Determination of diltiazem in the presence of timolol in human serum samples using a nanoFe₃O₄@GO modified glassy carbon electrode

Abstract

A new chemically modified electrode was constructed based on a magnetic graphene oxide modified glassy carbon electrode (nanoFe₃O₄@GO-GC). This is for the first time that an electrode was evaluated as an electrochemical sensor for the simultaneous determination of diltiazem and timolol in aqueous solutions. The measurements were carried out by the application of the differential pulse voltammetry method in a phosphate buffer solution with a pH of 6.00. The results revealed that nanoFe₃O₄@GO promotes the rate of oxidation by increasing the peak current. NanoFe₃O₄ loaded: on GO increases the anodic peak currents of diltiazem and timolol on an electrode surface. The electrostatic interaction between the diltiazem and timolol cations and the high electron density of the hydroxyl groups of nanoFe₃O₄@GO led to an increase in the concentration of diltiazem and timolol around the surface of the modified electrode and the peak current increased significantly. The prepared electrode shows voltammetric responses with good selectivity for diltiazem and timolol in optimal conditions, which makes it very suitable for the simultaneous determination of these drugs. The practical analytical utility of the modified electrode was illustrated by the simultaneous determination of diltiazem and timolol in spiked serum samples.

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1. Introduction

Electrochemical techniques have been used for the determination of a wide range of drug compounds, often without derivatization.¹ In addition, electrochemical techniques include the determination of the electrode mechanism of a drug.² The redox properties of drugs can provide insight into their metabolic fate, in vivo redox processes and pharmacological activity.^3,4 Therefore, it is important to find more sensitive sensors for the detection of drugs.

One of the best materials for the construction of electrochemical sensors is graphene oxide (GO). GO, as a basic material for the preparation of individual graphene sheets in bulk-quantity, has attracted great attention in recent years.^5–7 In addition, the incredibly large specific surface area (two accessible sides), the abundant oxygen containing surface functionalities, such as epoxide, hydroxyl, and carboxylic groups, and the high aqueous solubility afford GO sheets several benefits for numerous applications.^4–7 The intrinsic oxygen-containing functional groups were used as initial sites for the deposition of metal nanoparticles, such as Fe₃O₄, on the GO sheets, which opened up a novel route to multifunctional nanometer scaled catalytic, magnetic, and electronic materials.^8–11 However, to date, only few studies about drug monitoring by GO have been reported.

The determination and quantification of drugs in biological fluids and pharmaceutical samples are essential in pharmaceutical, toxicological, doping and clinical chemistry research.¹² Cardiovascular drugs encompass a large number of prescription medications that are used to control heart diseases. They are a complicated group of drugs with many being used for multiple heart conditions; thus it is necessary to obtain a sensitive sensor for the determination of these drugs in spiked serum samples.¹³ The therapeutic efficacy is related to the interference effect of cardiovascular drugs in biological fluids and pharmaceutical samples. Therefore, the simpler and more rapid methods for the simultaneous analysis of several cardiovascular drugs are interesting for therapeutic drug monitoring purposes.¹⁴ Despite their widespread applications and use, little work has been reported on the simultaneous determination of cardiovascular drugs by electrochemical and related sensing devices. Therefore, the simultaneous determination of these drugs is necessary for the better management of the pharmacotherapy of heart diseases.

Previous studies show the significant cardiovascular risk associated with hypertension and the control of hypertension has impressive effects on health status. The mono therapy approach to manage hypertension could not provide the desired results and less than one third of the hypertensive patients achieved the desired blood pressure.¹⁵ The combination of thiazides, β-blockers, acetyl choline esterase (ACE) inhibitors and calcium channel blockers are well-studied combination therapies, which showed that lowering the dose of these drugs by combining two or more will lead to a higher efficacy and lower side effects. Moreover, the newer classes such as angiotensin II receptor antagonists are also used in combination with other classes, but their effects are not well evaluated.¹⁶

The Quantification of cardiovascular drug was widely studied in combined drug therapy. Recently, the quantification of a set of drugs used in the combined drug therapy of cardiovascular diseases was studied with two different methods.^17,18 A review of the published papers showed that several analytical methods were developed for the simultaneous determinations of these drugs, which are usually used in combination therapy protocols; however, the simultaneous analyses of this family of drugs have been rarely studied.^17,18 It has been established that timolol (TM) increases the risk of an atrio-ventricular (AV) block and bradycardia when given with diltiazem (DT).¹⁹ With regards the different mechanism of actions of these drugs, their combination in different ways are among the interesting therapy strategies for cardiovascular diseases. As far as we are aware, there is no report on the determination of DT in the presence of TM by electrochemical method in biological samples. In the present work, we have constructed a new magnetic electrochemical sensor for the detection of DT in the presence of TM. The present study was an attempt on the development and application of modified electrodes, which are aimed at inspecting of the electrochemical processes of these important drugs. NanoFe₃O₄@GO was used as the modifier material, and we studied the electrochemical oxidation and determination of DT and TM at nanoFe₃O₄@GO modified glassy carbon electrode (NanoFe₃O₄@GO-GCE). To the best of our knowledge, this is the first report for the determination of DT in the presence of TM based on their direct electrochemical oxidation on graphene or its derivatives.

2. Experimental

2.1. Chemical and reagents

All the chemicals were purchased from Merck (Darmstadt, Germany) and used without further purification. The drugs involved were obtained as a gift from Sobhan Darou Co., (Rasht, Iran). The standard solution of the authentic drugs was prepared by dissolving an accurate mass of the bulk drug in an appropriate volume of 0.1 M phosphate buffer solution of pH 6.00 (PBS) (which was also used as the supporting electrolyte), and it was then stored in the dark place at 4 °C. Additional dilute solutions were daily prepared by accurate dilution just before the use. De-ionized (DI) water was used for the preparation of aqueous solutions.

2.2. Serum sample preparation

Drug-free serum samples were obtained from healthy volunteers and stored frozen until the assay. The serum samples were diluted (1 : 2) with the supporting electrolyte and filtered through a 5 μm filter. Various portions of the stock DT and TM solution were transferred into 10 mL volumetric flasks containing 3.0 mL of the serum sample. These solutions were diluted to the mark with the supporting electrolyte for the preparation of spiked samples (final dilution of 1 : 3 with the supporting electrolyte). The protein-free spiked serum solutions were directly analyzed by the calibration method, according to the proposed procedure.

2.3. Apparatus

Electrochemical measurements were carried out in a conventional three-electrode cell (from Metrohm) powered by an electrochemical system comprising of an AUTOLAB system with PGSTAT302N (Eco Chemie, Utrecht, The Netherlands). The system was run on a PC using the NOVA 1.7 software. The AC voltage amplitude used was 10 mV and the equilibrium time was 5 s. An Ag/AgCl-Sat'd KCl (from Metrohm) and a platinum wire were used as the reference and counter electrodes, respectively. The working electrode was a glassy carbon (GC) electrode (from Azar Electrode Co., Urumia, Iran) and the nanoFe₃O₄@GO-GC electrode, with an exposed geometric surface area of 0.0314 cm². For differential-pulse voltammetry (DPV) measurements, a pulse width of 25 mV, a pulse time of 50 ms, and a scan rate of 5 mV s⁻¹ were employed.

FT-IR spectra were recorded on a Shimadzu model FT-IR prestige 21 spectrophotometer using KBr discs. X-ray powder diffraction (XRD) measurements were performed using a Philips diffractometer manufactured by X'pert with monochromatized Cu Kα radiation.

2.4. Synthesis of the nanoFe₃O₄@GO

The GO was prepared from purified natural graphite using a modified Hummer's method,²⁰ where the graphite powder (0.5 g) was added to 50 mL of 98% H₂SO₄ in an ice bath, KMnO₄ (2 g) was then gradually added while stirring. The rate of addition was carefully controlled to avoid a sudden increase of temperature. The stirring was continued for 2 h at temperatures below 10 °C, followed by 1 h at 35 °C. Then, the reaction mixture was diluted with 50 mL of DI water in an ice bath, where the temperature was kept below 100 °C. The mixture was stirred for another 1 h, and further diluted to approximately 150 mL with DI water. Subsequently, 10 mL of 30% H₂O₂ was added to the mixture which changed its color to brilliant yellow. The resulting mixture was centrifuged and washed several times with aqueous solution of 5% HCl, and then with DI water until the pH of the supernatant became neutral. Finally, the resulting solid was dried at 60 °C for 24 h and a loose brown powder was obtained. The nanoFe₃O₄@GO composites were synthesized by the co-precipitation of FeCl₃·6H₂O and FeCl₂·4H₂O, in the presence of GO. An aqueous solution of ferric chloride and ferrous chloride was prepared in a 2 : 1 mole ratio. For the preparation of nanoFe₃O₄@GO, 40 mg of GO in 40 mL of water was ultrasonicated for 30 min, to which a 50 mL solution of FeCl₃ (110 mg), FeCl₂ (43 mg) and 20 mL 30% ammonia solution in double distilled water was added at room temperature. After the addition, nanoFe₃O₄@GO was obtained as a brown powder (Scheme 1).

Scheme 1 Synthesis procedure of nanoFe₃O₄@GO.

2.5. Characterization of nanoFe₃O₄@GO

The general morphologies of the synthesized nanoFe₃O₄@GO were observed by SEM and are shown in Fig. 1. A flat surface is shown in Fig. 1A, which demonstrates a structure feature of single atomic layer thickness. The properties of nanoFe₃O₄@GO are highly related to their micro structures, dispersity and the morphology of the Fe₃O₄ nanoparticles. The presence of Fe₃O₄ nanoparticles in the GO surface was confirmed by SEM (Fig. 1A). In contrast to the surface of GO, which was quite smooth, the added Fe₃O₄ nanoparticles to GO appeared as bright dots, which were spread on the surface of the GO. The morphology of nanoFe₃O₄@GO was examined by TEM (Fig. 1B), where the image of prepared composite showed Fe₃O₄ nanoparticles as nearly spherical and homogeneously distributed over the GO nanosheets.

Fig. 1 The SEM (A) and TEM (B) images of nanoFe₃O₄@GO.

In the FT-IR spectra, the band in the region of 400–650 cm⁻¹ was assigned to the stretching vibrations of the (Fe–O) bond in Fe₂O₃, thus confirming the existence of Fe₃O₄ (Fig. 2). Two additional bands at 1450 and 1376 cm⁻¹ appeared, suggesting some interaction between the carbonyl and hydroxyl groups of GO and Fe on the surface of the magnetic particles, and showing the bonding of the iron oxide nanoparticles to GO.

Fig. 2 FT-IR spectra of nanoFe₃O₄@GO.

The structural properties of the synthesized nanoFe₃O₄@GO were analyzed by XRD (Fig. 3). The diffraction peak of GO appeared at 23.94°, which originated from the diffraction on its (0 0 2) layer planes. Moreover, the synthetic nanoFe₃O₄@GO showed some low intensity diffraction peaks that were indexed to cubic Fe₃O₄. The XRD peaks of magnetic graphene-oxide were indexed to the (2 2 0), (3 1 1), (4 0 0), (4 2 2) and (5 1 1) planes of a cubic unit cell of magnetite, appearing at 35.01°, 43.27°, 53.22°, 58.46°, and 63.50°, respectively.

Fig. 3 XRD pattern of nanoFe₃O₄@GO.

2.6. Preparation of the nanoFe₃O₄@GO-GCE

Prior to the use, the nanoFe₃O₄@GO were sonicated in a 3 : 1 sulfuric–nitric acid solution for 6 h in an ultrasonic bath at room temperature, and then washed with distilled water. The obtained sample was separated, and dried overnight at 50 °C. Prior to the modification, the GC electrode was polished with a 0.05 μm alumina suspension on a polishing micro-cloth, followed by sonication for 5 min in an ultrasonic bath in a 1 : 1 ethanol–distilled water mixture. The electrode was then transferred to the 1.0 M sulfuric acid solution. A potential in the range of −1 to 1.5 V in a regime of cyclic voltammetry was applied for 20 cycles. Subsequently, the electrode was thoroughly rinsed with distilled water and dried in air (it should be noted that this pretreatment was also employed for the bare GC electrode for the experiments in which the unmodified electrode was tested). The nanoFe₃O₄@GO (50 mg) was dispersed into acetone/double distilled water with the aid of ultrasonic stirring for 6 h. A 10 μL aliquot of this dispersion (with a concentration of 1.0 mg mL⁻¹) was dropped on the GC electrode surface, and the surface was allowed to dry at room temperature (24 h). When not in use, the electrodes were stored at 4 °C.

3. Results and discussion

3.1. Electrochemical behaviors of DT and TM

Fig. 4A and B show the typical cyclic voltammograms (CV) of PBS containing 0.1 mM DT and TM using the GO-GC (A) and nanoFe₃O₄@GO-GC electrodes (B). Fig. 4A does not show oxidation signal on the GC electrode. This indicated the electro-inactivity of DT and TM on the GO-GC surface. A typical CV of the nanoFe₃O₄@GO-GC electrodes in 0.1 M PBS containing 1 mM DT and TM in the potential range of −1 to 1 V is shown as Fig. 4B, where the potential sweep rate of 100 mV s⁻¹ has been employed. DT and TM exhibit one oxidation peak, located at 0.98 and 0.62 using the nanoFe₃O₄@GO-GC electrode in PBS with pH = 6.00, respectively. In the reverse sweep, however, no peaks appeared, indicating an irreversible heterogeneous electron transfer process for the oxidation of DT and TM on the nanoFe₃O₄@GO-GC electrode surface. Furthermore, the peak currents significantly enhanced. These results indicated that the nanoFe₃O₄@GO film could accelerate the rate of electron transfer of DT and TM and have good electro-activity for the oxidation of these drugs. The possible reason for this is the electrostatic interaction between the modifier and the drugs. Therefore, the electrostatic interaction between DT and TM cations and the high electron density of hydroxyl groups of nanoFe₃O₄@GO would lead to an increase in the concentration of analytes around the surface of the modified electrode due to which the peak current significantly increased.

Fig. 4 Cyclic voltammograms of 0.1 M PBS containing 0.1 mM DT and TM at (A) GO-GC and (B) nanoFe₃O₄@GO-GC electrodes. Potential sweep rate was 100 mV s⁻¹. (C) Influence of pH on the oxidation potential of 0.01 mM TM (A) and DT (B).

The peak potential was closely dependent on the pH of the solution. It was found that the values of the peak potential shifted to negative values with the increase of pH (as shown in Fig. 4C). The peak potential (E_p) moved in a negative direction with the increasing pH and they showed the following relationship: E_p = −52.219 pH + 57.784 (R² = 0.9892) and E_p = −51.330 pH + 55.714 (R² = 0.9782) for DT and TM, respectively. The slope of −52.2 and −51.3 mV pH⁻¹ demonstrated that the number of electron and proton transferred in the electrochemical reaction of DT and TM were equal, and it is one.

The influence of the scan rate on the electrochemical response of DT at the modified electrode was investigated by CV. The oxidation peak currents exhibited a linear relation to the square root of the scan rate in the range between 10–700 mV s⁻¹ with the linear regression equation of I_pa (μA) = 0.225 (mV s⁻¹) + 2.390 (R² = 0.9935). The results in Fig. 5A indicated that the electron transfer reactions were diffusion-controlled processes. The anodic peak potential positively shifted with the increase in scan rate, indicating the irreversible nature of the electrode reaction. The results indicated that the modified electrode could accelerate the electron transfer reaction and exhibit a good electro-activity. The possible reaction mechanism was that DT existed as a cation with the hydroxyl group of nanoFe₃O₄@GO in pH 6.00 PBS (Scheme 2).

Fig. 5 (A) Cyclic voltammograms of nanoFe₃O₄@GO-GC electrode in the presence of DT at different scan rates (from inner to outer): 10, 20, 30, 40, 50, 70, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650 and 700 mV s⁻¹. Inset: peak current vs. scan rate. (B) Cyclic voltammograms of nanoFe₃O₄@GO-GC in the presence of TM at different scan rates (from inner to outer): 10, 20, 50, 70, 100, 150 and 300 mV s⁻¹. Inset: peak current vs. scan rate.

Scheme 2 Oxidation mechanism of DT (A) and TM (B).

A similar result was obtained for the electrooxidation of TM. The CVs of 1 μM of TM at the modified electrode in 0.1 M PBS (pH 6.00) at various scan rates were recorded to investigate the influence of the scan rate on the electrochemical response of TM. The results in Fig. 6B show that the anodic peak currents were proportional to the square root of scan rate in the range of 10–300 mV s⁻¹ with the linear regression equation of I_pa (μA) = 0.207 (mV s⁻¹) + 1.225 (R² = 0.991), indicating that the oxidation reactions were controlled by a diffusion process. In addition, the oxidation peak potentials shifted with the increase of the scan rate, which suggested that the electrode reaction of TM is irreversible. Moreover, the dependence of ln I_p on ln v is linear (Fig. 6A and B) and described by the following equations:

ln I_p = 0.489 ln v (V s⁻¹) − 5.102 mA (R² = 0.991) TM (1)

ln I_p = 0.543 ln v (V s⁻¹) − 6.466 mA (R² = 0.986) DT (2)

Fig. 6 (A and B) Dependence of anodic peak current on the neperian logarithm of scan rate of TM and DT, respectively. (C) Effects of pH on the peak current of DT and TM at the modified electrode. DT and TM concentration is 1 μM; scan rate: 100 mV s⁻¹.

Their slopes are 0.489 (for TM) and 0.543 (for DT), which indicate the diffusion control of the electron transfer process. A slope close to 0.5 is expected for a diffusion-controlled process.^21,22

3.2. Effect of pH on the voltammetric responses of DT and TM

Because the oxidation of the selected analytes is pH dependent, to optimize the operational conditions and obtain the best peak separation and higher response, the effect of pH on electrochemical response of the nanoFe₃O₄@GO-GC electrode towards the simultaneous determination of DT and TM solutions were investigated. The effects of pH on the oxidation responses of DT and TM at the modified electrode were studied over a pH range from 3 to 10. According to the experimental results in Fig. 6C, the modified electrode showed good electro-activity for the redox reactions of DT and TM in a wide pH range. The anodic peak currents increased with the increasing pH up to 6.00, and then the peak currents decreased. The maximum peak current value can be observed at pH 6.00 for DT and TM; thus, it was selected as the optimum pH for the simultaneous determination of DT and TM in this study.

3.3. Simultaneous detection of binary mixture of DT and TM

Fig. 5 illustrates the CVs of the mixture containing DT and TM at the modified electrode. As shown in Fig. 7 when the modified electrode was used, the mixture displayed a difference in peak potentials of about 170 mV. Therefore, the sharp and well-defined oxidation peaks with significantly enhanced peak currents were selected for the simultaneous detection of DT and TM.

Fig. 7 Cyclic voltammograms of nanoFe₃O₄@GO-GC electrode in 0.1 M PBS containing DT and TM with different concentrations: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1, 2, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45 and 50 mM. Scan rate: 100 mV s⁻¹, pH = 6.00.

The next attempt was to simultaneously detect DT and TM at the modified electrode by DPV because of its higher current sensitivity and better resolution than CV. Fig. 8 illustrates the DPV responses of the modified electrode while the concentrations of DT and TM was synchronously increased. It can be seen that the anodic peak currents for the two analytes linearly increased with increase of their concentrations. The analytical responses of the modified electrode toward simultaneous determination of DT and TM are listed in Table 1. The proposed method had a low limit of detection (LOD), wide linear range and good stability compared with other methods. As the results indicate, the modified electrode can be used for the sub-micromolar detection of the proposed analytes without any pretreatment or pre-concentration steps. The comparison of this method with other electrochemical methods for the simultaneous determination of DT and TM is listed in Table 2.^23–27 The analytical parameters of the modified electrode, such as the LOD and linear concentration range, are better than or even comparable with the reported data in the literature for the voltammetric or amperometric detection of the selected analytes with different modified electrodes.

Fig. 8 Differential pulse voltammograms of nanoFe₃O₄@GO-GC electrode in 0.1 M PBS containing DT and TM with different concentrations: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 and 1.5 μM. Scan rate: 100 mV s⁻¹, pH = 6.00. Inset: plot of peak current vs. DT and TM concentrations.

Table 1 Analytical parameters obtained from the determination of DT and TM on nanoFe₃O₄@GO-GC electrode

Method	Drug	Linear concentration range (μM)	LOD (μM)	Linear regression (R^2)
DPV	DT	0.1–100	0.06	0.9910
	TM	0.1–340	0.02	0.9907

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Table 2 Analytical parameters for the detection of DT and TM for several methods

Drug	Method	Linear concentration range (μM)	LOD (μM)	Reference
DT	Potentiometry	10 to 10 000	3.2	23
DT	CV	1–41 450	0.29	24
TM	SW-AdSV	0.1 to 1.5	0.0126	25
	AdS-SWV	0.001 to 0.12	0.006	26
		0.012 to 0.1
	SWP	0.04 to 3.0	0.025
TM	DPV	1 to 5	2.5	27
DT	DPV	0.1 to 100	0.06	This work
TM		0.2 to 340	0.02

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3.4. Analytical application

The practical analytical utility of the modified electrode was illustrated by the simultaneous determination of DT and TM in serum samples.

The generally poor selectivity of voltammetric techniques can pose problems in the analysis of biological samples, which contain oxidizable substances. However, no current due to the oxidation of the drugs in the serum samples appeared. 1.00 mL of DT (specified content of DT is 10 mg mL⁻¹) and 5.00 mL TM (specified content of TM is 1 mg mL⁻¹) injections were diluted to 50 mL with the buffer solution, respectively. Different capacity diluted solutions were pipetted into each of a series of 10 mL volumetric flasks and diluted to the mark with 0.1 M PBS (pH 6.00). Then, the test solution was placed in the electrochemical cell. The proposed DPV method was used for the simultaneous detection of DT and TM. The average determination results of DT and TM in the spiked serum samples were 9.44 and 0.93 mg mL⁻¹, which were almost corresponding to the values that were given by the injection specifications. This process was repeated 10 times, and the RSDs obtained for DT and TM were 1.80% and 2.13%, respectively.

To a series of 5, 10 and 20 mL measuring flasks, different capacity diluted DT and TM injection solutions was added and diluted to the mark with 0.1 M PBS (pH 6.00). The DPVs were recorded and the anodic peak currents were measured for DT and TM. The obtained results are shown in Table 3. It can be seen that the proposed method has a good precision and can be applied for the simultaneous determination of DT and TM.

Table 3 Determination of DT and TM in spiked serum samples (n = 6)

DT added (μM)	TM added (μM)	DT			TM
		Found (μM)	Recovery	RSD (%)	Found (μM)	Recovery	RSD (%)
5	5	5.08	101.6	2.4	4.78	95.6	3.0
10	10	10.6	106	2.0	9.50	95	3.21
20	20	19.44	97.2	3.1	19.49	97.4	3.0

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3.5. Reproducibility and stability

To verify the reproducibility, durability and long-term stability of the prepared sensor, consecutive amperograms in three concentrations of DT and TM were recorded. Fig. 9A and B shows the repeatability and reproducibility of the amperometric responses to the successive injections of DT and TM. The relative standard deviations (RSD) of the amperometry currents of 1, 1.5 and 2 μM of DT were 3.4%, 1.0% and 0.8% (n = 6), respectively, revealing an acceptable reproducibility of the method. In addition, the RSD for three replicate determinations of TM were 3.3%, 0.5% and 5.0% (n = 6) at concentrations of 1, 1.5 and 2 μM, respectively. The obtained RSD using the amperograms of DT and TM indicated that the fabrication method was reproducible and repeatable. In addition, the stability of the modified electrode was investigated by the amperometric method (Fig. 9C). When the modified electrode was stored in the atmosphere, the current response decreased ∼15% after 30 s. This indicates negligible fouling of the nanoFe₃O₄@GO surface by DT and TM and oxidation products. Therefore, nanoFe₃O₄@GO showed a high stability and strong mediation properties for the amperometric measurements of DT and TM.

Fig. 9 Typical amperometric signals obtained during the successive increments of DT (A) and TM (B) to 0.1 M PBS using the applied potential of 1.08 and 0.68 V, respectively (n = 6). (C) The recorded chronoamperogram for 1 μM of DT (selected drug) during 30 s.

3.6. Interference studies

To evaluate the selectivity of the developed DPV procedure, the influence of various interferences was examined. Considerable interference can be caused by co-existing surface-active compounds capable of competing with the analytes of interest for the adsorption site on the electrode surface, resulting in decreased or increased peak heights. The competitive co-adsorption interference was evaluated in the presence of various substances that usually occur in biological fluids. For these investigations, the interfering species were added at different concentrations (1, 2, 5 and 50-fold) higher than the concentration of DT and TM (1 μM). The addition of 50-fold of sucrose in the test drugs solution, caused the DPV peak current to decrease by about 8%. Apparently, these inhibition effects were caused by the working electrode surface blockage due to the adsorption of interferences. In contrast, the addition of 50-fold of lactose in the drug solution caused the DPV response of the drug to increase by about 16%. Moreover, the addition of 2-fold of some amino acids (Glycine, Cysteine, and Tyrosine) in the test drug solution (1 μM) caused the DPV peak current to decrease by about 10%, 10%, and 24%, respectively. Furthermore, the addition of ascorbic acid (5-fold) in the DT and TM solution caused the DPV response of the drug to decrease by about 14% and 10%, respectively.

4. Conclusions

In the present study, a sensitive, inexpensive, and rapid method for the simultaneous determination of DT and TM was proposed based on the electro-oxidation of the proposed analytes on nanoFe₃O₄@GO-GC electrodes. The proposed electrode was used for the determination of DT and TM in PBS, without the necessity for sample pretreatment or any time-consuming extraction or evaporation steps prior to the analysis, with a satisfactory recovery. The good stability, wide linear concentration range, low detection limit, and a distinct advantage of polishing in the event of surface fouling, suggest that this electrode is an attractive candidate as a transducer for practical applications. Moreover, due to the large surface area of GO and remarkable electroactivity properties of Fe₃O₄, the nanoFe₃O₄@GO-GC electrode can be used for the simultaneous determination of some other drugs that are necessary for the interference study.

Acknowledgements

This is a report of a database from the thesis entitled “Development of sensitive, routine and reliable methods for determination of cardiovascular drugs in biological samples” registered in Drug Applied Research Center. We gratefully acknowledge the financial support of this work by the Drug Applied Research Center, Tabriz University of Medical Sciences.

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