A new mechanistic approach to elucidate furosemide electrooxidation on magnetic nanoparticles loaded on graphene oxide modified glassy carbon electrode

Abstract

The electrochemical behavior of furosemide was investigated at the magnetic graphene oxide functionalized by chlorosulfonic acid-modified glassy carbon electrode (Fe₃O₄-GO-SO₃H-GCE) in phosphate buffer solution (PBS), pH 6.8. Cyclic voltammetric study indicated that the oxidation process is irreversible and diffusion controlled. The number of exchanged electrons in the electro-oxidation process was obtained, and the data indicated that furosemide is oxidized via two one-electron steps. The results revealed that Fe₃O₄-GO-SO₃H promotes the rate of oxidation by increasing the peak current. The diffusion coefficient and electron-transfer coefficient of furosemide were found to be 2.18 × 10⁻⁶ cm² s⁻¹ and 0.46, respectively. A sensitive, simple and time-saving differential-pulse voltammetric procedure was developed for the analysis of furosemide. The results show that by using the proposed method, furosemide can be determined with a detection limit of 0.11 μM. Finally, the applicability of the method to direct assays of spiked human serum and urine fluids is described.

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1. Introduction

Development of simple, sensitive, and accurate methods for detecting active ingredients is an important issue since drug analysis plays a key role in drug quality control, and this has a great impact on public health. Electrochemical techniques were used for the determination of a wide range of drug compounds. They have the advantage of not requiring, in most instances, derivatization, and they are less sensitive to matrix effects than other analytical techniques. Additionally, electrochemical techniques include the determination of the drug's electrode mechanism. Redox properties of drugs can provide insight into their metabolic fate, their in vivo redox processes and their pharmacological activity.^1–4

In hypercalcemia treatment of, rapid reduction of serum calcium is required. The procedure includes rehydration with saline and diuresis with furosemide.^5,6 Most patients presenting with severe hypercalcemia have a substantial component of prerenal azotemia owing to dehydration, which prevents the kidney from compensating for the rise in serum calcium by excreting more calcium in the urine. Therefore, the initial infusion of 500–1000 mL h⁻¹ of saline to reverse the dehydration and restore urine flow can by itself substantially lower serum calcium. The addition of a loop diuretic such as furosemide not only enhances urine flow but also inhibits calcium re-absorption in the ascending limb of the loop of Henle.^5,6 Therefore, careful medical supervision is necessary during treatment.

Monitoring of furosemide was requested in drug quality control and therapeutically drug monitoring investigations. Several methods have been used for the determination of furosemide in pharmaceutical formulations and biological fluids.⁷ Chromatography (especially LC/MS/MS) is now widely and routinely used for the analysis of furosemide.⁸ Chromatographic analyses are generally performed using expensive instruments. It requires extensive labor and analytical resources, and often results in a lengthy turn-around time. Given the low cost, ease of use, and sensitivity, electrochemical techniques are alternative methods for determining furosemide. But, surveying the literature revealed that electrochemical methods have been rarely applied to determination of pharmaceutical furosemide in serum and urine samples.^9–14 Therefore, development of simple, sensitive, rapid and reliable electrochemical methods/sensors for the determination of furosemide is of great importance.

One of the best materials for construction 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.^15–17 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 water solubility afford GO sheets great promise for many more applications.^15–17 The intrinsic oxygen-containing functional groups were used as initial sites for 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.^18–21 However, few studies about the drug monitoring by GO have been reported to date.

In the present work, we used Fe₃O₄-GO-SO₃H as modifier material and studied the electrochemical oxidation of furosemide at Fe₃O₄-GO-SO₃H modified glassy carbon electrode and developing a new electroanalytical procedure for quantification of furosemide in real samples. Also, a new mechanism based on the electrooxidation of furosemide was also investigated. To the best of our knowledge, this is the first report of the determination of furosemide based on their direct electrochemical oxidation on graphene or its derivatives.

2. Experimental

2.1. Chemicals

All chemicals used in this work were of analytical reagent grade, from Merck (Germany). Furosemide was obtained as a gift from Arasto Pharmaceutical Chemicals Inc. (Tehran, Iran). The standard solution of authentic furosemide was prepared by dissolving an accurate mass of the bulk drug in an appropriate volume of 0.1 M phosphate buffer solution, pH 6.80 (PBS) (which was also used as supporting electrolyte), and then stored in the dark place at 4 °C. Additional dilute solutions were prepared daily by accurate dilution just before use. Furosemide solutions were stable and their concentrations did not change with time.

Drug-free serum samples were obtained from healthy male volunteers and stored frozen until the assay. The serum samples were diluted (1 : 2) with the supporting electrolyte and filtrated through a 5 μm filter to produce protein-free human serum. Various portions of stock furosemide solution were transferred into 10 mL volumetric flasks containing 3.3 mL of the serum sample. These solutions were diluted to the mark with the supporting electrolyte for 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 our proposed procedure.

Urine samples were diluted (1 : 2) with PBS after adding an appropriate amount of standard furosemide solution. The resulting solution was then directly analyzed, according to the proposed procedure, without any pretreatment or extraction steps.

2.2. Instruments

Electrochemical measurements were carried out in a conventional three-electrode cell (from Metrohm) powered by an electrochemical system comprising of AUTOLAB system with PGSTAT302N (Eco Chemie, Utrecht, The Netherlands). The system was run on a PC using 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 reference and counter electrodes, respectively. The working electrode was a glassy carbon (GC) electrode (from Azar Electrode Co., Urumia, Iran) and Fe₃O₄-GO-SO₃H-GC electrode, exposing a 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 10 mV s⁻¹ were employed.

FT-IR spectra were recorded on a Shimadzu model FTIR 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 K_α radiation.

Prior to use, the Fe₃O₄-GO-SO₃H 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 taken, and dried overnight at 50 °C. Prior to the modification, the GC electrode was polished with 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–redistilled water mixture. The electrode was then transferred to the 1.0 M sulfuric acid solution. Potential in the range of −1–1.5 V in a regime of cyclic voltammetry was applied for 20 cycles. Afterward, the electrode was rinsed thoroughly with distilled water and dried in air. It should be noted that this pretreatment was also employed for the bare GC electrode in the experiments that the unmodified electrode was tested. Fe₃O₄-GO-SO₃H (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 concentration of 1.0 mg mL⁻¹) was dropped on the GC electrode surface, and the solvent was allowed to dry in air.

2.3. Synthesis of Fe₃O₄-GO-SO₃H

At first the (Fe₃O₄)-GO with loaded iron oxide nanoparticles was prepared according to the reported method in the literature.²² Then, to (Fe₃O₄)-GO (1 g), chlorosulfonic acid (0.5 g, 4.5 mmol) in 5 mL dichloromethane was added dropwise at room temperature within 30 min. After completion of the addition, the mixture was mechanically stirred for other 30 min until HCl was removed from reaction and (Fe₃O₄)-GO-SO₃H was obtained as brown powder (Scheme 1).

Scheme 1 Synthesis procedure of Fe₃O₄-GO-SO₃H.

The general morphologies of synthesized Fe₃O₄-GO were observed by SEM and shown in Fig. 1. A flat surface was shown in Fig. 1A, which demonstrating a single atomic layer thickness structure feature. The properties of Fe₃O₄-GO-SO₃H are highly related to their micro structures, their dispersity and the morphology of Fe₃O₄ nanoparticles. The presence of Fe₃O₄ nanoparticles in GO-SO₃H surface was confirmed by SEM. The added Fe₃O₄ nanoparticles to GO-SO₃H appeared as bright dots that were spread on the surface of (Fe₃O₄)-GO-SO₃H. In contrast to the surface of GO-SO₃H which was quite smooth, the added Fe₃O₄ nanoparticles to GO-SO₃H appeared as bright dots, which were spread on the surface of the GO-SO₃H. The morphology of Fe₃O₄-GO-SO₃H 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 Fe₃O₄-GO-SO₃H in different magnitude.

In FT-IR spectra the band in the region of 1036 and 1153 cm⁻¹ is attributed to the stretching vibrations of the (S=O) and a peak appeared at about 3367 cm⁻¹ is due to the stretching of OH groups in the SO₃H. Also, the band from 400–650 cm⁻¹ is assigned to the stretching vibrations of the (Fe–O) bond in Fe₂O₃ (Fig. 2).

Fig. 2 FT-IR spectra of Fe₃O₄-GO-SO₃H.

The structural properties of the synthesized Fe₃O₄-GO were analyzed by XRD (Fig. 2). The diffraction peak of GO appeared at 23.94° which originated from the diffraction on its (0 0 2) layer planes. Also, synthetic nanocatalysts showed some low intensity diffraction peaks that were indexed to cubic Fe₃O₄. The XRD peaks of magnetic graphene-oxide were indexed to (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).

Fig. 3 XRD pattern of Fe₃O₄-GO-SO₃H, inset: catalyst recovery at the reaction.

Coulometric measurements were performed on the Fe₃O₄-GO-SO₃H-GC electrode in 0.1 M PBS containing 1 μM furosemide for 1100 s, at two different fixed potentials of 0.98 V and 1.12 V respectively, surface area = 0.059 cm².

2.4. Preparation of modified Fe₃O₄-GO-SO₃H electrodes

Glassy carbon electrode (GCE, 2 mm in diameter) was polished to a mirror-like finish with 0.3 and 0.05 μm alumina slurry (Beuhler, USA) followed by rinsing thoroughly with double distilled water. Then it was successively sonicated in acetone and double distilled water, and allowed to dry at room temperature. The real area of the pretreated GC was 0.092 cm². 10 μL of Fe₃O₄-GO-SO₃H dispersed ethanol solution (0.4 mg mL⁻¹) is cast on the surface of GCE, and the electrode is heated for 7 min at 60 °C for solvent evaporation. After, the modified electrodes were rinsed with double distilled water. Finally, Fe₃O₄-GO-SO₃H modified GC electrodes were obtained. When not in use, the electrodes were stored at 4 °C.

The microscopic areas of the Fe₃O₄-GO-SO₃H-GC and the bare GCEs were obtained by CV using 1 mM K₄Fe(CN)₆ as a redox probe at different scan rates. For a reversible process, the anodic peak current i_pa can be calculated as follows:²³

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

where n, A, C* and D are the number of exchanged electrons, real surface area of the working electrode, bulk concentration and diffusion coefficient of the electro-reactant species, respectively. For K₄Fe(CN)₆, n = 1 and D = 7.60 × 10⁻⁶ cm s⁻¹.²⁴ Therefore, from the slope of the i_pa vs. ν^0.5 relation, the microscopic areas were determined to be 0.03 cm² for the bare GC and 0.059 cm² for the Fe₃O₄-GO-SO₃H-GC electrodes. Evidently, the modified electrode had an increased surface area of nearly 180%. All studies were carried out at room temperature.

3. Results and discussion

Fig. 4A shows typical cyclic voltammograms of PBS containing 5.0 mM furosemide using GC As seen in Fig. 4A any oxidation signal did not appear on the GC electrode. This indicated the electroinactivity of furosemide on the GC surface. Typical cyclic voltammograms of Fe₃O₄-GO-SO₃H-GC electrodes in 0.1 M PBS and in the potential range of −0.6–1 V is shown as Fig. 4B (absence of furosemide; curve a) and (presence of furosemide; curve b) where potential sweep rate of 2 mV s⁻¹ has been employed. In the absence of furosemide, no peak appears at the Fe₃O₄-GO-SO₃H-GC. While furosemide exhibits two oxidation peaks, located at 0.98 and 1.12 mV (indicated as I and II, respectively), at a potential sweep rate of 2 mV s⁻¹. The results indicate that the redox peaks are ascribed to the electrochemical reactions of furosemide which is according with other report.¹⁰ In the reverse sweep, however, no peaks appeared, indicating an irreversible heterogeneous electron transfer process for the oxidation of furosemide on the Fe₃O₄-GO-SO₃H-GC electrode surface. From cyclic voltammograms recorded at different potential sweep rates (Fig. 5A and B) using the Fe₃O₄-GO-SO₃H-GC electrode, it can be inspected that both peak currents I and II depend linearly on the square root of the potential sweep rate (Fig. 5C). Therefore, both peaks I and II are related to two consecutive diffusion-controlled processes. It should be noted that the peak currents appeared in the voltammogram represented in Fig. 5A and B did not decrease upon consecutive potential cycling; so good stability was achieved using Fe₃O₄-GO-SO₃H-GC electrode. It can be supposed that Fe₃O₄-GO-SO₃H with nano-meter dimensions are stably distributed and assembled on the GC electrode which is fully and easily accessible to analytes, and consequently can be readily and completely used as an electrochemical sensing unit, yielding a higher sensitivity.²⁵ Moreover, GOs have a particular electronic structure, high electrical conductivity and topological defects present on their surfaces.²⁵ GOs bear both basal plane sites and edge plane like sites/defects in their structures which may have caused the electrocatalytic efficiency during the electro-oxidation process.^26,27 Therefore, Fe₃O₄-GO-SO₃H-GC catalyzed the oxidation of furosemide.

Fig. 4 Cyclic voltammograms of 0.1 M PBS containing 1 μM furosemide using GC (A) and Fe₃O₄-GO-SO₃H-GC (B) electrodes (curve a) in the absence of furosemide Potential sweep rate was 2 mV s⁻¹.

Fig. 5 Cyclic voltammograms for PBS containing 1 μM furosemide using the Fe₃O₄-GO-SO₃H-GC electrode recorded at lower (A) and upper (B) potential sweep rates. Potential sweep rates in A and B are: 2, 5, 10, 20 and 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300 mV s⁻¹, respectively. (C) Dependency of peaks I and II currents on the corresponding square root of the potential sweep rate using the Fe₃O₄-GO-SO₃H-GC electrode. (D) Dependency of peak potential on the corresponding natural logarithm of potential sweep rate.

The significant enhancement of the furosemide electrooxidation at Fe₃O₄-GO-SO₃H-GC electrode can be explained according to the catalysis mechanism. Because the GOs had a high surface area, it boosted the faradic currents of the sluggish reaction as well as increased the amount of chemisorbed layer of the furosemide. This behaviour catalyzed the electron transfer of furosemide in the solution. The more active functional groups on the surface of the Fe₃O₄-GO-SO₃H can easily form hydrogen bonds with the amine groups of the furosemide, which weaken the –NR bond energies; the electrons would be transferred through SO₃H⋯N–R. In addition, the density of the electron cloud was lower from furosemide to intermediate to product, and therefore, their electroactivity decreased and they were less easily oxidized which is in accordance with the experimental results (Scheme 2).

Scheme 2 Oxidation mechanism of furosemide.

The electrocatalytic activity of Fe₃O₄-GO-SO₃H can be ascribed to the combination of the electron-acceptor ability of SO₃H and confinement effects due to the attachment to the Fe₃O₄-GO matrix. On the basis of the previous considerations, the electrocatalytic activity of Fe₃O₄-GO-SO₃H must result from the superposition of the effect associated with more external SO₃H units and that due to boundary-associated ones. On the other hand, since the catalytic effect associated with Fe₃O₄-GO-SO₃H consists essentially of an enhancement of the anodic peak for furosemide oxidation, the catalytic effect can tentatively be associated with a redox reaction in solution phase between SO₃H units and any intermediate species resulting from the initial electron transfer of furosemide.

On the basis of CV results, it is found that we have two rate determination steps in the electro-oxidation of furosemide on Fe₃O₄-GO-SO₃H-GC electrode. The consecutive oxidation of single substance with two rate determination steps in a potential scan experiment is complicated. For the stepwise oxidation of a single substance O, that is, A → B + n₁e (E₁), B → C + n₂e (E₂), the situation is similar to the two-component case, but it is more complicated. In the elucidation of the mechanism and rate determination step in a multi-step reaction, the scan rates play a prominent role. In order to further clarify the electrochemical oxidation mechanism of furosemide on Fe₃O₄-GO-SO₃H-GC electrode, effect of scan rate on cyclic voltammetry response is investigated. The results of CV studies for electro-oxidation of furosemide were obtained in two ranges of potential sweep rates. The first study was at low sweep rates (2–20 mV s⁻¹) and the second investigation was at higher sweep rates from 20–300 mV s⁻¹. The effects of scan rate on the voltammograms recorded for Fe₃O₄-GO-SO₃H-GC electrode in 0.1 M PBS containing 1 μM furosemide are shown in Fig. 5A and B. It is clear that the current ratio of peak II/peak I (I_pII/I_pI) increase with rising scan rate until to 20 mV s⁻¹. In addition at higher scan rate (>20 mV s⁻¹) only peak II is observed while peak I almost disappeared. On the other hand only one peak is found in the voltammogram recorded at high scan rate. Additionally the ratio (I_pII/I_pI) increase with increasing scan rate. Hence one can conclude if:

• ν < 20 mV s⁻¹, two separate waves are observed which the first wave corresponds to oxidation of A to B in an n₁-electron reaction, with B diffusing into the solution as the wave is traversed. At the second wave, B continues to be oxide directly at the electrode. The voltammogram for this case presented in to Fig. 5A. Base on these results the following mechanism of furosemide on lower scan rates follow as:

• ν > 20 mV s⁻¹, one wave is observed which the A was oxide directly at the electrode. The voltammogram for this case presented in to Fig. 5B and the mechanism in higher scan rates follow as:

• according to results and also above mechanisms, it can be considered that at low sweep rates, desorption step of the intermediated (according to mechanism I-produced from firs electron transfer step) and formation of product (produced from second electron transfer step in first mechanism) becomes more probable as the rate-determination step. Such behaviour for the electrochemical oxidation of furosemide has not been observed up to now.

• In summary, in the case of electrooxidation of furosemide on Fe₃O₄-GO-SO₃H-GC electrode the nature of the i–E curve depends on scan rates. When v is between 2 and 20 mV s⁻¹, the individual waves are merged into a broad wave whose E_p is dependent of scan rate. When v is between 20 and 200 mV s⁻¹, a single peak is found therefore the second step is easier than the first, a single wave characteristic of a irreversible 2e⁻ oxidation (A → C + 2e⁻) is observed.

Voltammetric parameters for furosemide oxidation on the Fe₃O₄-GO-SO₃H-GC electrode surface were also studied. The effect of potential sweep rate was studied, in the range of 2–200 mV s⁻¹. CVs recorded at different potential sweep rates for 0.1 M PBS containing 1 μM furosemide, using the Fe₃O₄-GO-SO₃H-GC electrode, are depicted in Fig. 5A and B along with the potential sweep rate increase, both peak currents increased and the peak potentials shifted to positive values, confirming the irreversible nature of the reaction processes. For an irreversible diffusion-controlled process, the peak potential (E_p) is proportional to the logarithm of potential sweep rate (ν) within the following equation:²⁸

On the basis of this equation, the slope of the E_p vs. log ν linear plot is b/2, where b indicates the Tafel slope. The dependency of peak II potential on log ν for the oxidation of furosemide on the Fe₃O₄-GO-SO₃H-GC electrode is depicted in Fig. 5D value of b = 55.1 mV was obtained, which indicates that for the oxidation of furosemide, the charge-transfer coefficient, α, was 0.46.

From the CVs shown in Fig. 5C, both peaks I and II currents can be seen to depend linearly on the corresponding square root of potential sweep rate. This dependency indicates that a mass transport process has occurred in the oxidation process, via diffusion. From the slope of the linear dependency of peak II current on the square root of potential sweep rate, and using the Randles–Sevcik equation for totally irreversible electron-transfer processes, we can calculate the diffusion coefficients of furosemide using eqn (1). Using the proposed method, the diffusion coefficient of furosemide was found to be 2.18 × 10⁻⁶ cm² s⁻¹. The values of the diffusion coefficients obtained using both electrodes are nearly the same, since diffusion of electro-reactant species in the bulk of the solution does not depend on the target surface in a semi-infinite linear diffusion process.

Also, plotting the current function (peak current divided by the square root of the potential sweep rate) against the square root of the potential sweep rate (Fig. 6A) revealed negative slope confirming the electrocatalytic nature of the process.

Fig. 6 (A) Current function vs. ν^0.5 for 0.1 M PBS containing 1 μM furosemide. (B) Dependency of peak potentials with respect to the solution pH.

The effect of pH on the electrochemical behavior of furosemide was investigated by CV, using 0.1 M buffer at various pH values ranging from 1.00 to 12.00. Fig. 6B shows the changes of peak potential II with respect to the solution pH. The plot shows two linear segments which is accordance with other reports.^9,29

Coulometry was performed in PBS containing 1 μM furosemide at 0.98 and 1.12 V, corresponding to the potentials of I and II peaks using the Fe₃O₄-GO-SO₃H-GC electrode. Electrolysis progress was carried out using CV. The extrapolated charge consumption for total electrolysis of the solution after corrections for background/charging currents was derived, with the number of exchanged electrons for peaks I and II obtained as 1.1 ≈ 1 and 0.91 ≈ 1, respectively. Hence, furosemide is oxidized via two one-electron steps on the Fe₃O₄-GO-SO₃H-GC electrode surface (Fig. 7).

Fig. 7 Coulometric response of Fe₃O₄-GO-SO₃H-GC electrode in 0.1 M PBS containing 1 μM furosemide. T= 1100 s, E₁ = 0.98 V and E₂ = 1.12 V, surface area = 0.059 cm².

The above analysis for the electrochemical oxidation mechanism of furosemide is further supported by chronoamperometric measurements indicated in Fig. 8. The potentials applied are 0.98 and 1.12 V vs. Ag/AgCl which are close to those of anodic peaks I and II, respectively. Fig. 8 shows that compared in the presence of various concentrations of furosemide, much large increase in the current at 1.12 V is observed than that at 0.98 V.

Fig. 8 Chronoamperometric responses on the Fe₃O₄-GO-SO₃H-GC electrode in 0.1 M PBS at E = 0.98 V and E = 1.12 V with different furosemide concentrations: 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), and 10 μM (f). Inset: dependency of transient current on t^−1/2.

The plot of net current versus t^−1/2 which has been obtained by removing the background current by the point-by-point subtraction method gives a straight line, Fig. 8 (inset). This indicates that the transient current must be controlled by a diffusion process. The transient current is due to catalytic oxidation of atenolol which increases as the atenolol concentration is raised. No significant cathodic current was observed when the electrolysis potential was stepped to 0.51 vs. (Ag/AgCl)/V indicating the irreversible nature of the oxidation of drugs by using the slopes of these lines; we can obtain the diffusion coefficients of the drugs according to the Cottrell equation:²⁸

I = nFAD^1/2C*π^−1/2t^−1/2 (3)

where D is the diffusion coefficient, and C* is the bulk concentration. The mean value of the diffusion coefficients of atenolol was found to be 2.80 × 10⁻⁶ cm² s⁻¹.

The calibration curve for furosemide in PBS was obtained by differential-pulse voltammetry (DPV). Fig. 9A shows typical DPV curves for different concentrations of furosemide in PBS using Fe₃O₄-GO-SO₃H-GC electrode. The dependency between peak current and drug concentration was rectilinear using Fe₃O₄-GO-SO₃H-GC electrode for peak I (Fig. 9B).

Fig. 9 (A) DPV curves for different concentrations of furosemide in PBS using the Fe₃O₄-GO-SO₃H-GC electrode. From inner to the outer: 0.01, 0.02, 0.04, 0.06, 0.07, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.9, and 1 μM. (B) Dependency of peak I and II current on the concentration of furosemide.

The analytical characteristics of furosemide in PBS using both electrodes are reported in Table 1. The limits of detection (LOD) and quantitation (LOQ) of the procedure were calculated according to the 3 SD/m and 10 SD/m criteria, respectively, where SD is the standard deviation of the intercept and m is the slope of the calibration curves.³⁰ The LOD and LOQ are reported in Table 1; their values confirm the sensitivity of the proposed procedure for the determination of furosemide. The precisions of the method are calculated as the relative standard deviation (RSD). The procedure was repeated on the same day on the same spiked solutions at concentrations in the range of the standard series. The intra-assay RSDs of the proposed method, determined on the basis of peak current for 10 replications. The accuracy of the proposed method was determined by spiking serum and urine samples with different concentrations of furosemide. Good percentage recoveries were obtained from both samples (see Table 2).

Table 1 Parameters determined for calibration curves of furosemide on Fe₃O₄-GO-SO₃H-GC electrode

Analytical parameters	Peak I	Peak II
a Each value was obtained from ten experiments.b LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation.
Linear range (μM)	15–340	17–100
Slope (μA M^−1)	0.964	1.0291
Intercept	0.174	0.271
RSD (%)^a	2.17	2.19
LOD (μM)^b	0.10	0.11
LOQ (μM^−1)^b	0.61	0.50

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Table 2 Results obtained for furosemide analysis from spiked human serum and urine samples using Fe₃O₄-GO-SO₃H-GC electrode

Analytical parameters	Medium
	Serum		Urine
	Peak I	Peak II	Peak I	Peak II
a Each value was obtained from ten experiments.b LOD, limit of detection; RSD, relative standard deviation.c Recovery value is the mean of ten experiments.
Linear range (μM)	20–100	26–100	18–720	18–500
Slope (μA M^−1)	5.73	4.06	1.89	1.80
Intercept	0.6	0.61	4.0	0.93
RSD (%)^a	3.5	4.0	3.6	3.6
LOD (μM)^b	1.1	1.0	1.5	1.5
Recovery (%)^c	94.1	95	103.5	102.0

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The comparison of Fe₃O₄-GO-SO₃H-GC electrode with other sensors for the furosemide determination was listed in Table 3. It can be seen that the Fe₃O₄-GO-SO₃H-GC electrode offered reasonable linear range for furosemide detection and the detection limit was lower than some of previous reports. These results indicated that Fe₃O₄-GO-SO₃H-GC electrode is an appropriate platform for the determination of furosemide. On the other word, the prepared electrode shows voltammetric responses with low detection limit and high linear range for furosemide in optimal conditions, which makes it very suitable for determination of this drug. It is found that, by modification of on GC surface via Fe₃O₄-GO-SO₃H, a novel strategy for developing an efficient and robust electrochemical sensing platform was established. The electrochemical sensor showed high sensitivity and simplicity for detection of furosemide.

Table 3 Analytical parameters for detection of furosemide at several methods

Electrode type	Method	Linear range/(μM)	LOD/(μM)	Ref.
HMDE	DPV	6.05–54.42	2.57	11
Graphite-polyurethane composite electrode		3–9	0.96	12
Multi-walled carbon nanotubes paste electrode		8–200	0.29	13
Gold electrode		6–800	0.4	14
Fe3O4-GO-SO3H-GC electrode		20–100 (serum)	0.1 (serum)	This work
		18–720 (Urine)	0.11 (urine)

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The applicability of the proposed method for the determination of furosemide in biological fluids was examined by measuring the peak I and II currents as functions of the bulk concentration of the drug in urine and serum samples using the Fe₃O₄-GO-SO₃H-GC electrode. The serum and urine samples were diluted 10 and 20 times with PBS before taking the measurements, to prevent the matrix effect of real 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 oxidation of the compounds in either the serum or urine samples appeared. The results obtained from the proposed technique for determining furosemide in serum and urine samples are listed in Table 2. The percentage recovery of furosemide was determined by comparing the peak currents of a known drug concentration in both media with their equivalents in calibration curves; these results are also summarized in Table 2. Good recoveries of furosemide were achieved from these matrices, meaning that application of our proposed voltammetric method to the analysis of furosemide in biological fluids could be easily assessed.

The effect of some common interfering species on the determination of 0.1 mM furosemide at Fe₃O₄-GO-SO₃H-GC was investigated using the optimum measurement conditions. The oxidation peaks of interferents should not appear where the peak corresponds to furosemide appears. Therefore, in order to investigate the effect of some interferent's substances such as glucose, starch, dextrose, sucrose etc. on the voltammetric response of furosemide, this study was carried out. Table 4 shows the tolerance limit for each interferents. The tolerance limit is defined as the concentrations of interfering species which give an error of ≤15% in the determinations of furosemide. The data show that interferences are only significant at relatively high concentrations, confirming that the proposed method is free from common interfering species.

Table 4 Maximum tolerable concentration of interfering species on 0.1 mM furosemide

Interfering species	Interference concentration/mM	ΔI/μA (%)
Oxalic acid	0.1	+7.4
Lactic acid		+7.0
Citric acid		+2.2
Sucrose		−10
Glucose		+5.0
Dextrose		−12.2

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The repeatability of the modified electrode was also investigated. When the modified electrode was stored in the atmosphere, the current response decreased 7.5% after ∼20 days. Furthermore, the current response of the modified electrode in buffer solution, pH 6.8, after 100 rounds of cyclic scanning was almost unchanged. It proved the good stability of the modified electrode. To check the inter-electrode reproducibility of the modified electrode, five modified electrodes were tested simultaneously by recording CVs in buffer solution, pH 6.8, and containing 10 μM of furosemide at a scan rate of 50 mV s⁻¹. The average catalytic peak current is 7.11 μA with a relative standard deviation of 6.0%. The GC electrode modified with Fe₃O₄-GO-SO₃H imparts a higher stability onto voltammetric measurements of furosemide.

4. Conclusion

A glassy carbon electrode was modified with abrasive casting of Fe₃O₄-GO-SO₃H. The electrocatalytic oxidation of furosemide was studied in phosphate buffer solution, pH 6.80, on Fe₃O₄-GO-SO₃H-GC electrode surface. The kinetics of furosemide oxidation on the modified surface was enhanced. This electrocatalytic effect was attributed to the nano-size, special electronics and structure of Fe₃O₄-GO-SO₃H. A differential-pulse voltammetry procedure was optimized and successfully applied for quantification of furosemide in human biological fluids. This sensor can be used as an amperometric detector for routine analysis of furosemide in flow systems when coupled to chromatographic and electrophoretic separation systems.

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. The authors are also grateful to Prof. Jue Wang for his fruitful comments.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra46973e

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