Highly selective differential pulse voltammetric determination of warfarin in
pharmaceutical and biological samples using MnFe 2 O4 /MWCNT modified
carbon paste electrode

M. Taei, F. Hasanpour, F. Basiri, N. Tavakkoli, N. Rasouli

PII: S0026-265X(16)30104-7
DOI: doi: 10.1016/j.microc.2016.06.022
Reference: MICROC 2512

To appear in: Microchemical Journal

Received date: 30 December 2015

Revised date: 19 June 2016
Accepted date: 22 June 2016

Please cite this article as: M. Taei, F. Hasanpour, F. Basiri, N. Tavakkoli, N. Rasouli,
Highly selective dierential pulse voltammetric determination of warfarin in pharmaceu-
tical and biological samples using MnFe2 O4 /MWCNT modied carbon paste electrode,
Microchemical Journal (2016), doi: 10.1016/j.microc.2016.06.022

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 ACCEPTED MANUSCRIPT

Highly selective differential pulse voltammetric determination of warfarin in

pharmaceutical and biological samples using MnFe2O4/MWCNT modified

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carbon paste electrode

M. Taei1, F.Hasanpour1, F.Basiri1, N.Tavakkoli1, N.Rasouli1

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Chemistry Department, Payame Noor University, 19395-4697 Tehran, I.R. of IRAN.

Abstract

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A chemically modified electrode was prepared by incorporating MnFe2O4 into multi-walled carbon nanotubes
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paste matrix (MWCNTs/MnFe2O4/CPE) to determine warfarin. Cyclic voltammetry, differential pulse
voltammetry, chronoamperometry, and electrochemical impedance spectroscopy were used to investigate
the electrochemical behavior of warfarin at the chemically modified electrode. According to the results,
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MWCNTs/MnFe2O4/CPE showed high electrocatalytic activity for warfarin oxidation, producing a sharp
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oxidation peak current at about +0.91 vs Ag/AgCl reference electrode at pH 4.0. The peak current was
linearly dependent on warfarin concentration over the range of 0.10- 447.0 mol L-1 with the detection
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limit (3) of 0.08 mol L-1. The proposed method was successfully applied as a rapid, highly selective,
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simple, and precise one to determine warfarin in biological fluids.

Keywords MnFe2O4; Multi-walled carbon nanotubes; Warfarin; Electrochemical impedance spectroscopy; Paste
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electrode

1. Introduction

In recent years, magnetic nanoparticles (MNPs) have attracted attention in electrochemical sensors and

biosensors. One of the important classes of magnetic materials is spinel structure ferrites with general

formula MFe2O4 (M is transition metal). Among the ferrites, MnFe2O4 has been focused owing to its

Coresponding Author: Tel. +98-313-4507421; Fax: +98 -315-3669078; E-mail: m.taei@ch.iut.ac.ir;

m_taei57@yahoo.com

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excellent magnetic property, high electronic conductivity, high thermal stability, and effective catalytic

activity [1-2]. As a new form of carbon material, Carbon nanotubes (CNT) are among the best candidates

for the preparation of electrocatalytic nanocomposite materials [3]. The reason for this may be due to the

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nanometer dimensions of MWCNTs, the electronic structure and the topological defects (edge plane-like

sites/defects) present on MWCNTs surfaces. Meanwhile, the MWCNTs increase the effective area of the

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electrode[4].Therefore, they can incorporate into electrochemical sensors offering unique advantages

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including low detection limits, high sensitivities, reduction of over-potentials, and resistance to surface

fouling [5,6].

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Warfarin is a blood anticoagulant that inhibits the function of Vitamin K dependent coagulation.
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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
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and bleeding, nausea, vomiting, stomach pain, bloating, gas, or altered sense of taste, and its most serious
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drawback is hemorrhage that can be life-threatening even causing death. Therefore, detection of warfarin

in biological and clinical samples is very important [79]. To date, various methods such as micellar
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electrokinetic chromatography electrospray ionization mass spectrometry (MEKC-ESI-MS) [10],

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supercritical fluid chromatography tandem mass spectrometry (SFCMS/MS) [11], high performance
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liquid chromatography (HPLC) using ultraviolet or fluorescence detections[1218] and capillary zone

electrophoresis (CZE) [1921] have been developed to determine warfarin. However, these methods

suffered from serious problems such as the need for expensive instruments and toxic solvents and using

large biological fluid volumes.In addition, they usually have complicated sample pretreatment that is

laborious and time-consuming. Electrochemical methods have also received much interest due to their

higher selectivity, lower cost, and faster operation than other methods. Ghoneim, and Tawfik [22]

investigated the use of square-wave adsorptive cathodic stripping voltammetry on the surface of hanging mercury

drop electrode to determine warfarin.Although this technique is sensitive, the toxicity of mercury is an essential

problem. Recently, several papers have introduced new modified electrodes to determine warfarin in

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pharmaceuticals and biological fluids like urine and plasma [23-25]. In 2015, Gholivand et al. reported an

electrochemical sensor based on Fe3O4 magnetic nanoparticles modified carbon paste electrode to

determine warfarin [23]. Although this method is sensitive, due to stability limitation, its application is

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difficult. In fact, Fe3O4 magnetic nanoparticles are unstable in the external environment and may undergo

rapid degradation and self aggregation easily. Therefore, a modifiable material is needed to make them

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more stable and to avoid their aggregation. Taking into account the good performance of CNTs in

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electrochemical sensors, this work developed the synthesis of manganese ferrite nanoparticles and

incorporation of MWCNT for the modification of carbon paste electrode (CPE). The newly developed

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electrode exhibits excellent electrocatalytic activity towards warfarin. Table 1 presents the advantages and
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disadvantages of the proposed modified electrode to determine warfarin as compared to other analytical

methods reported in the literature.

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2. Experimental
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2.1. Apparatus
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Voltammetric measurements were carried out using an electrochemical system

comprising the Autolab PGSTAT101with NOVA software (Ecochemie, Utrecht, The

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Netherlands), a conventional three-electrode cell assembly containing an Ag/AgCl electrode as

the reference electrode, a platinum wire as the counter electrode and the

MWCNTs/MnFe2O4/CPE as the working 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 XRD (Holland Philips Xpert, X-ray diffractometer with Cu-K radiation)

and FE-SEM (Hitachi S-4160). FT-IR was recorded using a JASCO FT-IR (680 plus). The

analysis of chemical composition of the modified electrode was performed using an energy

dispersive spectrometer (EDXA).

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2.2. Chemicals

All chemicals were of analytical reagent grade and were purchased from Merck

(Darmstadt, Germany) except otherwise stated. A stock solution of 0.01 mol L1 warfarin was

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prepared by dissolving suitable amount of sodium warfarin (from Sigma-Aldrich) in water, and

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the solution was diluted to 25 mL with water in a 25-mL volumetric flask. Working solutions

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were prepared by diluting the stock solution with deionized water.

Phosphate buffer solutions (0.10 mol L1) with different pH values were used. Pure

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graphite powder (particle size <50 m) and MWCNTs (>90% MWCNTs basis, with a diameter
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of 2030 nm and a length of 515 m) were prepared from Irans Research Institute of

Petroleum Industry. High-viscosity paraffin (d = 0.88 kg L1) was used to prepare paste
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electrodes.
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2.3. Preparation of MnFe2O4magnetic nanoparticles

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MnFe2O4 samples were prepared by wet chemical route. In a typical procedure, the aqueous
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solutions of Fe(NO3)3.9H2O (4 g, 10 mmol) and Mn(NO3)2.6H2O (1.15 g, 5 mmol) were mixed

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and stirred together. After a digestion time of 30 min, 0.5 g of CTAB was added into the above

solution, followed by the addition in dropwise of an aqueous solution of NaOH (V= 15 mL, 4

M), then the emulsion became black and the precipitate appeared. The pH of the solution was

continuously monitored and maintained in the range of 11-12. At this stage, the solution changed

the color indicating the formation of MnFe2O4 nanoparticles. The solution was heated to 80 C

and was constantly stirred for 60 min. The solution was then cooled to the room temperature. A

blackish precipitate was separated and several times washed with water and ethanol. The

precipitate which was dried for 12 h at 110 C appeared black in color. The samples were heated

in the furnace at 600 C for 2 h [26].

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2.4. Preparation of MWCNTs/ MnFe2O4 modified electrode

To eliminate metal oxide catalysts within the nanotubes, multi-walled carbon nanotubes were

refluxed in the 2.0 M HNO3 for 15 h, and then they were washed with twice-distilled water and

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dried at room temperature. Nitric acid usually causes a significant destruction of carbon

nanotubes and introduces COOH groups at the ends or at the sidewall defects in the nanotube

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structure.The MWCNTs/MnFe2O4 modified electrode was prepared by mixing 75 mg of

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MnFe2O4,150 mg of MWCNTs and 800 mg of graphite powder. Then, diethyl ether was added

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and mixed to obtain a uniform mixture. After the evaporation of diethyl ether, 200 mg paraffin

oil was added and the solid was mixed with mortar and pestle to get a uniformly wetted paste.
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Then, a portion of the wetted paste was packed into a glass tube with a copper wire which had

been placed into the glass tube for electrical connection. The MWCNTs/carbon-paste electrode
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(CNPE) was prepared by mixing,150 mg of MWCNTs and 800 mg of graphite powder in the

same procedure (without adding MnFe2O4).The unmodified CPE was prepared by mixing
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graphite powder with the appropriate amount of paraffin and thorough hand mixing in a mortar
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and pestle
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2.5. Real samples preparation

Urine samples were stored in a refrigerator immediately after their collection. A 10 mL

of the sample was centrifuged for 10 min at 2000 rpm. The supernatant was filtered using a 0.45

m filter and then it was diluted 5-times with PBS pH 4.0. The solution was transferred into the

voltammetric cell to be analyzed without any further pretreatment. Standard addition method was

used to determine the warfarin content of the sample.

Serum samples were obtained and stored frozen until the analysis. In order to precipitate

proteins in the plasma samples,1.0 mL of the samples was treated with 2 mL acetonitrile. Then,

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the mixture was vortexed for a further 30 s and after that it was centrifuged at 3000 rpm for 10

min. The supernatant was transferred to a small flask and evaporated with the stream of nitrogen.

The dry residue was diluted with phosphate buffer solution of pH 4.0 and transferred into the

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voltammetric cell (10 mL) to be analyzed without any further pretreatment. Standard addition

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method was used to determine warfarin in the samples.

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For tablet analysis, 5 tablets of warfarin (labeled 5 mg of warfarin per tablet, Orion Co),

were completely ground and homogenized. Then, 2.31 g of the powder was accurately weighed

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and dissolved with ultrasonication in 25 ml of water. After mixing completely, the mixture was
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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 (equal to 0.01 mol

L1 warfarin). Then, 200 L of the solution plus 5 mL of the buffer (pH 4.0) was diluted with
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water in a 10mL volume flask, and the resulting solution was used for analysis. After that, the

diluted sample solutions were placed in an electrochemical cell to determine their concentrations
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using the DPV method.

To analyze injection solutions, 300.0 L of each ampoule (2.0 mg/mL warfarin

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sodium,Bristol-Myers Squibb) was added into a 5-mL buffer solution (0.1 mol L1 of pH 4.0),

and it was diluted with water in a 10mL volume flask. The test solution was transferred into the

electrochemical cell to determine warfarin levels.

Results and discussion

3.1. Morphological characterization of MnFe2O4 nanoparticles

X-ray powder diffraction analysis was used to identify the crystal structure of nanoparticles.

Fig.1A shows the XRD patterns of the obtained MnFe2O4 sample. The XRD pattern of MnFe2O4

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is well matched with that of pure MnFe2O4 (JCPDS card No.75-0034). No peak for another

crystal phase was detected, indicating that no crystal phase impurity exists in the obtained

sample. The crystallite size of the sample was calculated using the highly intense peak by

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Scherers formula:

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D (hkl) = Eq. (1)

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Where D is the size of the axis parallel to (hkl) plane, K is a constant with a typical value of 0.89

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for spherical particle, is the wavelength of radiation (0.15405 nm for Cu-K), is the full

width half maximum (FWHM) in radians and is the position of the diffraction peak maximum.
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The calculated crystallite size of MnFe2O4 was 124 nm.The FT-IR spectra were recorded to
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identify the functional groups available in MnFe2O4 nanoparticles as presented in Fig.1B. The
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broad peak observed at 3442.31 cm-1 is attributed to the stretching vibration of hydroxyls

absorbed on the surface and O-H groups in absorbed water, indicating the adsorbed moisturer.
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The peak at around 1640 cm-1 is assigned to H-O-H bending vibration in the water molecule. The

absorption bands in the range 578.34 cm-1 can be assigned to Mn-O vibrations, and in the range
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456 cm-1, it represents Fe-O vibration of spinel ferrite (MnFe2O4). The surface morphology and

the microstructure were observed with a scanning electron microscopy (SEM). SEM

representative micrographs for the nano-crystalline MnFe2O4 prepared by co-precipitation

method and annealed at 600 C are shown in Fig.1C.The SEM photograph revealed maximum

dimensions of the particles to be always less than 100 nm. This is an experimental proof of the

theoretical calculation of particle size by Debye Scherrer equation from XRD data. Figure1

3.2. Characterization of MWCNTs/MnFe2O4/CPE

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Fig. 2A shows the morphology of the electrode surface for MWCNTs/ MnFe2O4/CPE.

This picture shows that the MnFe2O4 and MWCNTs were distributed in the carbon paste and

filling the pores between the graphite particles. Energydispersive X-ray analysis (EDXA) was

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also employed to determine the chemical composition of the MnFe2O4 as shown in Fig.2B.

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Results from EDXA spectra show that the sample contains Mn, Fe and O for MnFe2O4. The

atomic ratio is in agreement with the expected stoichiometry in the sample.Figure2A&2B

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Electrochemical impedance spectroscopy (EIS) gives insight into properties of electrode

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surface and electrolyte. The Nyquist plots of electrodes were obtained in ac frequency range of

0.1 Hz to 100 KHz in 10-3 mol L-1 Fe (CN)63-/4- solution at polarization potential 0.15 V (see
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figure S1). The Nyquist plots were fitted with Rs(Q[Rct w]). Where Rs refers to the electrolyte
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resistance, Rct is the charge transfer resistance, Q is the constant phase element, while w is
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Warburg impedance coupled to Rct, which is related to Nernstian diffusion. According to the

impedance data in Table 2, the charge transfer resistance (Rct) of Fe2+/Fe3+ at the bare electrode,
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CNPE, MnFe2O4/CPE, and MWCNTs/MnFe2O4/CPE are 1211 , 746.0 , 391 and 206 ,

respectively. This demonstrates that MWCNTs and MnFe2O4 accelerate the interfacial electron
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transfer between the electrode surface and the analyte. Figures(2C& S1)&Table 2

In order to illustrate that the prepared MWCNTs/MnFe2O4/CPE could increase the surface area

of the sensor, real surface area (A) of CPE and MWCNTs/ MnFe2O4/CPE was determined by the

Randles-Sevcik formula:

Ipa = 2.69 105 n3/2 A C0 DR1/2 Eq. (2)

Where Ipa (A) refers to the anodic peak current, n is the electron transfer number, A is the

surface area of the electrode, DR is the diffusion coefficient, C0 (mol cm3) is the concentration

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of K3Fe(CN)6 and is the scan rate. For 1mmol L-1 K3Fe(CN)6 in the 0.1mol L-1 KCl electrolyte:

n = 1 and DR =7.6106 cm2 s1, then from the slope of the Ipa 1/2
relation, the real surface

area was calculated. The results showed that the electrode surface area was 0.0456 cm2

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(Figure S2) for the unmodified carbon paste electrode and 0.1003cm2 for

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MWCNTs/MnFe2O4/CPE, which is 2.2 times greater than that for the CPE (Figure S3).

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3.3. Electrochemical behavior of warfarin at the surface of modified electrodes

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Figure 3A shows DP voltammograms of warfarin at modified and unmodified electrodes. As it
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can be seen, in the presence of each carbon nanotube and MnFe2O4 nanoparticle the oxidation

current has increased. However, by application of both nanoparticles maximum oxidation current
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has been obtained. This current change at MWCNTs/MnFe2O4/CPE is not only due to the
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surface area, but also due to the synergic effect of MnFe2O4 and MWCNTs on the oxidation of
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warfarin at pH 4.0. In fact, the isoelectric point, i.e., the pH value at which the particle surface
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carries no net electric charge, is 7.0 when NaOH is used as mineralizer for the synthesis of

MnFe2O4 [27]. Therefore, the surface of nanoparticles is positively charged under pH 7.0. The
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synergic effect of MWCNTs and MnFe2O4 nanocomposite may be due to the electrostatic

interaction between negatively charged MWCNTs and positively charged MnFe2O4at pH 4.0

[27].

The electrochemical behavior of warfarin was also investigated by CVs in the ranges of

0.70 to 1.20 V (Fig.3B). A significant enhancement in peak currents was obtained at

MWCNTs/MnFe2O4/CPE, which demonstrated an increase in quantity of electrons transferred

per time. In addition, the effect of scan rate on the anodic peak current of warfarin at MWCNTs/

MnFe2O4/CPE was also investigated. The results showed that by increasing the scan rate, the

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anodic peak current (IPa) is gradually increased. The linear relation between peak current and

square root within a scan rate of 10 170 mV s1 indicated that a diffusion controlled oxidation

reaction occurs at the surface of MWCNTs/MnFe2O4/CPE ( Figure S4).Figure 3&S4

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3.4. Optimization of measurement conditions

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In order to find the optimum conditions with the highest sensitivity for the determination

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of warfarin, the influence of various parameters such as DPV parameters, the mass of

nanoparticles to MWCNTs and pH on the peak current were studied.

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DPV technique was used to determine warfarin because of higher sensitivity and
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resolution than CV. The DPV parameters changed when the concentration of warfarin was 400

mol L1. The results showed that the maximum peak current was obtained with a pulse
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amplitude of 100 mV, a pulse time of 50 ms, and a voltage step time of 0.1 s. These values were
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selected for further study.

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To obtain maximum current with suitable potential of the proposed electrode toward
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warfarin, the effect of the mass of MnFe2O4 to MWCNTs was investigated. The amount of
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MWCNTs was optimized when the percentage of MnFe2O4 nanoparticles was fixed at 6.0% of

the entire amount of paste material. Apparently, the maximum oxidation current can be obtained

at the electrode containing 12.2 % of MWCNTs of the total amount of paste material. By further

increase in MWCNTs ratio, the electrode became rigid and it was hard to polish. Therefore, the

renewed surface cannot be obtained easily. To optimize the amount of MnFe2O4 nanoparticles in

the paste, the percentage of MWCNT was fixed at its optimized value, and then, the percentage

of MnFe2O4 nanoparticles was changed. In this case, the maximum current was observed in 6.1%

of MnFe2O4 nanoparticles. Therefore, the ratio of 1/2 for MnFe2O4/MWCNTs of the total

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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.

Since proton contributes in the proposed oxidation mechanism of warfarin, the pH of the

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solution can affect the oxidation current. Accordingly, the effect of pH of the solution on the

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oxidation current was studied by recording DP voltammograms of 250.0 mol L1 warfarin with

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different pHs at MWCNTs/ MnFe2O4/CPE. The results showed that at pH 4.0, maximum

oxidation current obtained; so, this value was selected as the optimum pH for further

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experiments(Figure 4). Furthermore, it can be seen that the oxidation peak potential of warfarin
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shifts negatively with rising pH demonstrating that deprotonation is involved in the oxidation

process. The value of proton in the electrode reaction was estimated by [28]:
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Ep=E0/-0:059p/n pH Eq. (3)

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The plot of Ep versus pH gives a linear equation as: Ep(V)=-0.027 pH+1.016. The slope of
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dE/dpH indicates the number of electrons is twice as many as the protons that participated in the
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oxidation process. As previously proved [23], the oxidation mechanism of warfarin contains two
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electrons and one proton exchange. Fig 4 shows a definite break corresponding to the apparently

pKa value 6.0. The value of pKa is in excellent agreement with the literature [29].Figure 4

3.5. Chronoamperometric studies

The typical single step chronoamperometry of MWCNTs/MnFe2O4 modified the

electrode with successive addition of warfarin solution (pH=4.0) by setting working potential at

+0.95 V vs. Ag/AgCl electrode. A plot of I versus t-1/2 for different concentrations of warfarin

(in the ranges of 0.30 to 2.0 mmol L-1) was given a straight line, which proved this current is

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controlled by Cottrellian behavior (Figure S5). The slope of such lines can be used to estimate

the diffusion coefficient (D) of warfarin. The mean value of the D was found to be 2.46 10-6

cm2 s-1. The catalytic reaction rate constant (Kh) can be evaluated by chronoamperometry

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according to the method described in the literature [30]:

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IC / IL = 1/21/2 = 1/2(Kh Cb t)1/2 Eq. (4)

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Where IC is the catalytic current of warfarin at MWCNTs/MnFe2O4/CPE, IL is the limited current

in the absence of warfarin and = Kh Cbt (Cb is the bulk concentration of warfarin, mol cm-3) is

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the argument of error function. The Kh is the catalytic rate constant (cm3 mol-1 s-1). From the
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slope of IC/IL versus t1/2 plot the value of Kh can be calculated for a given concentration of

warfarin. The value of Kh was calculated as 5.2 102 cm3 mol-1 s-1, which demonstrates the sharp
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feature of the catalytic peak observed for the oxidation of warfarin at the surface of
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MWCNTs/MnFe2O4/CPE.Figure S5
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3.6. Figures of merit

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DP voltammetry (with pulse amplitude of 100 mV, pulse time of 50 ms, sweep rate of 50
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mV s1) was applied to obtain calibration curve. The relationship between oxidation current and

concentration was linear in the range of 0.10 to 477.0 mol L1 warfarin

(I (A) =0.067C warfarin (0.004)(mol L1)+2.364(0.341), R2=0.9981) (Figure S6). The lower

detection limit of the method was determined using the equation Cm=3Sb/m, where Sb is the

standard deviation of the blank response (A) and m is the slope of the calibration curve. Using

the data from the calibration curve and measurements of blank, the lower detection limit was

obtained to be 0.08 mol L1 of warfarin.Figure S6

The stability of MWCNTs/MnFe2O4/CPE was checked out over a four-week period using

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10 mol L-1of warfarin. The DPV of warfarin at the surface of the modified electrode (stored in

the laboratory at room temperature) shows that the oxidation peak current of warfarin was

decreased less than 4.7% of the initial value without any alteration in the peak potential. The

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reproducibility of the modified electrode was investigated by comparing the current of DPV

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response to warfarin at six modified electrodes prepared independently. The relative standard

deviation (RSD) of 3.3% was obtained at 10 mol L1warfarin.The repeatability of

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MWCNTs/MnFe2O4/CPE was investigated by measuring relative standard deviation (RSD%) of

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ten successive assays of 10 mol L1 warfarin. The oxidation current was almost unchanged, and

RSD of 1.1% was observed. These results indicate that MWCNTs/MnFe2O4/CPE has a good
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stability, repeatability and reproducibility.Figure S7
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3.7. Interference study

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The effects of potentially interfering species, found in biological fluids or

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pharmaceuticals, were investigated to determine 10.0 mol L-1 warfarin. The maximum
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concentration of the potential interfering species, which causes an error less than 5%, was

considered as tolerance limit. The results show that 1000-fold concentration of glucose, ascorbic
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acid, dopamine, alanine, phenylalanine, methionine, sucrose; 500-fold concentration of glycin,

uric acid, did not affect the oxidation current of warfarin. However, the oxidation current of 10

mol L-1 warfarin was decreased to 91% of its initial currents in the presence of 200 mol

L-1 cysteine. Despite its interference, cysteine is not present at significant levels in real samples.

4.Analysis of real samples

4.1. Assay of warfarin in tablets

The proposed method was successfully applied for the analysis of wrafarin tablets.

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According to the obtained results in table 3, a good agreement can be seen between the proposed

method and the standard method [16]. The average determination results of warfarin in the

tablets samples were quite corresponding to the value given by drug specification. This produce

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was repeated five times, and the relative standard deviation obtained was 1.94%. Different

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standard concentrations of warfarin were added to the diluted warfarin tablet, and the recovery

was between 97.5 and 99.2 % for five measurements. In addition, we tested the applicability of

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MWCNTs/MnFe2O4/CPE to determine warfarin in the warfarin sodium injection sample. The

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results in table 3 indicate that the modified electrode is an effective sensor for determining

warfarin and that it can be applied for its detection in pharmaceutical samples.
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4.2. Assay of warfarin in human serum and urine

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To check the capability of the proposed sensor for the determination of warfarin in a
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complex matrix of biological samples, a spike method was chosen. Three measurements were
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performed for each concentration. As seen in Table 3, good recoveries and RSD were obtained
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indicating that the proposed sensor in this work is suitable for warfarin determination in Serum

and urine samples with high sensitivity and precision. To further ensure the accuracy in this

result, the amounts of warfarin in Serum samples of two patients (two men), subjects on

warfarin, were measured. Sampling was made after 3.5 h from consumption of the tablet. A

statistical comparison was also made between the proposed method and the standard method

(liquid chromatography with ultraviolet detection at =320 nm) using Students t-test (for

accuracy), variance ratio, and F-test (for precision) at the 98% confidence level [16]. The results

confirm that there are no significant differences between the results of the proposed sensor and

the standard method. Table 3

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5. Conclusions

The results show that the combination of MWCNT and manganese iron oxide

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nanoparticles can cause excellent electrocatalytic oxidation of warfarin. The MWCNTs/MnFe2O4

modified CPE had antifouling properties with desirable electrochemical features such as high

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sensitivity, selectivity, low detection limit, high stability and reproducibility. Other advantages of

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the proposed method are technical simplicity, and rapid preparation of the sensor. The proposed

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method is free from interference of common oxidizable substances existing in biological fluids

and pharmaceutical samples. MA

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Legend for the figures:

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Fig. 1. (A) The XRD pattern for cubic spinel MnFe2O4 nanoparticles, (B) FT-IR spectra of MnFe2O4

nanoparticles, (C) SEM image of MnFe2O4, and (D) Particle size distribution diagram of MnFe2O4

nanoparticles

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Fig. 2. SEM image of (A) MWCNTs/MnFe2O4/CPE, (B) The corresponding EDXA spectrum taken from

the whole area of (A), and (C) Impedance spectra in 1.0 mmol L1 [Fe(CN)6]3-/4- containing 0.1 mol L1

KNO3 at (a) CPE, (b) CNPE,(c) MnFe2O4/CPE, and (d) MWCNTs/MnFe2O4/CPE. Conditions:

polarization potential: 0.15 V, frequency: 0.1 to 105 Hz.

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Fig. 3.(A): Differential pulse and; (B) cyclic voltammograms of warfarin (250.0 mol L1) at (a) CPE,

(b) CNPE,(c) MnFe2O4/CPE, and (d) MWCNTs/MnFe2O4/CPE; DPV experimental conditions: pulse
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amplitude of 100 mV, pulse time of 50 ms, sweep rate of 50 mV s1; phosphate buffer (0.1 mol L1, pH
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Fig. 4. Dependence of oxidation peak potential of warfarin with pH at the modified CPE.

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Graphical abstract
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Table 1. Comparison of some characteristics of the different modified electrodes for the determination

of warfarin

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Electrode Limit of Linear Toxic materials RSD(%) Ref. Ref.
detection dynamic

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range
(mol L1)

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(mol L1)

Hanging mercury drop 0.001- Mercury Not 22 21

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electrode 0.004 0.01 reported

Fe3O4/CPE 0.5 - 1000 - 2.3 23 22

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0.21

CdS-quantum 0.0580 Cadmium 2.3 24 23

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dots/MWCNT/chitosan/GCE 0.008
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MWCNT/Molecular 0.0006 0.0001- DMF, 3.2 25 24

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imprinting/GCE
Methanol and
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Phenylene
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diamine

MWCNTs/MnFe2O4/CPE 0.10- _ 1.1 This This

0.08 447.0 work work

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Table 2. Comparison of the electrochemical impedance data in 1.0 mmol L1 [Fe(CN)6]3-/4- containing 0.1
mol L1 KNO3. Conditions: polarization potential: 0.15 V, frequency: 0.1 to 105 Hz.

Electrode Electrochemical Impedance Parameter

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RS() Rct(k) n Q(F) W10-2 (sn
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CPE 70.8 1.211 0.88 0.12 0.20

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CNPE 70.2 0.746 0.90 0.88 0.18

MnFe2O4/CPE 84.3 0.391 0.89 0.76 0.16

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MWCNTs/MnFe2O4/CPE 89.2 0.206 0.83 0.45 0.22

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Table 3. Determination of warfarin in real samples.

Sample Added Warfarin expected Proposed Relative error RSD% Recovery Standard method (mol L1
(mol L1) method (%) (%) texp. ttab.(98%)
(mol L

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Urine a

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- <Limit of - - <Limit of detection
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250.0 - -2.6 2.01 97.4 249.2(2.5)

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243.4(4.9) 2.36 3.75
15.0 - -3.2 2.46 96.8
256.4(6.3)

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Urine b - <Limit of - - <Limit of detection
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15.0 - +2.0 5.9 102.0 15.08(1.4)
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15.3(0.9) 0.30 3.75
40.0 - +0.18 4.9 100.2
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110.0 - -5.72 1.4 94.3 101.1(3.3)
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103.70(1.5) 1.61 3.75

30.0 - -0.93 3.4 99.1
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138.7( 4.7)
200.0 -4.73 1.7 95.3
323.9( 5.4)
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Serumd - <Limit of - - <Limit of detection

detection
450.0 - -1.80 1.2 98.2 446.8 ( 4.0)
441.8( 5.3) 1.69 3.75
20.0 - -1.04 2.0 99.0
465.1(9.2)

Serume - - 2.73( 0.1) - 3.7 - 2.64( 0.24)

0.79 3.75

50.0. 54.3( 1.3) - 2.4 103.1

Serumf - 2.23( 0.11) - 4.9 - 2.08( 0.3)

1.03 3.75

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10.0 11.43( 0.70) - 6.1 94.6

g
Ampoule - 197.8 201.0(2.90) +1.6 1.4 - 198.3(4.5)
1.13 3.75

100 297.8 303.2(4.7) +1.8 1.6 101.8

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10.0 307.8 309.1(9.2) +0.42 3.0 100.4

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Tableth - 201.0 200.1(3.90) -0.45 1.9 - 201.3(2.6)

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0.58 3.75

100.0 301.0 293.7(5.4) -2.40 1.8 97.5 -

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10.0 311.0 307.2(8.7) -1.2 2.8 98.7 -

50.0 361.0 358.4(7.1) -0.72 2.0 99.2 -

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a&b
Sampling was made after 3.5 h from a man who is safe and not used warfarin
c&d
Sampling was made after 3.5 h from a man who is safe and not used warfarin
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e&f
Sampling was made after 3.5 h from a man who is patient and used warfarin
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g
2.0 mg/mL warfarin sodium,Bristol-Myers Squibb
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warfarin tablet (labeled 5.0 mg of warfarin per tablet, Orion Co.)
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shows the standard deviation for five replicate measurements

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1-A Spinel-structured MnFe2O4 nanoparticles was synthesized by precipitation method.

2- The structure of the MnFe2O4 was characterized by using XRD, FE-SEM and FT-IR.

3- A novel warfarin sensor has been developed based on MWCNTs modified with MnFe2O4.

4-The proposed electrode was very easy to fabricate and eco-friendly for analysis.

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