electrooxidation of perphenazine is irreversible, the peak potential is a criterion of the redox potential of this drug on the GC/GO electrode. An negative shift of the peak potential upon increment in the solution pH indicates that the redox potential of the drug shifts in the negative direction with increase in the solution pH; hydrogen ions are produced during the electrooxidation of perphenazine. From the slope of the linear dependency of the peak potential on the solution pH, it can be deduced that perphenazine was oxidized on the GC/GO surface through the same electron and proton process.

Fig. 3 Linear sweep voltammograms of electrooxidation of perphenazine on the GC/GO electrode surface at different pH values. The potential sweep rate was 50 mV s⁻¹. Inset: dependencies of the anodic peak potential and current of perphenazine electrooxidation on the solution pH.

The electrooxidation reaction of perphenazine on the GC/GO electrode, based on the results presented in Fig. 3, previous studies on the phenothiazines, and specially the piperazine derivatives in moderated pH values,^20,47 the electrooxidation reaction can be written as shown in Scheme 1.

Scheme 1 Electrooxidation reaction of perphenazine.

The cyclic voltammograms of 4.0 mmol L⁻¹ perphenazine solution recorded at different potential sweep rates from 5 to 600 mV s⁻¹ are shown in Fig. 4A. The peak current increased with increase in the potential sweep rate, and the peak potential shifted to more positive values. The latter observation further confirmed the irreversible nature of the electrooxidation process. The electron transfer coefficient of the electrooxidation reaction can be obtained using the linear dependency of the anodic peak current on the natural logarithm of the potential sweep rate, as shown in Fig. 4B, and using the following equation:⁴⁸

E_pa = (RT/2αF)ln ν (2)

where E_pa, α and ν are the anodic peak potential, electron transfer coefficient and the potential sweep rate, respectively, and the other variables have their usual meanings. The electron transfer coefficient was obtained as 0.52. In addition, the heterogeneous electron transfer rate constant and the electron transfer coefficient can also be obtained using the linear dependency of the natural logarithm of the anodic peak current on (E_pa − E⁰′) values, as shown in Fig. 4C, and using the following equation:⁴⁵

I_pa = 0.227FAC*k⁰ exp[−(αF/RT)(E_pa − E⁰′)] (3)

where I_pa, n, A, C*, k⁰ and E⁰′ are the anodic peak current, the number of exchanged electrons, the electrode surface area, the bulk concentration of perphenazine, the standard rate constant, and the formal potential (equal to the extrapolated peak potential at potential sweep rate of zero), respectively. The electron transfer coefficient and the standard rate constant were obtained to be 0.52 and 2.45 × 10⁻⁴ cm s⁻¹, respectively. Moreover, the peak currents in the voltammograms presented in Fig. 4A depend linearly on the square root of the potential sweep rate, as shown in Fig. 4D.

Fig. 4 (A) Cyclic voltammograms of 4.0 mmol L⁻¹ perphenazine solution at different potential sweep rates of 5, 10, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 and 600 mV s⁻¹. (B) Dependency of the anodic peak current on the natural logarithm of the potential sweep rate. (C) Dependency of the natural logarithm of the anodic peak current on (E_pa − E⁰′). (D) Dependency of the peak currents of the voltammograms presented in (A) on the square root of the potential sweep rate.

Using the Randles–Sevcik equation:⁴⁵

I_pa = (2.99 × 10⁵)α^1/2n^3/2AC*D^1/2ν^1/2 (4)

where D is the diffusion coefficient of the drug and the slope of the line presented in Fig. 4D, the diffusion coefficient of perphenazine was obtained as 4.78 × 10⁻⁶ cm² s⁻¹.

In order to develop an analytical method for the analysis of perphenazine, DPV was employed. The differential pulse voltammograms of different perphenazine concentrations recorded using the GC/GO electrode are displayed in Fig. 5, and the corresponding calibration curve is shown in the inset. The limits of detection (LOD) and quantitation (LOQ) of the procedure were calculated according to the 3SD/m and 10SD/m criteria, respectively, where SD is the standard deviation of the intercept and m is the slope of the calibration curve.⁴⁹ The determined parameters for the calibration curve of perphenazine, accuracy, precision, LOD, LOQ and the slopes of calibration curves for the DPV measurements are presented in Table 1.

Fig. 5 Differential pulse voltammograms of different perphenazine concentrations recorded using a GC/GO electrode. Inset: the corresponding calibration curve.

Table 1 The determined parameters for the calibration curve of perphenazine and accuracy and precision using GC/GO

Method	DPV	Amperometry
a The value was reported for 1.0 mol L^−1 perphenazine.
Linear range/mmol L^−1	0.7–7.0	0.8–8.0
Sensitivity (slope)/mA mol^−1 L cm^−2	36.54	35.23
Intercept/μA cm^−2	17.16	0.43
R^2	0.9988	0.9991
Standard error of slope (P = 0.005)	2.63 × 10^−4	2.08 × 10^−4
Standard error of intercept (P = 0.005)	0.045	0.053
LOD/μmol L^−1	38.4	46.6
LOQ/μmol L^−1	128.0	155.3
RSD%	3.03	3.18
Bias^a%	−2.18	−1.73

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In order to develop a more simple procedure for the analysis of perphenazine, amperometry was also employed. Typical amperometric signals obtained during the successive increments of perphenazine to PBS are presented in Fig. 6. Gentle stirring was performed for a few seconds to promote the homogenization of the solution after each injection of drug solution. The electrode responses were quite rapid and proportional to the perphenazine concentration, as shown in the inset. The determined parameters for the calibration curve of perphenazine using the amperometric method are shown in Table 1.

Fig. 6 Typical amperometric signals obtained during the successive increments of perphenazine to PBS. The potential was 650 mV. Inset: the corresponding calibration curve.

In order to develop an electrochemical procedure for the determination of perphenazine in biological fluids, the application of the amperometric method was attempted for the analysis of perphenazine in human serum blood, urine and breast milk. Amperometric signals were recorded for perphenazine-spiked serum and urine samples. The results obtained from the amperometric technique for the determination of perphenazine in the biological fluids are presented in Table 3.

Table 2 Comparison of analytical methods for the determination of perphenazine

Method	Linear range/μM	Detection limit/μM	Reference
a DPV technique.b Amperometry technique.
Spectrophotometry	—	0.12	49
Molecular imprinting-post chemiluminescence	0.25–25	0.07	50
Chemiluminescence sensor based on molecular imprinting	0.12–25	0.05	51
Capillary electrophoresis	—	0.15	52
Kinetic spectrophotometry	10–160	5.3	53
Flow-injection chemiluminescence	0.25–170	0.2	54
Stripping voltammetry	—	5	55
This study	700–7000	38.4	This study^a
This study	800–8000	46.6	This study^b

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Table 3 The determined parameters for the calibration curve of perphenazine in human serum blood, urine and breast milk using GC/GO

Medium	Serum blood	Urine	Breast milk
Linear range/mmol L^−1	0.8–8.0	0.8–8.0	0.8–8.0
Sensitivity (slope)/mA mol^−1 L cm^−2	30.79	27.09	28.63
Intercept/μA cm^−2	0.29	0.41	0.57
R^2	0.9988	0.9992	0.9987
Standard error of slope (P = 0.005)	2.17 × 10^−4	3.29 × 10^−4	2.69 × 10^−4
Standard error of intercept (P = 0.005)	0.074	0.083	0.065
LOD/μmol L^−1	53.7	88.5	62.4
LOQ/μmol L^−1	179.0	295	208
RSD%	3.17	4.96	3.72

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The applicability of the proposed amperometric method for the sample dosage form was examined by analyzing the perphenazine tablets. It was found that the drug amounts determined using this method are along the same lines as the reported values. The values of the analytical parameters obtained for the drug tablets according to this method are reported in Table 4.

Table 4 Determination of perphenazine in commercial tablets

Tablet	Amount labeled/mg	Amount found/mg	Bias%
A	2.00	1.97	−1.5
B	8.00	7.85	−1.9

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The selectivity of the amperometric responses for the perphenazine assay was examined in the presence of some common excipients in the same ratios usually used in pharmaceutical preparations (for example, microcrystalline cellulose, colloidal silicon dioxide, hydroxypropylmethylcellulose, titanium dioxide, gelatin, talc, starch, and magnesium stearate). The results showed no significant interference from the excipients of the perphenazine tablets. Therefore, perphenazine assay in the presence of excipients is possible using this procedure; hence, it can be considered selective.

A comparison of different analytical methods for the determination of perphenazine is made in Table 2.

Conclusion

Graphene oxide nanosheets were first synthesized and then applied to a glassy carbon surface to fabricate a graphene-modified electrode. The modified electrode showed efficient activity in the electrooxidation of perphenazine in physiological medium, resulting from the special size, shape and structure of graphene. The electrooxidation process was diffusion-limited without adsorption and surface fouling. Kinetic parameters involved in the electrooxidation reaction, such as diffusion coefficient, electron transfer coefficient and heterogeneous electron transfer rate constant were obtained with classical electrochemical analyses. Voltammetric and amperometric responses generated by the electrode confirmed that the drug can be determined with high sensitivity in real samples and biological fluids. The electrode can be applied in the routine analysis of the drug; moreover, it has the potential to be used for the successful determination of perphenazine in pharmaceutical and clinical preparations. The modified electrode may be applicable in the analysis of some other drugs and biologically active compounds.

Acknowledgements

We would like to thank the Research Council of Shiraz University of Medical Sciences (92-01-57-6468) and also the Iran National Science Foundation (INSF) for supporting this research.

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