Potentiodynamic Poly(resorcinol)-Modified Glassy Carbon Electrode as a Voltammetric Sensor for Determining Cephalexin and Cefadroxil Simultaneously in Pharmaceutical Formulation and Biological Fluid Samples.

This study covers the development of a fast, selective, sensitive, and stable method for the simultaneous determination of cephalosporins (cephalexin (CLN) and cefadroxil (CFL)) in biological fluids and tablet samples using potentiodynamic fabrication of a poly(resorcinol)-modified glassy carbon electrode (poly(reso)/GCE). The results of cyclic voltammetry and electrochemical impedance spectroscopy supported the modification of the GCE by a polymer layer that raised the electrode surface area and conductivity. At the poly(reso)/GCE, an irreversible oxidative peak with four- and fivefold current enhancement for CLN and CFL, respectively, at a substantially lower potential demonstrated the catalytic action of the modifier. Under optimized solution and parameters, the peak current response at the poly(reso)/GCE revealed a linear dependence on the concentration of CLN and CFL within the range 0.1-300 and 0.5-300 μM, respectively, with a limit of detection (LoD) of 3.12 and 8.7 nM, respectively. The levels of CLN in four selected tablet brands and CFL in two tablet brands were in the vicinity of 91.00-103.65% and 97.7-98.83%, respectively, of their nominal values. The recovery results for CLN in pharmaceutical samples were in the range of 99.00-100.67% and for CFL 97.9-99.75% and for blood serum and urine samples 99.55-100.55% and 99.33-100.34% for CLN and 97.13-100.60% and 96.73-102.50% for CFL, respectively. Interference recovery results with errors less than 4.81%, lower LoD, wider dynamic range, excellent recovery results, and good stability of the modifier compared to those for the previously reported methods validated the use of the poly(reso)/GCE for determining CLN and CFL simultaneously in various real samples.

Introduction

Cephalosporins are a class of β-lactam antibiotics, containing a β-lactam ring.[1,2] They work by interfering in the formation of the bacterial cell wall.[1−3]

Cephalexin (CLN) ((6R,7R)-7-{[(2R)-2-amino-2-phenylacetyl]amino}-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid) (Scheme S1A) and cefadroxil (CFL) (6R,7R)-7-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid) (Scheme S1B) belong to the first-generation cephalosporins. They are effective against bacterial infections in the urinary and respiratory tracts (sinusitis, otitis media, pharyngitis, tonsillitis, and bronchitis), stomach pain, vomiting, nausea, diarrhea, joint pain, unpleasant taste, restlessness, and vaginal itching, caused by Gram-positive and Gram-negative bacteria.[4−6] They are semisynthetic broad-spectrum antibiotics administered orally against infections caused by Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli.[4]

According to the World Health Organization, cephalosporins are considered the most common drugs that have developed bacterial resistance.[7] Development of a fast, selective, sensitive, and stable method for the detection of CLN and CFL is essential.

Spectrophotometry,[8,9] chromatography,[10,11] and hyphenated methods[12,13] are among the common techniques reported for the determination of CLN and CFL individually in different samples. Unfortunately, all these methods need tedious sample preparation, are time-consuming, require complex analytical equipment, and are non-ecofriendly.[14,15] On the other hand, owing to their simplicity, low cost, reproducibility, high sensitivity, fast analytical responses, and environmental friendliness, electrochemical techniques are more appealing than conventional ones.[14,15]

Resorcinol (1,3-dihydroxybenzene) (Scheme S2) is ascribed as one of the azo-dyes, which has two hydroxyl groups available for coordination and electropolymerization.[16]

Attempts have been made for application of voltammetric methods for determining CLN and CFL separately in different real samples.[17−19] Because CLN and CFL are both cephalosporins (β-lactam drugs) that share common structures and properties, the development of a sensitive method for their simultaneous determination is vital. In this regard, no electrochemical methods have been reported for the simultaneous determination of CLN and CFL. The electrochemical performance of a working electrode is usually improved with surface modification by enhancing its compatibility, electron conductivity, and surface area.[20,21] Conducting polymers,[19,22] metal-based nanomaterials,[23] and transition metal complexes[21,24−26] are among the commonly reported electrode-modifying materials which are deposited on the surface of the electrode by potentiostatic electropolymerization,[27] potentiodynamic electropolymerization,[21,24] and casting techniques.[28] Hence, this work demonstrated the development and evaluation of an electrochemical sensor based on the poly(reso)/GCE for the simultaneous determination of CLN and CFL in tablet formulations, human urine, and human serum samples using a square wave voltammetric method (preparation explained in the experimental section of the Supporting Information).

Results and Discussion

Fabrication of the Poly(reso)/GCE

For the potentiodynamic deposition of a modifying material onto the electrode surface, the potential scan range was the most important parameter to be optimized. Figure S1 shows the response of a poly(reso)/GCE deposited in various potential scan windows for simultaneous equimolar (1.0 mM) concentration of CLN and CFL.

The poly(reso)/GCE fabricated in the potential window −0.8 to +1.8 V showed the highest catalytic effect toward the oxidation of CLN and CFL interns of the current enhancement with the highest slope change (insets A and B of Figure S1).

Similarly, controlling the film thickness during a potentiodynamic deposition of a modifying material onto the electrode surface is important. In this work, the current response of the modified electrode for the simultaneous determination of equimolar solutions of CLN and CFL as a function of the number of scan cycles (10–30) was investigated (Figure S2). The inset of the figure shows that the anodic peak current of CLN and CFL increased with the number of scan cycles up to 20. However, with a further increase in the number of cycles beyond 20, the current response decreased, which was attributed to the saturation of the active sites in the poly(reso)/GCE (inset). Hence, a poly(reso)/GCE synthesized from reso in pH 7.0 PBS scanned between −0.8 and +1.8 V for 20 scan cycles (Figure ) was selected to determine CLN and CFL simultaneously in tablet formulations, human blood serum, and human urine samples.

Figure 1

Repetitive CVs of GCE in pH 7.0 PBS containing 1.0 mM reso scanned between −0.8 and + 1.8 V at a scan rate of 100 mV s–1 for 20 cycles. Inset: CVs of stabilized (a) bare GCE and (b) poly(reso)/GCE in 0.5 M H2SO4 between −0.8 and +0.8 V at 100 mV s–1.

In the poly(reso)/GCE, the broad cathodic peak (peak a′), which is centered at about −445 mV, and the oxidative peak (peak a) (Figure ) are thought to be the result of the deposited polymer film through radical cation-induced polymerization as a consequence of electron delocalization within the reso ring,[16] and the oxidative peak (peak b) is anticipated to be in response to the oxidation of water.

However, the bare GCE cyclic voltammogram (CV) in 0.5 M H2SO4 containing no analyte (inset curve a) showed only a broad reductive peak above −600 mV ascribed to molecular oxygen reduction, and the appearance of multiple oxidative and reductive peaks from the poly(reso)/GCE (curve b of the inset) further verified redox polymer film deposition on the electrode surface.

Electrochemical Characterization of the Poly(reso)/GCE

The surface of the poly(reso)/GCE was characterized by cyclic voltammetry in 0.1 M KCl solution containing 10.0 mM Fe(CN)6)3–/4– as a redox probe. The peak improvement of both anodic and cathodic peak currents with narrower peak-to-peak separation potential (ΔE = 109 mV) for poly(reso)/GCE compared to GCE (curve a) (ΔE = 415) indicate better electrocatalytic property and increased electron transfer capability of the poly(reso)/GCE (curve b) (Figure ). From the slope value of the plot of Ipa versus ν1/2 of the CV response of each electrode for Fe(CN)63--/4– (Figure S3A and B) using the Randles–Sevcik equation (eq ), the active surface areas of the bare GCE and poly(reso)/GCE were calculated (Table ).where Ipa stands for anodic peak current (A), n the number of electrons transferred (n = 1), A the active surface area of the electrode (cm2), D the diffusion coefficient of the probe (D = 7.6 × 10–6 cm2/s), Co the bulk concentration of the probe, and ν the scan rate (V/s). As shown in Table , the effective surface areas of the two electrodes are in agreement with the trend observed for the current response in Figure . Thus, the presence of reso improved the effective surface area nearly 3.9 times compared to that of the bare GCE.

Figure 2

CVs of (a) GCE and (b) poly(reso)/GCE containing 10.0 mM (Fe(CN)6)3–/4– and 0.1 M KCl in pH 7.0 PBS at a scan rate of 60 mV s–1.

Table 1

Summary of the Calculated Effective Surface Area of the Unmodified GCE and Poly(reso)/GCE

electrode	slope of Ipa vs ν1/2	effective surface area (cm2)
unmodified GCE	4.0	0.054
poly(reso)/GCE	15.5	0.210

Electrochemical Impedance Spectroscopic Characterization

Electrochemical impedance spectroscopy (EIS) was used to evaluate the electron conductivity of the modifier material and verify the electrode modification. Figure presents the Nyquist plots for bare GCE and poly(reso)/GCE. Both electrodes exhibited a semicircle in the high-frequency region with varying diameter and at the low-frequency region a line at about 45° representing the diffusion of the electroactive species from the bulk to the solution–electrode interface.

Figure 3

Nyquist plot of (a) bare GCE and (b) poly(reso)/GCE in pH 7.0 PBS containing 10.0 mM [Fe(CN)6]3–/4– and 0.1 M KCl in the frequency range 0.01–100000 Hz, amplitude 0.01 V, and potential 0.23 V. Inset: proposed equivalent circuit, where Zd stands for the Warburg diffusion constant.

Table S1 lists a summary of the circuit components for the two electrodes, including solution resistance (Rs), charge transfer resistance (Rct), and double layer capacitance (Cdl), as determined from the corresponding Nyquist plot using eq .[21]where CdI is the double layer capacitance, f the frequency corresponding to the maximum imaginary impedance value, and Rct the charge transfer resistance.

The Rct value for the poly(reso)/GCE was lower, demonstrating that the poly(reso) film deposited on the surface of the GCE greatly enhanced the conductivity, and hence the electron transfer rate between the substrate and the analyte increased, which could be attributed to the conductive nature of the polymer film. The electrode surface roughness (RF) and the apparent heterogeneous electron transfer rate constant (k0) of poly(reso) could be calculated using eqs and 4.[24,29,30]where RF is the surface roughness, CdI and CS are the electrochemical double layer capacitance of a planar and smooth electrode surface of the same material measured under the same conditions, respectively, and R is the molar gas constant (8.314 J mol–1 K–1), T is the temperature (298 K), F is the Faraday constant (96485 C mol–1), A is the surface area, Rct is the charge transfer resistance of the electrode, and C is the concentration of [Fe(CN)6]3–/4– (10.0 mM).

From the slopes of Ipa versus ν1/2 for unmodified and poly(reso)-modified electrode surfaces (Figure S3A and B), a roughness factor of 3.9 of the poly(reso)/GCE compared to the bare GCE is found.

The k0 values obtained for the poly(reso)/GCE are 4.0 times higher than those for the bare GCE (Figure ), clearly indicating that the electrochemical activity of the redox probe is improved on the poly(reso)/GCE. Therefore, this reso-modified electrode has the potential for application as a most sensitive electrochemical sensor.

Cyclic Voltammetric Investigation of CLN and CFL

Electrochemical Behavior of CLN and CFL

Cyclic voltammograms of an equimolar concentration (1.0 mM) of CLN and CFL in pH 7.0 PBS at the bare GCE and poly(reso)/GCE were investigated (Figure ). Both CLN and CFL showed irreversible oxidation at both the unmodified GCE and poly(reso)/GCE with different current intensities and peak potentials. In contrast to the weak and broad oxidation peak at the unmodified GCE (inset curve a), the presence of an oxidative peak with four- (CLN) and five- (CFL) fold of current enhancement, respectively, at reduced potential at the poly(reso)/GCE (inset curve b) indicated the modifier catalytic effect toward the simultaneous oxidation of CLN and CFL (Figure ).

Figure 4

Blank-corrected CVs of unmodified GCE (a–c) and poly(reso)/GCE (d–f) in pH 7.0 PBS containing 1.0 mM CFL (a and d), CLN (b and e), and an equimolar mixture of CFL and CLN (c and f) at a scan rate of 100 mV s–1. Inset: background-subtracted CVs of (a) bare GCE and (b) poly(reso)/GCE.

Effect of pH on Ipa and Epa of CLN and CFL

The dependence of the peak current and peak potential of CLN and CFL on the pH of the PBS was evaluated to rationalize whether a proton has participated in the reaction, to determine the proton electron ratio, and to explain the type of interaction between the analyte and the surface of the electrode. The peak potential of oxidation of both CLN and CFL shifted to a more negative direction with an increase in the pH of the PBS from 4.0 to 8.0 (Figure A), showing the proton participation during the oxidation of CLN and CFL at the poly(reso)/GCE. The slope of the plot of Epa versus pH for CLN and CFL was 0.04 and 0.05 V/pH, respectively, showing the involvement of an equal number of electrons and protons.[14,18] Moreover, the oxidative peak current of CLN and CFL at the surface of the poly(reso)/GCE increased with pH value from pH 4.0 to 6.0 and then decreased beyond 6.0 (curve a of Figure B and C). Therefore, pH 6.0 was chosen as the optimum value for the next experiments. This trend might be ascribed to the Coulombic forces of interaction exhibited between the modifier (pKa 9.20 and 10.60)[31,32] and CLN, which has two pKa values (2.56 and 6.88),[33−35] and CFL (pKa 2.48, 7.37, and 9.64, corresponding to the carboxylic acid, amine, and phenol functional groups, respectively).[36]

Figure 5

(A) Blank-corrected SWVs of the poly(reso)/GCE in PBS of various pH values (a–i: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively) containing an equimolar (1.0 mM) mixture of CLN and CFL. Plots of (mean ± %RSD) (B) CLN and (C) CFL: (a) Ip vs pH and (b) Ep vs pH in the entire pH range.

Effect of the Scan Rate on the Ipa and Epa of CLN and CFL

The effect of the scan rate on the peak potential and peak current during the simultaneous determination of CLN and CFL at the poly(reso)/GCE was studied. Figure S4 presents voltammograms of an equimolar mixture (1.0 mM) of CLN and CFL in pH 6.0 PBS at the poly(reso)/GCE in the scan rate from 20 to 300 mV s–1. The observed potential shift with increasing scan rate for both CLN and CFL confirmed the irreversibility of the oxidation of CLN and CFL (Figure S4A). The plot of oxidation peak currents of CLN and CFL against the square root of the scan rate has a better correlation value (R2 = 0.99279 and 0.99621, respectively) than for the scan rate (R2 = 0.97601 and 0.95707, respectively) (Figure S4B and C), suggesting that mass transport kinetics of the oxidation of CLN and CFL at the polymer-modified electrode was predominantly diffusion-controlled.[37,38]

The number of electrons involved (n) and the electron transfer coefficient (α) for the oxidation of CLN and CFL at the poly(reso)/GCE were determined from cyclic voltammetry data and calculated using eqs and 6:[36,39]where α is the electron transfer coefficient, n the number of electrons transferred, EP the peak potential, E° the formal potential, k0 (s–1) the electrochemical rate constant, and the other parameters have their usual meanings.

Ep and Ep1/2 are taken for the cyclic voltammogram at a scan rate of 100 mV s–1 for CLN (1378 and 1433 mV) and CFL (930 and 846 mV), respectively. Accordingly, the value of αn was calculated from eq as 0.87 (CLN) and 0.57 (CFL). Considering α for a totally irreversible electrode process of 0.50,[40] the number of electrons (n) transferred in the electrochemical oxidation of CLN and CFL at the surface of the poly(reso)/GCE was estimated to be 1.74 (∼2.0) and 1.14 (∼1.0), respectively.

Moreover, from the slope of the plot of Ep versus ln(ν) for both CLN and CFL (Figure S5) (slope = 0.013/n(1 – α)) of 0.035 for CLN and 0.032 for CFL, the value of n(1 – α) at the experimental temperature of 25 °C calculated using eq was 0.37 and 0.41, respectively. Taking the two and one electron for oxidation of CLN and CFL, respectively, calculated using eq , the electron transfer coefficient (α) was estimated to be 0.81, and 0.59, respectively, confirming the irreversibility of the simultaneous oxidation of CFL and CLN at the surface of the poly(reso)/GCE.[39]

On the basis of the computed kinetic parameters (n and α) and proton to electron ratio (1:1), a reaction mechanism was proposed (Scheme S3), which is consistent with the literature.[18,41]

SWV Investigation of CLN and CFL at Poly(reso)/GCE

Cyclic voltammetry is less effective than square wave voltammetry at separating the Faradaic current from the non-Faradaic current.[39,42] It was selected to determine CLN and CFL simultaneously in human serum and tablet samples. Figure shows square wave voltammograms (SWVs) of an equimolar mixture (1.0 mM) of CLN and CFL in pH 6.0 PBS at both unmodified GCE and poly(reso)/GCE.

Figure 6

SWVs of unmodified GCE (a and b) and poly(reso)/GCE (c and d) in pH 6.0 PBS containing no CLN and CFL (a and c) and an equimolar (1.0 mM) mixture of CLN and CFL (b and d) at step potential 4 mV, amplitude 25 mV, and frequency 15 Hz. Inset: blank-corrected SWVs of (a) unmodified GCE and (b) poly(reso)/GCE.

In contrast to the oxidative peak for CLN and CFL at the unmodified electrode (curve a of the inset), the appearance of well-shaped oxidative peaks with about 4- and 3.5-fold of current enhancement at great overpotential reduction at the poly(reso)/GCE (curve b of inset) confirmed the catalytic role of the poly(reso) film in the oxidation of CLN and CFL.

Optimization of Square Wave Voltammetric Parameters

The dependence of the SWV peak current response of the poly(reso)/GCE for CLN and CFL in pH 6.0 PBS on square wave parameters such as step potential, amplitude, and frequency was investigated. The peak current increment of CLN and CFL with the increase of each SWV parameter is theoretically expected. The step potential of 8 mV, the amplitude of 35 mV, and the frequency of 20 Hz were chosen as the ideal values as a compromise between the current increment and related capacitive current (Figure S6A–C).

Calibration Curve for the Simultaneous Determination of CLN and CFL and the Detection Limit

A calibration curve to determine CLN and CFL simultaneously was evaluated by varying their concentrations under optimized conditions. Moreover, a calibration curve for the simultaneous determination of CLN and CFL was obtained for each in the presence of a constant amount of the other. The SWV displayed two peaks that were suitably separated and well-shaped peaks for CLN and CFL whose current grew as the concentration did over the entire range (0.1–300.0 μM) (Figure A). Similarly, under the optimized SWV parameters, the CFL current response at a fixed concentration (80.0 μM) of CLN (Figure B) showed a linear dependence in the concentration range of 5.0 × 10–7–3.0 × 10–4 M. While, the current response for CLN (Figure C) varied with its concentration in the range 1.0 × 10–7–3.0 × 10–4 M at a fixed concentration of CFL (80.0 μM CFL). Interestingly, it was observed that no significant change occurred in the current response of CLN with successive additions of different concentrations of CFL and vice versa.

Figure 7

Corrected for blank SWVs of poly(reso)/GCE in pH 6.0 PBS containing various concentrations of (A) equimolar mixtures of CLN and CFL (a–m: 0.1, 0.5, 1.0, 5.0, 10.0, 20.0, 40.0, 80.0, 120.0, 160.0, 200.0, 250.0, and 300.0 μM, respectively), (B) CFL (a–l: 0.5, 1.0, 5.0, 10.0, 20.0, 40.0, 80.0, 120.0, 160.0, 200.0, 250.0, and 300.0 μM, respectively) and 80.0 μM CLN, and (C) CLN (a–m: 0.1, 0.5, 1.0, 5.0, 10.0, 20.0, 40.0, 80.0, 120.0, 160.0, 200.0, 250.0, and 300.0 μM, respectively) and 80.0 μM CFL at step potential 8 mV, amplitude 35 mV, and frequency 20 Hz. Insets: respective plot of oxidative peak current (%RSD as an error bar) vs concentration.

The limit of detection (LoD = 3 s/m, for n = 7) was 3.12 and 8.7 nM, and the limit of quantification (LoQ = 10 s/m) was 10.4 and 29.0 nM, for CLN and CFL, respectively. The low %RSD values (below 4.1% for CLN and 3.7% for CFL for n = 3) associated with current measurement for the standard CLN and CFL solutions showed better precision and hence confirmed the applicability of the proposed method based on the poly(reso)/GCE for the simultaneous determination of CLN and CFL.

Simultaneous Determination of CLN and CFL in Real Samples Using Poly(reso)/GCE

Tablet Samples

The applicability of the proposed sensor for the simultaneous determination of CLN and CFL content in each tablet sample listed under the experimental section found in Supporting Information was investigated. The square wave voltammograms for tablet samples with 20.0 and 40.0 μM nominal CLN and CFL concentration for each tablet brand are presented in Figure S7A and B. The voltammograms showed the absence of CFL in the CLN tablets and CLN in the CFL tablet samples.

However, the detected CLN and CFL content was in the range 91.00–103.65% and 97.7–98.83%, respectively, of what was expected with %RSD value below 3.5% and 3.0% for CLN and CFL, respectively (Table S2). Very good agreement between the detected CLN and CFL amounts with the company’s label showed the accuracy and precision of the developed method.

Human Blood Serum Sample

The proposed method was also employed for the determination of both CLN and CFL in a human blood serum sample. The absence of a visible peak at the characteristic potential of the two analytes (curve a of Figure S8) indicates the absence of CLN and CFL in the analyzed human blood serum sample at a detectable level.

Human Urine Sample

The developed method was also used to determine CLN and CFL in a human urine sample. The SWV for the unspiked urine sample (curve a of Figure S9) showed a visible peak far from the CLN and CFL characteristic potential (∼620 mV), which was assigned as creatinine, indicating the absence of CLN and CFL at a detectable level in the analyzed urine sample, which concurs with our earlier report.[19,23]

Validation of the Developed Method

Spike Recovery Studies

Human Blood Serum Sample

Figure S8 presents the recovery analysis of human blood serum samples spiked with 40.0, 60.0, and 80.0 μM equimolar mixtures of CLN and CFL. As can be seen from the figure, the unspiked serum sample showed no peak for either CLN or CFL (curve a), and the spiked samples revealed two peaks; one for CLN and the other for CFL at respective potentials. Excellent recovery results (Table S3) in the range of 99.55–100.55% for CLN and 97.13–100.60% for CFL confirmed that the developed method for the simultaneous determination of CLN and CFL in biological fluids such as human blood serum was accurate. Moreover, a similar current for an equal concentration of CLN and CFL in the samples showed the reproducibility of the results.

Human Urine Sample

Urine samples spiked with 40.0, 60.0. 80.0 and 100.0 μM equimolar mixtures of CLN and CFL were analyzed. As shown in Figure S9, the spiked urine samples revealed two new peaks at the respective potentials of CFL and CLN, confirming initially the absence of CLN and CFL in the analyzed urine samples, while the peak intensity for the peak a′ remained constant. The spike recovery results presented in Table S4, in the range 99.33–100.34% for CLN and 96.73–102.50% for CFL, showed the accuracy of the proposed method and hence validated the method for the simultaneous determination of CLN and CFL in urine samples.

Tablet Samples

The applicability of the developed method for the determination of CLN and CFL in real samples was evaluated by a spiking experiment. In previously analyzed Felexin, Salexin, and Drox brand tablet samples spiked with various concentrations of standard solutions of CLN and CFL, an increment in the peak current was observed in proportion to the amounts spiked (Figure S10A–C). The recovery results were in the range of 99.00–100.67% for CLN and 97.9–99.75% for CFL in both tablet samples (Table S5), further confirming the accuracy of the developed method based on the poly(reso)/GCE for its applicability for determining CLN and CFL simultaneously in real samples.

Interference Study

The selectivity of the sensor (poly(reso/GCE) for the simultaneous detection of CLN and CFL in the presence of potentially interfering substances, such as ascorbic acid (AA), ampicillin (AMP), cloxacillin (CLOX), and glucose (Glu), was examined (Figure S11). Because both CLN and CFL tablets contained only themselves, a mixture of the two tablet samples was analyzed in the recovery section with equimolar concentrations (40 μM) of Felexin and Drox brands when various levels (20.0–80.0 μM) of the selected potential interferents were present. The detection of the claimed CLN and CFL with an associated error of less than 4.81% even in the presence of the potential interferents at their different levels (Table S6) confirmed the validity of the proposed method for the selective determination of CLN and CFL in the presence of the interferents.

Stability and Reproducibility Studies

The stability and repeatability of the modified electrode and the reproducibility of the results were evaluated by recording five successive SWVs of the modified electrode in pH 6.0 PBS containing an equimolar (1.0 mM) mixture of CLN and CFL at an interval of 2 h in a day with an error of 1.86 and 2.28% (%RSD), respectively (Figure S12A). Also with an associated error of 2.95% for CLN and 3.46% for CFL, five SWV measurements in 20 days recorded at an interval of 4 days (Figure S12B) showed the stability of the modifier and hence the reproducibility of the results. In general, the precision, accuracy, selectivity, reproducibility of the results, and stability of the novel electrode modifier were validated for the simultaneous determination of CLN and CFL in real samples.

Comparison of the Present Method with Previously Reported Methods

The effectiveness of the developed method in this work was compared with literature-reported methods considering the linear dynamic range, limit of detection, nature of the substrate, and type of modifier.

The present method based on the poly(reso)/GCE provides the least limit of detection with a wider linear dynamic range than the other reported methods (Table ). Therefore, the present method using the reso modifier can be an excellent candidate for the simultaneous determination of CLN and CFL in various real samples.

Table 2

Comparative Performance of the Developed Sensor to the Selected Reported Works

substrate	modifier	method	analyte	dynamic range (μM)	LOD (μM)	ref
GCE	nano-Ag-APME	SW-AdSV	CFL	0.033–0.304 and 10–70	0.01 and 0.03	(17)
HgE		polarographic	CLN	0.1–25.0	0.05	(18)
GCE	MPTS-MWCNT	DPV	CLN	0.5–50.0	0.12	(37)
GCE	AuNP/MWCNT	amperometry	CFL	2.0–10.0	0.22	(41)
CPE	NiONPs	amperometric	CLN	2.5–35	1.3	(43)
				65–1230
GCE	poly(reso)/GCE	SWV	CLN	0.1–300	0.00312	this work
			CFL	0.5–300	0.0087

Conclusions

Cyclic voltammetry and electrochemical impedance spectroscopy were employed to verify the deposition of an electroactive poly(reso) film on the surface of a glassy carbon electrode. The excellent electrocatalytic activity of poly(reso)/GCE toward the simultaneous oxidation of CLN and CFL in PBS might be due to the increased effective surface area, electrical conductivity, and surface roughness of the electrode. The SWV application of poly(reso)/GCE for determining CLN and CFL simultaneously in four and two selected brands of tablet formulation, respectively, human blood serum and human urine samples, is reported for the first time.

The CLN and CFL content of the examined tablet samples ranged between 91.00 and 103.65% and between 97.7 and 98.83% of their nominal labels, respectively, under optimized solution and SWV parameters, confirming the efficiency of the developed method. Excellent spike recovery results of CLN and CFL in the range 99.55–100.55% and 97.13–100.60% in human blood serum samples, 99.33–100.34% and 96.73–102.50% in urine samples, and 99.00–100.67%, and 97.9–99.75% in tablet samples, respectively, showed the accuracy of the method.

Excellent precision, a wide dynamic concentration range, reasonably good spike, and interference recovery results, low detection limit, high stability of the developed sensor, and reproducibility of the results confirmed the practicality of the developed method for the simultaneous determination of CLN and CFL in tablet and blood serum samples, making the present method an excellent candidate for determining CFL and CLN simultaneously in real samples with a complex matrix including pharmaceutical, urine, and serum samples.