Electrochemical sensitive determination of acetaminophen in pharmaceutical formulations at iron oxide/graphene composite modified electrode

2.1 Apparatus and chemicals

CHI 660E electrochemical workstation (CH Instruments, USA) was used for all electrochemical measurements. All the electrochemical measurements were carried out with GCE or a modified GCE (working electrode), an Ag/AgCl (3.0 M NaCl) electrode (reference electrode) and a platinum wire (axillary electrode). For morphological studies of prepared materials, ZEISS® energy-dispersive X-ray spectroscopy (EDX) coupled with field emission-scanning electron microscopy (FE-SEM) Ultra Plus (Germany) was employed. Preparation of buffer solutions was carried out using pH 60 pH pen (Extech Instruments).

Acetaminophen (Merck, South Africa), potassium chloride (Merck, South Africa), ferric chloride hexahydrate (Merck, South Africa), potassium ferricyanide (Merck, South Africa), graphite powder (Merck, South Africa), potassium ferrocyanide (Sigma-Aldrich, China), magnesium sulfate (RLFCI, India), sucrose (Merck, South Africa), hydrazine hydrate (Sigma-Aldrich, China), glucose (ACE, South Africa), L-lactose (Sigma-Aldrich, Germany) and sodium chloride (Sigma-Aldrich, USA) were used as usual without any further pre-treatment. De-ionised water (DI water) was employed throughout this experiment. Na₂HPO₄·2H₂O (Sigma-Aldrich, Germany) and NaH₂PO₄·2H₂O (Merck, South Africa) were used to prepare buffer solutions.

2.2 Synthesis of Fe₂O₃/RGO composite

Graphene oxide (GO) was synthesized through modified Hummers method (Hummers and Offeman, 1958). In a typical synthesis procedure of Fe₂O₃/RGO, 20 mg of GO was dispersed in 40 mL of DI water and sonicated for 10 min. To this, 0.2 g of ferric chloride (FeCl₃·6H₂O) was added and stirred for 30 min. Then, 15 mL of hydrazine hydrate was added to the above solution and stirred again for 3 h. The resultant product was centrifuged and washed with DI water several times to get Fe₂O₃/RGO. In a similar way, RGO was synthesized in absence of Fe₂O₃.

2.3 Preparation of Fe₂O₃/RGO/GCE

Prior to GCE modification, the surface of GCE was polished using smoothing pads with 0.3 and 0.05 µm of γ-alumina powder. 10 mg of Fe₂O₃/RGO was dispersed into 5 mL of DI water to make a suspension. Then, 6 µL of the Fe₂O₃/RGO suspension was dropped onto the surface of GCE and dried under IR lamp for 5 min. This electrode was denoted as Fe₂O₃/RGO/GCE and used as a working electrode. RGO/GCE was also prepared in a similar way.

3.1 Characterization of nanocomposite

The morphological characterization of RGO and Fe₂O₃/RGO was carried out using FE-SEM. As can be seen in Fig. 1(A), RGO displays the wrinkles of graphene nanosheets. This structure is advantageous to offer a large surface area. Fig. 2(B) displays the morphology of Fe₂O₃/RGO, which reveals that a large number of spindles of Fe₂O₃ were incorporated on the surface of RGO. Such structures prevent the aggregating of RGO and improve the conductivity of Fe₂O₃, which is advantageous for enhancing the electrochemical detection of AC. The energy-dispersive X-ray spectrum (Fig. 1(C)) reveals an elemental composition of Fe₂O₃/RGO nanocomposite. Therefore, it confirms the formation of Fe₂O₃/RGO.

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Fig. 1

Surface characterization of (A) RGO, (B) Fe₂O₃/RGO and (C) EDX spectra of Fe₂O₃/RGO.

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Fig. 2

EIS spectra of 0.1 M KCl containing 2.5 mM [Fe(CN)₆]^3−/4− at modified and bare GCEs. Inset: Randles equivalent circuit model.

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3.2 Electrochemical impedance study and electrode surface area

Electrochemical impedance spectroscopy (EIS) is used to study the charge transfer resistance at the electrode/electrolyte interfaces (Palakollu et al., 2017b). By using this technique, the charge transfer properties of various electrodes were studied in 0.1 M KCl having 2.5 mM [Fe(CN)₆]^3−/4−. The resulting Nyquist plots for modified electrodes and bare GCE were shown in Fig. 2. The obtained impedance data was fitted into the corresponding electronic elements based on the Randle’s equivalent circuit model for estimating the charge transfer resistance (R_ct). From the Randles equivalent circuit model, the charge transfer resistance (R_ct) (the semicircle) was found to be 106.3 Ω at bare GCE and defines least interfacial charge transfer. The R_ct at RGO/GCE was found to be 38.3 Ω, demonstrating the creation of surface with low resistance onto the RGO/GCE that would promote the electron flow rate. However, the R_ct was found to be 10.28 Ω at Fe₂O₃/RGO/GCE, describes least charge transfer resistance and implying that the combination of RGO and Fe₂O₃ could demonstrate the synergistic effect which effectively enhances their electrical conductivity. Therefore, the study results reveal that Fe₂O₃/RGO/GCE facilitates the least charge transfer resistance and high conductivity than RGO/GCE and bare GCE.

The surface area of modified electrodes were calculated using CV response at a 0.1 M KCl containing 2.5 mM K₃[Fe(CN)₆]. For a reversible reaction, the Randles-Sevcik equation (Bard and Faulkner, 2001) is $${\text{i}}_{\text{p}}=\left(2.69\times {10}^5\right)\text{A}{\text{D}}^{1/2}{\text{n}}^{3/2}{\upsilon }^{1/2}\text{C}$$ where i_p is the peak current (Amp), A is the surface area of the electrode (cm²), D is diffusion coefficient of K₃[Fe(CN)₆] (taken to be 7.60 × 10⁻⁶ cm² s⁻¹), n is the number of electrons involved (n = 1 for K₃[Fe(CN)₆]), υ is the scan rate (V s⁻¹) and C is the concentration of K₃[Fe(CN)₆] (mol cm⁻³). By considering the slope of the plots of anodic peak currents vs square root of scan rate (Fig. S1 (A, B)), the surface area of RGO/GCE and Fe₂O₃/RGO/GCE was found to be 0.08216, 0.09981 cm², respectively. Therefore, the surface area of Fe₂O₃/RGO/GCE was higher than RGO/GCE and theoretical geometrical area of GCE (0.07 cm²).

3.3 Electrocatalytic activity of AC

In order to verify the electrocatalytic response of Fe₂O₃/RGO/GCE, cyclic voltammetric (CV) responses of 1 mM AC in 0.1 M PBS (pH 4.0) were recorded at various modified electrodes. As shown in Fig. 3, AC shows a quasi-reversible redox system with relatively poor redox peaks at GCE, indicating slow electron transfer of the redox system of AC at GCE. However, a well-defined redox system of AC with increased peak currents and decreased over potentials was noticed at RGO/GCE suggesting better electron transfer rate at RGO/GCE due to the large surface area of graphene sheets. Interestingly, an enhancement in the peak currents of redox system with a decreased peak to peak separation potential (ΔE_p) of AC was observed at Fe₂O₃/RGO/GCE, which could be attributed to the electronic conductivity and electrocatalytic activity of Fe₂O₃/RGO and their synergy. The intercalation of Fe₂O₃ with RGO decreases the stacking of RGO as well as improves the electronic conductivity and electron transfer ability of the Fe₂O₃/RGO/GCE for AC detection. This recommends that oxidation of AC is extremely favourable at Fe₂O₃/RGO/GCE than the other electrodes. The schematic illustration of electrochemical sensing of AC at Fe₂O₃/RGO/GCE is presented in Scheme 1.

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Fig. 3

CVs of 1 mM AC in 0.1 M PBS (pH 4.0) at a scan rate of 100 mV s⁻¹.

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Scheme 1

Schematic illustration of electrochemical sensing of AC at Fe₂O₃/RGO/GCE.

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The apparent diffusion coefficient (D_app) of AC at modified and bare GCEs were calculated using above said Randles-Sevcik equation (Bard and Faulkner, 2001). The estimated D_app values of AC are given in Table 1. As can be seen in Table 1, it has been concluded that the D_app value at Fe₂O₃/RGO/GCE was higher when compared to bare GCE and RGO/GCE. The higher D_app of AC at Fe₂O₃/RGO/GCE might be due to the good electrocatalytic activity for electro-oxidation of AC at Fe₂O₃/RGO/GCE. This observation is evidence of the electrocatalytic activity of Fe₂O₃/RGO/GCE (Palakollu and Karpoormath, 2018).

3.4 Effect of scan rate

The effect of scan rate on the peak currents of AC in 0.1 M PBS (pH 4.0) at Fe₂O₃/RGO/GCE was investigated from 5 to 250 mV s⁻¹. Fig. 4(A) displays the cyclic voltammograms of AC at different scan rates. From Fig. 4(B), the redox peak currents of AC (i_p) were linearly proportional to the square root of scan rate (ν^1/2) in the range from 5 to 250 mV s⁻¹ indicating diffusion-controlled process. The corresponding linear regression equation(s) can be written as follows. $${\text{i}}_{\text{pa}}\left(\mu \text{A}\right)=-2.21739+1.20566{\nu }^{1/2}{\left(\text{mV}{\text{s}}^{-1}\right)}^{1/2}{\text{R}}^2=0.99154$$ $${\text{i}}_{\text{pc}}\left(\mu \text{A}\right)=0.20224-0.3949{\nu }^{1/2}{\left(\text{mV}{\text{s}}^{-1}\right)}^{1/2}{\text{R}}^2=0.99683$$

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Fig. 4

(A). CVs of 1 mM AC in 0.1 M PBS (pH 4.0) at various scan rates (5–250 mV s⁻¹) at Fe₂O₃/RGO/GCE. (B). Calibration plot of the redox peak currents vs. square root of scan rate.

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3.5 Effect of solution pH

The pH of supporting electrolyte plays an important role in the peak current of an analyte. Moreover, it also provides an insight to the number of protons and electrons involved in electro redox reactions. The electrochemical response of AC at pH 3.5–9.0 (0.1 M PBS) were carried out at Fe₂O₃/RGO/GCE using cyclic voltammetry. Fig. 5(A) displays the cyclic voltammograms of AC at various pH. Moreover, as an increase in the pH of PBS, the anodic peak potentials of AC shifted negatively. From Fig. 5(B), good linearity was attained for pH vs. peak potential of AC. Therefore, the electro-oxidation of AC is pH-dependent behaviour at Fe₂O₃/RGO/GCE. $$\text{E}\left(\text{V}\right)=0.74934-0.04947\text{pH}{\text{R}}^2=0.9922$$

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Fig. 5

(A). CVs of AC at various pH. (B). The plot of i_pa of AC vs. pH (3.5–9.0) and E_pa of AC vs. pH (3.5–9.0) at a scan rate of 100 mV s⁻¹.

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The slope of the calibration curve, 0.049 V/pH is close to Nernstian value 0.592 V/pH at room temperature and recommends that an equal number of electrons and protons are participating in the electrocatalytic-oxidation of AC at Fe₂O₃/RGO/GCE. From the cyclic voltammetric results, the highest peak current and the best peak shape were observed in pH 4.0. Thus, pH 4.0 of PBS was selected for the determination of AC.

3.6 Electrochemical determination of AC

The scope of enhancement in the electrochemical response of Fe₂O₃/RGO/GCE was examined through the determination of AC in 0.1 M PBS (pH 4.0) using DPV technique. From Fig. 6, the peak current was found to increase linearly with the successive addition of AC. Fig. 6 (inset) displays the calibration curve and the corresponding linear regression equation can be written as follows. $${\text{i}}_{\text{p}}\left(\mu \text{A}\right)=0.24183+1.38399\left[\text{AC}\right]\mu \text{M}{\text{R}}^2=0.99368$$

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Fig. 6

DPVs of AC with various concentrations in 0.1 M PBS (pH 4.0). Inset-Calibration plot of concentration of AC vs i_pa.

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The detection limit of AC using the proposed method was found to be 21 nM at a signal-to-noise ratio of 3 in a linear range from 1.0 × 10⁻⁷ to 74 × 10⁻⁶ M. A comparison of the limit of detection obtained using Fe₂O₃/RGO/GCE with recently reported methods has been summarized in Table 2 and obviously specifies an enhancement over the stated values.

3.7 Interference study

The influence of several foreign species in the quantification of AC was studied at Fe₂O₃/RGO/GCE under the experimental conditions (Fig. S2). The displayed results reveal that the presence of K⁺, Ca²⁺, Na⁺, Mg²⁺, SO₄²⁻ and Cl⁻ have not considerably influenced the peak current of AC. The tolerance limit was taken as the maximum concentration of the foreign substances, which caused an approximately ±5% relative error in the AC detection (Palakollu et al., 2017a). Moreover, a 10-fold of uric acid did not affect the peak current of AC. Therefore, the redox peaks of AC could obviously be separated from each other.

3.8 Stability and reproducibility

Long-term stability of the proposed sensor was studied for 7, 14, 21 and 28 days in 50 µM AC in 0.1 M PBS (pH 4.0) (Fig. S3). The responses, from electrodes measurements, were 96.14% (for 7 days), 94.45% (for 14 days), 91.76% (for 21 days) and 87.54% (for 28 days) when compared with day one response. Thus, good long-term stability of the proposed sensor was achieved. Additionally, in order to verify the reproducibility of the nanocomposite-based sensor, 3 Fe₂O₃/RGO composite modified electrodes were prepared under the same experimental conditions to test the cyclic voltammetric response of 50 µM AC in 0.1 M PBS (pH 4.0) (Fig. S4); the RSD was found to be 2.3%, indicating that the sensor has good reproducibility.

3.9 Real sample analysis

To confirm the applicability of Fe₂O₃/RGO nanocomposite modified GCE, the determination of the content of AC in commercially available tablet (Dolo − 650®) sample was carried out at this proposed sensor. The tablet sample was diluted with 0.1 M PBS (pH 4.0) to get the concentration of tablet sample within the linear range. The quantity of AC in the pharmaceutical formulation was estimated using standard addition method (Fig. S5). From Table 3, it can be concluded that the results attained using the proposed sensor displayed a satisfactory agreement with the labelled amount. Moreover, to examine the feasibility of the proposed sensor, the determination of AC in the urine sample was carried out. Prior to the analysis, the collected urine sample was diluted with 0.1 M PBS (pH 4.0). Then, the urine sample was added with an unknown concentration of AC and a series of a known standard sample of AC were spiked into the real sample using standard addition method (Fig. S6). From the calibration curve, the results were summarized in Table 4. The recovery studies reveal that the proposed sensor is reliable and affordable for real sample and pharmaceutical formulations analysis.