Warfarin is a blood anticoagulant that inhibits the function of vitamin K dependent coagulation. Warfarin is used to inhibit the coagulation of blood to reduce or prevent the chance of developing heart attacks, strokes, and venous and other blood clots. The common side effects of warfarin are easy bruising and bleeding, nausea, vomiting, stomach pain, bloating, gas, or altered sense of taste. The most serious drawback of using warfarin is hemorrhage, which can be life-threatening and even cause death. Therefore, detection of warfarin in biological and clinical samples is very important [1, 2, 3]. Several analytical techniques have been developed for determination of warfarin in biological fluid including micellar electrokinetic chromatography electrospray ionization mass spectrometry (MEKC-ESI- MS) [4], supercritical fluid chromatography-tandem mass spectrometry (SFCMS/MS) [5], high performance liquid chromatography (HPLC) using ultraviolet or fluorescence detection [6, 7, 8, 9, 10, 11, 12], capillary zone electrophoresis (CZE) [13, 14, 15] and square-wave adsorptive cathodic stripping voltammetry with hanging mecury electrode [16]. However, these methods suffer from serious problems such as expensive chemical materials for electrode modification, low stability, and high toxicity, and they are time consuming. Recently, several papers have introduced new modified electrodes for the determination of warfarin in pharmaceuticals and biological fluids like urine and plasma [17, 18, 19].

Chemically modified carbon paste electrodes (CPEs) are endowed with many good qualities, such as ease of handling and applicability to anodic oxidations. Different modifiers for carbon paste have been reported in the last years for the electrochemical analysis of drugs. In recent years, magnetic nanoparticles (MNPs) have attracted a growing interest in the development and fabrication of sensors and biosensors. MNPs exhibit the best performance at sizes of 10-20 nm owing to supermagnetism, which makes them especially suitable when looking for a fast response, large surface area, and high mass transfer [20, 21]. In 2015, Gholivand et al. [17] reported an electrochemical sensor based on Fe₃O₄ MNPs modified CPE for the determination of warfarin. Although this method is sensitive, its application is difficult because of stability limitations. Therefore, a modified material is needed to make them more stable and to prevent their aggregation.

In this work, we describe a novel strategy for the determination of warfarin using a paste electrode containing multiwalled carbon nanotubes (MWCNTs) plus a new magnetic nanoparticles (ZnCrFeO₄). The modified electrode has a catalytic effect on the oxidation current of warfarin and it shows advantages in terms of high sensitivity, reproducibility, and selectivity. Moreover, the stability is enhanced greatly compared with previously modified electrodes owing to the introduction of spinel-structured ZnCrFeO₄ nanoparticles. The analytical feasibility of the approach is examined by measuring warfarin content in different real samples with satisfactory results.

All voltammetric measurements were carried out using an electrochemical system comprising a Metrohm instrument (Herisau, Switzerland), Model 797 VA and a conventional three-electrode cell assembly containing an Ag/AgCl electrode as the reference electrode, a platinum wire as the counter electrode and the MWCNT/ZnCrFeO₄/CPEs as the working electrode. All of the potentials were measured and reported vs Ag/AgCl reference electrode. The pH of the solutions was controlled with a Corning pH meter (model 146). The structure and morphology of the product were characterized by using X-ray diffraction (XRD) (Holland Philips Xpert, X-ray diffractometer with Cu-K_α radiation) and field emission scanning electron microscope (FE-SEM) (Hitachi S-4160) with gold coating. Fourier-transform infrared spectroscopy (FT-IR) was recorded using a JASCO FT-IR (680 plus). The spectra of solids were obtained using a KBr pellet. The analysis of the chemical composition of the modified electrode was performed using an energy dispersive spectrometer (EDX).

All chemicals were of analytical reagent grade and purchased from Merck (Darmstadt, Germany) except where otherwise stated. A stock solution of 0.01 mol/L warfarin was prepared by dissolving a suitable amount of sodium warfarin (Sigma-Aldrich) in water and the solution was diluted to 25-mL with water in a 25 mL volumetric flask. Working solutions were prepared by diluting the stock solution with deionized water.

Phosphate buffer solutions (0.10 mol/L) with different pH values were used. Pure graphite powder (particle size <50 µm) and MWCNT (>90% MWCNT basis, with a diameter of 20-30 nm and a length of 5-15 µm) were prepared from Iran’s Research Institute of Petroleum Industry. High-viscosity paraffin (d=0.88 kg/L) was used for the preparation of paste electrodes.

The synthesis of ZnCrFeO₄ nanoparticles used for this study followed a method reported by Hamed et al. [21, 22]. Fe(NO₃)₃·9H₂O (8.07 g), Zn(NO₃)₂·6H₂O (5.94 g), and Cr(NO₃)₃·9H₂O (8.00 g) were mixed with 100 mL methanol to form a sol and the mixed solution was adjusted to pH~9 with ammonium hydroxide solution. After stirring the mixture for 20 min at 80℃, stirring was continued for 24 h at room temperature. The product was washed with twice-distilled water several times and dried at 60℃. After that, heat treatment of the product was carried out for 1 h at 700℃, then for another 2 h at 900℃.

To eliminate metal oxide catalysts within the nanotubes, MWCNTs were refluxed in 2.0 mol/L HNO₃ for 15 h, and then washed with twice-distilled water and dried at room temperature. HNO₃ usually causes a significant destruction of carbon nanotubes and introduces -COOH groups at the ends or at the sidewall defects in the nanotube structure.

The MWCNT/ZnCrFeO₄-modified electrode was prepared by mixing 30 mg of ZnCrFeO₄, 120 mg of MWCNT, and 850 mg of graphite powder. Diethyl ether was added and mixed to get a uniform mixture. After evaporation of the diethyl ether, 200 mg paraffin oil was added and the solid was mixed with mortar and pestle to obtain a uniformly wetted paste. The paste was then packed into a glass tube. Electrical contact was made by pushing a copper wire down the glass tube into the back of the mixture. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing it on weighing paper. The multiwalled carbon nanotubes/ carbon-paste electrode (CNPE) was prepared in the same way, but without adding ZnCrFeO₄. The unmodified CPE was prepared by mixing fine graphite powder with the appropriate amount of paraffin and thorough hand mixing in a mortar and pestle.

MWCNT/ZnCrFeO₄/CPEs was polished with white and clean paper. To prepare a blank solution, 10.0 mL of buffer solution (PBS, pH 4.0) was transferred into an electrochemical cell. The initial and final potentials were adjusted to 0.70 and 1.15 V vs Ag/AgCl, respectively. The differential pulse voltammogram (DPV) was recorded with pulse amplitude of 100 mV, pulse time of 50 ms, and sweep rate of 50 mV/s to give the blank signal, and labeled as I_pb. Then, different amounts of warfarin solution were added to the cell using a micropipette, and the DPV was recorded again to get the analytical signal (I_ps). The difference in current (I_ps-I_pb) was considered as a net signal (∆I) for each of the species. Calibration graphs were prepared by plotting the net peak currents versus warfarin concentrations in solution.

Urine samples were centrifuged (2000 r/min for 5 min) and the supernatant was filtered using a 0.45-μm filter. Then, 1.0 mL of the sample solution and 9.0 mL of 0.10 mol/L buffer (pH 4.0) were transferred into the cell to measure the warfarin content using the standard addition method.

Serum samples were obtained and stored frozen until the analysis. For preparation of serum samples, 1.0 mL of each sample was diluted to 10.0 mL in a voltammetric flask by phosphate buffer solution (pH 4.0). Then, 5 mL of this solution was transferred to the voltammetric cell, and were diluted to 10 mL with phosphate buffer solution. To this solution, different amounts of warfarin was added and the recovery percent was obtained by DPV technique and standard addition method.

The pharmaceutical samples were chosen from a warfarin tablet (labeled 5 mg of warfarin per tablet, Orion Co.), which was completely ground and homogenized, 2.31 g of which was accurately weighed and dissolved with ultrasonication in 25 mL of water. After mixing completely, the mixture was filtered with an ordinary filter paper. Then, 10 mL of the filtered solution was transferred into a 100 mL volumetric flask and the solution was diluted to the mark with water. Then, 200 µL of the solution plus 5 mL of the buffer (pH 4.0) was diluted with water in a 10-mL volumetric flask and the resulting solution was used for analysis. Then, the diluted sample solutions were placed in an electrochemical cell to determine their concentrations using the DPV method.

Sensing strategies based on MNPs offer advantages in terms of analytical figures of merit, such as enhanced sensitivity, low limit of detection (LOD), high signal-to-noise ratio, and shorter time of analysis than non-MNP-based strategies [23]. In addition, MNPs enhance sensitivity and stability of sensors and biosensors [23]. Therefore, a variety of spinel ferrite nanoparticles such as CoFe₂O₄, Fe₃O₄, MgFe₂O₄, ZnFe₂O₄, NiFe₂O₄, and ZnCrFeO₄ were used in the preparation of the modified electrode. The best electrocatalytic effect for determination of warfarin is obtained with ZnCrFeO₄ nanoparticles (Fig. 1). The excellent conductivity of ZnCrFeO₄ makes it a suitable mediator in the determination of warfarin. Furthermore, ZnCrFeO₄ nanoparticles are important kinds of magnetic materials with unique properties such as supermagnetic, nontoxicity, biocompatibility, and ease of synthesis [21, 22].

Fig. 1. Effect of spinel ferrite nanoparticles types in the determination of 1000 µmol/L warfarin. (1) MgFe2O4; (2) NiFe2O4; (3) CoFe2O4; (4) Fe3O4; (5) CuFeO4; (6) ZnFe2O4; (7) ZnFeCrO4.

The structure and morphology of the product were characterized by using XRD, FE-SEM, and FT-IR spectroscopy. Fig. 2(a) shows characteristic peaks that occur at 2θ of 30.08°, 35.36°, 37.08°, 43.08°, 53.27°, 56.93°, and 62.44°, which are marked by their corresponding crystallographic planes (220), (311), (222), (400), (422), (511), and (440), respectively. The XRD pattern confirms the formation of ZnCrFeO₄ spinel phase (cubic, JCPDS 43-0554).The FE-SEM image of the product shows that the morphology of the sample is spherical with nanometer dimensions (Fig. 2(b)).

Fig. 2. (a) XRD pattern of the ZnCrFeO4 nanoparticles, (b) FE-SEM image of ZnCrFeO4, and (c) FT-IR spectrum of ZnCrFeO4 nanoparticles.

Fig. 2(c) shows the FT-IR spectrum of ZnCrFeO₄. It clearly shows three principle absorption bands of Fe³⁺-O²⁻, Cr³⁺-O²⁻, and Zn²⁺-O²⁻ located at approximately 417, 490, and 615 cm⁻¹, respectively, for all the calcined samples. These bonds are associated with ZnCrFeO₄, and this indicates the formation of spinel ZnCrFeO₄ nanocrystals, as suggested by previously published data [24, 25]. The peaks observed at approximately 3415 and 1544 cm⁻¹ are ascribed to the stretching modes and H-O-H bending vibration of the free or absorbed water molecules on the surface of the ZnCrFeO₄ [21, 26]. In addition, the broad band centered at 3415 cm⁻¹ can be also assigned to a hydrogen bonded O-H stretching arising from surface hydroxyl groups on the spinel nanoparticles [27]. The absence of the peaks at 1000-1300 and 2000-3000 cm⁻¹ in the samples confirmed that the O-H and C-O stretching mode of organic sources in the calcined samples is not present [21, 28].

The real surface areas of the MWCNT/ZnCrFeO₄/CPEs, MWCNT/CPE, and CPE were obtained by cyclic voltammetry (CV) using 1 mmol/L K₃Fe(CN)₆ as a probe at different scan rates. For a reversible process, the Randles-Sevcik formula,

I_pa=2.69 × 10⁵ n^3/2 A C₀ D_R^1/2 ν ^1/2 (1)

was used, where I_pa (A) refers to the anodic peak current, n is the electron transfer number, A is the surface area of the electrode, D_R represents the diffusion coefficient, C₀ (mol/mL) stands for the concentration of K₃Fe(CN)₆, and ν is the scan rate. For 1 mmol/L K₃Fe(CN)₆ in the 0.1 mol/L KCl electrolyte, n=1 and D_R=7.6×10⁻⁶ cm²/s, and then from the slope of the I_pa-ν ^1/2 relation, the real surface areas were calculated. The results showed that the electrode surface area was 0.0426 cm² for the CPE, 0.0664 cm² for the MWCNT/CPE, and about 0.0721 cm² for the MWCNT/ZnCrFeO₄/CPEs. This means that the surface areas of the MWCNT/CPE and MWCNT/ZnCrFeO₄/CPEs are 1.56 and 1.69 times higher than that of CPE, respectively.

The direct electrochemistry of warfarin at the surface of the modified electrode was investigated using DPVs. Fig. 3 shows DPVs of different electrodes in a buffer solution (pH=4.0) in the absence or presence of 900 μmol/L warfarin. In the absence of warfarin no electrochemical responses were obtained on the bare carbon paste electrode, indicating that no electroactive substance existed on the electrode surface. For the carbon paste electrode, an electrochemical signal of warfarin was obtained with an I_pa of 12.2 μA and an oxidation potential of +0.99 V (Fig. 3, curve (5)). In contrast, at ZnCrFeO₄/CPE, an I_pa of 16.4 μA and an oxidation potential of 0.970 V was obtained (Fig. 3, curve (6)). These small changes in the peak potential indicated that ZnCrFeO₄ showed a small catalytic effect. The oxidation current of warfarin at CNPE was enhanced by 1.74 times compared with a bare electrode (Fig. 3, curve (7)). The reason for better performance of the MWCNT modified electrode may be as a result of the nanometer scale dimensions of the MWCNT, the electronic structure, and the topological defects (edge plane-like sites/defects) present on the MWCNT surfaces. Meanwhile, the MWCNT increase the effective area of the electrode. However, the largest oxidation current (30.0 µA at +0.97 V ) indicates that the synchronous introduction of MWCNT and ZnCrFeO₄ would amplify the peak current of warfarin compared with other electrodes. These results show that a further 2.40 times increase in the peak current of the analyte at the surface of the MWCNT/ZnCrFeO₄/CPEs is not only as a result of the surface area, but also from the synergic effect of the MWCNT and ZnCrFeO₄ nanoparticles. In acidic media (pH 4.0), hydroxyls are known to be present on the surface of spinels used as catalysis [29, 30]. In fact, H adsorption sites of spinel create hydroxyl groups on the surface of spinel [29]. The synergic effect of MWCNT and ZnCrFeO₄ could be from electrostatic interaction between the carboxyl groups attached to the nanotubes [31] and these hydroxyl groups, leading to enhancement in the oxidation current of warfarin.

Fig. 3. (a) Differential pulse voltammograms of the blank solution at (1) CPE, (2) ZnCrFeO4/CPE, (3) CNPE, and (4) MWCNT/ZnCrFeO4/CPEs; (b) Differential pulse voltammograms of warfarin (900.0 µmol/L) at (5) CPE, (6) ZnCrFeO4/CPE, (g) CNPE, and (8) MWCNT/ZnCrFeO4/CPEs. DPV experimental conditions: pulse amplitude of 100 mV, pulse time of 50 ms, sweep rate of 50 mV/s; phosphate buffer (0.1 mol/L, pH 4.0).

The effect of the scan rate on the anodic peak current of warfarin at MWCNT/ZnCrFeO₄/CPEs film coated GC electrode was studied. The results showed that by increasing the scan rate the I_pa increased gradually (Fig. 4). The good linear relationship holding between ν^1/2 and I_pa within a scan rate of 10-130 mV/s confirms a diffusion-controlled process on the modified electrode (R²=0.9991).

Fig. 4. Cyclic voltammograms of 800 µmol/L warfarin at MWCNT/ZnCrFeO4/CPEs with various scan rates. (1) 10 mV/s; (2) 30 mV/s; (3) 50 mV/s; (4) 70 mV/s; (5) 90 mV/s; (6) 130 mV/s.

To find the optimum conditions with the highest sensitivity for determination of warfarin, the influence of various parameters such as DPV parameters, the mass ratio of nano particles to MWCNT, and pH on the peak current was studied.

Because DPV has a much higher sensitivity and resolution than CV, DPV was used for determination of warfarin. The DPV parameters changed when the concentration of warfarin was 400 µmol/L. The results showed that the maximum peak current was obtained with a pulse amplitude of 100 mV, a pulse time of 50 ms, and a voltage step time of 0.1 s. These values were selected for further study.

To obtain a maximum current with suitable potential of the proposed electrode toward warfarin, the effect of the mass ratio of ZnCrFeO₄ to MWCNT was examined. The amount of MWCNT was optimized when the percentage of ZnCrFeO₄ nanoparticles was fixed at 10% of the entire amount of paste material. Consequently, the maximum oxidation current was obtained in the electrode containing 10% of MWCNT of the total amount of paste material. If the proportion of MWCNT is increased further, the electrode becomes rigid and hard to polish. Therefore the renewed surface cannot be obtained easily. To optimize the amount of ZnCrFeO₄ nanoparticles in the paste, the percentage of MWCNT was fixed at its optimized value and then, the percentage of ZnCrFeO₄ nanoparticles was changed. In this case the maximum current was observed with the electrode with 2.5% of ZnCrFeO₄ nanoparticles. So, the ratio of 2.5/10 for ZnCrFeO₄/MWCNT of the total amount of paste material was selected as the optimized ratio. It is worth mentioning that the peak potential of warfarin was not shifted significantly in all constructed electrodes.

The influence of the solution pH on the oxidation peak potential and peak current was investigated by DPV in the pH range 3.0-8.0. As shown in Fig. 5, the maximum current obtained in pH range 3.0-5.0, whereas at pH values higher than 5, a decreased warfarin peak current was observed. Therefore, for the determination of warfarin, a pH of 4.0 (PBS, 0.1 mol/L) was selected for further study. Furthermore, it can be seen that the oxidation peak potential of warfarin shifts negatively with rising pH, demonstrating that deprotonation is involved in the oxidation process. The value of proton charge in the electrode reaction was estimated by [32]:

E_p=E₀-0.059p/n pH (2)

Fig. 5. Dependence of oxidation peak potential of warfarin with pH at the modified CPE.

The plot of E_p versus pH gives a linear equation as E_p(V)=−0.039pH+1.178. The slope of dE/dpH indicates that the number of electrons is two times more than the proton that participated in the oxidation process. As previously proven [17], the oxidation mechanism of warfarin contains two electrons and one proton exchange. Fig. 5 shows a definite break corresponding to the apparent pK_a value 6.0. The value of pK_a is in excellent agreement with the literature [33]. Therefore, according to the above results the oxidation mechanism of warfarin at the surface of the modified electrode is proposed in Scheme 1. In the ZnCrFeO₄ molecules Zn(+2), Cr(+3), and Fe(+3) (Cr₂O₇²⁻+14H⁺+6e⁻→ 2Cr³⁺+7H₂O, E⁰=1.33 V irriversible) cannot be oxidized at the surface of the electrode in the range of 0.0 to +1.3 V and we cannot see any oxidation peak of ZnCrFeO₄ in buffer solution. The electrocatalytic effect of these magnetic nanoparticles is related to the synergic effect (between MWCNT and ZnCrFeO₄) as a result of the increase of the microscopic surface area and facilitation of electron transfer on the surface of the electrode.

Scheme 1. The oxidation mechanism of warfarin at the surface of the modified electrode.

Fig. 6 compares the morphological features of CNPE and MWCNT/ZnCrFeO₄/CPEs using FE-SEM. Significant differences in the surface structure of the two electrodes were observed. In fact, ZnCrFeO₄ at MWCNT did not change the morphology of MWCNT. In addition, it can be clearly seen that MWCNT and ZnCrFeO₄ dispersed on the paste. A quantitative EDX analysis of ZnCrFeO₄ demonstrates that the molar ratio of Cr, Zn, and Fe atoms is about 1:1.1, which is consistent with the stiochiometric composition of ZnCrFeO₄ nanoparticles. The EDS dot-mapping of elements in MWCNT/ZnCrFeO₄/CPEs also shows that spinel nanoparticles relatively uniformly distributed on the modified electrode (Fig. 6(d)).

Fig. 6. SEM images of (a) CNPE and (b) MWCNT/ZnCrFeO4/CPEs; (c) The corresponding EDX spectrum taken from the whole area of (b); (d) EDX dot-mapping of Zn, Cr, Fe, and O at the modified electrode.

Electrochemical impedance spectroscopy (EIS) gives insight into properties of the electrode surface and electrolyte. The Nyquist plots of the electrodes were obtained in ac frequency range of 0.1 Hz to 100 kHz in 1 mmol/L [Fe(CN)₆]^3−/4− solution (Fig. 7). The charge transfer resistance (R_ct) of Fe²⁺/Fe³⁺ at the bare electrode, ZnCrFeO₄/CPE, CNPE, and MWCNT/ZnCrFeO₄/ CPEs are 1100, 700, 580, and 420 Ω, respectively. This demonstrates that MWCNT and ZnCrFeO₄ accelerate the interfacial electron transfer between the electrode and the detection solution.

The typical single step chronoamperometry of MWCNT/ ZnCrFeO₄-modified electrode was performed with successive addition of warfarin solution (pH=4.0) by setting working potential at +0.70 V vs Ag/AgCl electrode. A plot of I versus t^−1/2 for different concentration of warfarin (in the range of 0.30-1.0 mmol/L) gave a straight line (Fig. 8), which proved that this current is controlled by Cottrellian behavior. The slope of such lines can be used to estimate the diffusion coefficient (D) of warfarin. The mean value of D was found to be 7.41 × 10⁻⁶ cm²·s. The catalytic reaction rate constant (K_h) can be evaluated by chronoamperometry according to the method described in the literature [34]:

I_C/I_L=π^1/2γ^1/2=π^1/2(K_hC_bt)^1/2 (3)

Fig. 7. Impedance spectra in 5.0 mmol/L [Fe(CN)6]3−/4− containing 0.1 mol/L KNO3. Conditions: polarization potential 0.15 V, frequency 5.0×10−3 to 1.0 × 105 Hz.

where I_C is the catalytic current of warfarin at MWCNT/ ZnCrFeO₄/CPEs, I_L is the limited current in the absence of warfarin, and γ=K_hC_bt (C_b is the bulk concentration of warfarin, mol/cm³) is the argument of error function. K_h is the catalytic rate constant (cm³ mol^-1 s^-1). The value of K_h can be calculated from the slope of I_C/I_L versus t^1/2 plot for a given concentration of warfarin. The value of K_h was calculated as 1.4 × 10² cm³ mol^-1 s^-1, which demonstrates the sharp feature of the catalytic peak observed for oxidation of warfarin at the surface of MWCNT/ZnCrFeO₄/CPEs.

Fig. 8. Chronoamperometric studies of (1) 0, (2) 300, (3) 500, and (4) 1000 µmol/L warfarin with pH 4.0 at MWCNT/ZnCrFeO4/CPEs.

Under the optimized experimental condition, DPV was applied to generate a calibration graph. The plot of I_pa against warfarin concentration is linear over the range of 0.02 to 920.0 µmol/Lwith the equation of I_pa (µA)=(0.0105±0.001) C_warfarin+(0.8146±0.05) (R²=0.9943, n=5), where C_warfarin is warfarin concentration in µmol/L and I_pa is its anodic oxidation current in µA. The limit of detection was obtained as 0.003 µmol/L based on S/N=3.

The stability of the MWCNT/ZnCrFeO₄/CPEs was checked using DPV, in 0.1 mol/L PBS (pH 4.0) containing 10.0 µg/mL of warfarin stored in the refrigerator for three weeks. The peak heights of the DPV showed no significant change over a one-week period. The anodic peak current only decreased gradually to 97.0% of the initial value after three weeks of storage. The reproducibility of the modified electrode was investigated by comparing the current of DPV response to warfarin at six modified electrodes prepared independently. The relative standard deviation (RSD) of 3.4% was obtained at a warfarin concentration of 10 µmol/L. The repeatability of the sensor was investigated by measuring relative standard deviation (RSD) of ten successive assays of 10 µmol/L warfarin. The oxidation current was almost unchanged and an RSD of 2.2% was observed.

The analytical selectivity of the proposed method was evaluated by determination of warfarin (50.0 µmol/L) in the presence of various foreign species. The tolerance limit was considered as the maximum concentration of the interfering substance yielding a relative error less than 5% of the oxidation signal of warfarin. The results are presented in Table 1 and show that many anions and cations have no considerable effect on the determination of warfarin. However, cysteine at more than 150-fold interfered with the warfarin signal. Despite its interference, it is not present at significant levels in real samples.

Table 1 Interference study for the determination of 50.0 µmol/L warfarin under the optimized conditions. Species Tolerance limit (molSubstance/ molPhenaopyridine) Na+, Cl-, F-, Ca2+, K+, NO3-, Mg2+, ClO4-, histidine Ascorbic acid, methionine, uric acid Fructose, sucrose, urea, aspartic acid, glycine, epinephrine, glucose Cysteine 1000 * 500 300 150 * Maximum concentration of the substances used.

Table 1 Interference study for the determination of 50.0 µmol/L warfarin under the optimized conditions.

The analytical utility of fabricated sensor for determination of warfarin in real samples was evaluated. The standard addition method was used for analysis and the results of the proposed method were confirmed by the standard method [10]. The results are presented in Table 2. The statistical analysis of the results using Student’s test and the variance ratio F-test was used to compare the two methods for their accuracy and precision, which revealed the potential applicability of the proposed method. In addition, representative DPVs for determination of warfarin under optimized conditions in real samples with standard addition method are also illustrated in Fig. 9.

Table 2 Determination of warfarin in real samples. Real sample Added (µmol/L) Proposed method (µmol/L) Recovery (%) Standard method (µmol/L) Fexp. Ftab.(0.05);4,4 Spooled texp. ttab.(98%) Urine 1 — 350.0 15.0 <Limit of detection 349.8(±5.1) 357.7(±7.1) — 99.94 98.0 <Limit of detection 343.2(±4.7) — 1.18 9.6 4.9 2.1 3.75 Urine 2 — 15.0 70.0 <Limit of detection 15.2(±0.7) 85.4(±3.2) — 101.3 100.5 <Limit of detection 14.7(±0.4) — 3.06 9.6 0.57 1.39 3.75 Serum 1 — 140.0 50.0 200.0 <Limit of detection 133.7(±1.3) 184.5(±3.1) 388.9(±4.9) — 95.5 97.1 99.7 <Limit of detection 136.9(±1.6) 1.51 9.6 1.45 3.48 3.75 — — Serum 2 — 450.0 20.0 80.0 <Limit of detection 435.1(±6.1) 483.3(±9.2) 551.3(±8.7) — 96.7 102.8 100.2 <Limit of detection 446.8 (±5.3) 1.32 9.6 5.71 3.23 3.75 — — Tablet a — 198.1(±3.4) — 201.1(±4.6) 1.83 9.6 4.04 1.17 3.75 60.0 300.0 255.7(±3.4) 523.7(±8.3) 97.9 93.3 — — a Warfarin tablet (labeled 5.0 mg of warfarin per tablet, Orion Co.).

Table 2 Determination of warfarin in real samples.

Fig. 9. Determination of warfarin in real samples. (a) Spiked urine sample with the corresponding standard addition plot; (b) Spiked plasma sample with the corresponding standard addition plot; (c) Warfarin tablet sample with corresponding standard addition plot. Pulse amplitude of 100 mV, pulse time of 50 ms, sweep rate of 50 mV/s, phosphate buffer (0.1 mol/L, pH 4.0).

A novel sensor based on a MWCNT/ZnCrFeO₄-modified carbon paste electrode was developed for determination of warfarin. The carbon nanotube paste electrode modified with ZnCrFeO₄ was quite stable. The electrochemical behavior of MWCNT/ZnCrFeO₄/CPEs has been studied by cyclic voltammetry and chronoamperometry in both the absence and presence of warfarin. It was found that the electrode modification with MWCNT and ZnCrFeO₄ led to an enhanced sensitivity of warfarin voltammetric detection. In fact, edge plane-like sites/defects of MWCNT along with the electrocatalytic effect of ZnCrFeO₄ are responsible for amplifying the warfarin oxidation signal. The excellent conductivity of ZnCrFeO₄ makes the modified electrode a suitable mediator in the determination of warfarin. In fact, the performance of this sensor in the presence of ZnCrFeO₄ offers advantages in terms of analytical figures of merit such as enhanced sensitivity, low limit of detection, and wider dynamic range than non-MNP modified electrodes. To optimize the electrocatalytic response of the sensor to warfarin oxidation, the present electrochemical sensor was optimized in terms of composition of the electrode, pH, and DPV parameters. The DPV peak current is increased linearly with the concentration of warfarin in the range of 0.02-920.0 μmol/L with a very low detection limit of 0.003 μmol/L. The proposed sensor can serve as a useful, simple, and sensitive protocol for determination of warfarin in clinical laboratories.