Comparative study of normal, micro- & nano-sized iron oxide effect in potentiometric determination of fluconazole in biological fluids

Abstract

Three novel fluconazole (FLU) selective electrodes were investigated with di-octyl phthalate as a plasticizer in a polymeric matrix of polyvinyl chloride and 2-hydroxypropyl-β-cyclodextrin as an ionophore. The potentiometric strategy was based on functionalized magnetic iron oxide particles. A mixture of equal volumes of 1 × 10⁻² M FLU and 1 × 10⁻² M KCl was used as an internal reference solution for sensor 1. An aqueous dispersion of magnetic micro iron oxide particles was introduced into an internal reference solution for sensor 2, whereas an aqueous dispersion of magnetic nano iron oxide particles was introduced for sensor 3. By applying a magnetic field, the iron oxide particles could be attached to the surface of an ionophore-free polymeric membrane. FLU was accurately determined in spiked human plasma and spiked cow milk.

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1 Introduction

Fluconazole, 2-(2,4,-difluorophenyl)-1,3-bis(1H-1,2,4,-triazol-1-yl)propan-2-ol] (Diflucan), is a first line antifungal drug that is used in the treatment of supercritical and systemic candidiasis and in the treatment of cryptococcus in patients with acquired immunodeficiency syndrome (AIDS). It acts by blocking the synthesis of ergosterol, an essential component of the fungal cell membrane.¹

Measuring its concentrations in the plasma of patients is of clinical relevance when pharmacokinetics is unpredictable. FLU concentrations in plasma can be measured by HPLC or by bioassay. However, because of its robustness, HPLC remains the reference method and is required to validate the development of any bioassay.^2,3 Prior to HPLC analysis, careful sample preparation to extract FLU from plasma needs labour intensive procedures using organic solvents and/or solid-phase extraction.⁴ However, some of these methods need expensive equipment and/or are time-consuming. Moreover, several spectroscopic methods have been developed for the determination of FLU,^5–9 but they need pretreatment steps before application in biological fluids.

In recent years, there has been a growing need for constructing chemical sensors for the fast and economical monitoring of pharmaceutical compounds.^10–14 The present work describes the use of functionalized iron oxide micro and nano particles with 2-hydroxypropyl-β-cyclodextrin as an ionophore for the development of novel sensors for the determination of FLU in bulk powder, different pharmaceutical formulations, biological fluids (plasma, urine and milk), and in the presence of other co-administrated drugs such as Tinidazole.

2 Experimental

2.1 Apparatus

A Jenway pH meter 3310 pH/mV/°C meter with Orion, reference electrode (Ag/AgCl, double junction) model 63178 USA 314-771-5750, and a Jenco digital ion analyzer model 6209 were used for potential measurements. Jenway pH glass electrode (UK) and Bandelin Sonorox, Rx 510 S, and magnetic stirrer (Budapest, Hungary) were used for pH adjustment. Malvern Zetasizer (United Kingdom) and JEOL JEM-2100 transmission electron microscope (München, Germany) were used for the characterization of iron oxide particles.

2.2 Chemicals and reagents

Fluconazole, 100%, was obtained from Eipico Co. (Egypt). Treflucan® tablets (nominally containing 150 mg of fluconazole per tablet, Eipico Co., Cairo, Egypt) were used in this work.

All chemicals and reagents used were of analytical reagent grade, and water was bi-distilled.

• Polyvinyl chloride carboxylate (PVC carboxylate) and 2-hydroxypropyl-β-cyclodextrin (β-CD) were obtained from Fluka Chemie Gmbh (Steinheim, Germany).

• Di-octyl phthalate (DOP) was purchased from Aldrich (Steinheim, Germany).

• Tetrahydrofuran (THF) was obtained from Merck (Dermstadt, Germany). Iron oxide NPs (5 nm diameter) were prepared by and purchased from Nanotech Egypt for photo electronics Co. (Dreamland, 6th October City, Egypt).

• Ferric chloride hexahydrate (FeCl₃·6H₂O) and ferrous sulphate (FeSO₄) were obtained from Aldrich (Steinheim, Germany).

• Potassium chloride, ammonium sulphate, methanol, citric acid, disodium hydrogen phosphate, sodium hydroxide, hydrochloric acid, zinc sulphate (ZnSO₄), magnesium sulphate (MgSO₄), glycine, L-cysteine, ethylene diamine tetra-acetic acid (EDTA), sodium lauryl sulphate (SLS), and cetyl trimethyl ammonium bromide (CTAB) were obtained from Prolabo (Pennsylvania, USA).

• Tinidazole (99.50%) was kindly provided by Medical Union Pharmaceuticals (Ismailia, Egypt).

• Plasma and urine were supplied by VACSERA (Giza, Egypt) and used within 24 h.

• Cow milk was purchased from the market.

2.3 Procedures

2.3.1 Membrane fabrication. In a 5 cm Petri dish, 0.04 g 2-hydroxypropyl-β-CD was thoroughly mixed with 0.19 g PVC and 0.35 ml DOP, and the mixture was then dissolved in 5 ml THF till complete homogeneity was obtained. The petri dish was covered with filter paper and left to stand overnight to allow solvent evaporation at room temperature, and a master membrane of 0.1 mm thickness was obtained.

2.3.2 Preparation and characterization of iron oxide MPs. Deionized water was deoxygenated by bubbling N₂ gas for 10 minutes prior to the use. FeCl₃·6H₂O (1.28 M) and FeSO₄ (0.64 M) stock solutions were prepared as a source of iron by dissolving the respective chemicals in deionized water under vigorous stirring. A stock solution of 1.5 M NaOH was prepared as the alkali source and 0.4 M HCl as the acid source. A solution of 0.01 M HCl was prepared for surface neutralization.

Aqueous dispersion of magnetic MPs was prepared by alkalinizing an aqueous mixture (equal volumes) of ferric and ferrous salts with NaOH. N₂ gas was blown through the reaction medium during synthesis operation in a closed system, where 25 ml of iron source was added drop-wise into 250 ml of alkali source under vigorous mechanical stirring (2000 rpm) for 30 min at room temperature.

The precipitated powder was isolated by applying an external magnetic field and the supernatant was removed from the precipitate by decantation. Deoxygenated deionized water was added to wash the powder and the supernatant was decanted after centrifugation at 3500 rpm. After washing the powder 4 times, 0.01 M HCl was added to neutralize the anionic charge on the particle surface. The cationic colloidal particles were separated by centrifugation and peptized by adding deoxygenated deionized water.

The obtained iron oxide MPs were characterized by measuring particle size using a Malvern Zetasizer, as shown in Fig. 1.

Fig. 1 Determination of synthesized iron oxide particles' size by a Malvern Zetasizer.

2.3.3 Characterization of iron oxide NPs. Purchased iron oxide NPs were characterized under JEOL JEM-2100 transmission electron microscope, as shown in Fig. 2.

Fig. 2 Nano iron oxide particles under JEOL JEM-2100 transmission electron microscope.

2.3.4 Functionalization of iron oxide micro and nano ferrofluids. 0.1 g 2-hydroxypropyl-β-CD and 0.45 ml DOP were successively dissolved in 5 ml THF, to which 5 ml of the prepared iron oxide MPs magnetic fluids were added.

By means of ultrasonic treatment for 30 min, a stable magnetic fluid solution was obtained. Through the evaporation of THF from the mixed solution for 24 h, the final magnetic fluids were obtained. The product was collected and stored at room temperature for use in the assembly of sensor 2.

The same procedure was repeated using 5 ml of 0.5 g ml⁻¹ iron oxide NPs aqueous dispersion in the case of sensor 3. Charts 1 and 2.

Chart 1 Schematic representation for the steps of preparation of the membrane.

Chart 2 Schematic representation for the functionalization of iron oxide nano particles.

2.3.5 Electrode assembly. A disk of an appropriate diameter (about 8 mm) was cut from the previously prepared master membrane using a cork borer and cemented using THF to an interchangeable PVC tip that was previously clipped into the end of an appropriate glass outer casting for the three proposed sensors. A mixture of equal volumes of 1 × 10⁻² M FLU and 1 × 10⁻² M KCl was used as an internal reference solution for sensor 1, into which an Ag/AgCl wire (1 mm diameter) was immersed as an internal reference electrode.

For sensor 2 and 3, 0.2 ml of magnetic MPs and NPs ferrofluids were added to a mixture of equal volumes of 1 × 10⁻² M FLU and 1 × 10⁻² M KCl, respectively, and used as an internal reference solution, into which a Ag/AgCl wire (1 mm diameter) was immersed as an internal reference electrode.

The sensors were conditioned by soaking in 10⁻² M aqueous FLU solution for 24 h, and they were stored in the same solution when not in use.

2.3.6 Sensors calibration. The conditioned sensors were calibrated by separately transferring 50 ml aliquots of solutions (10⁻¹¹ to 10⁻² M) of FLU into a series of 100 ml beakers. The membrane sensors, in conjunction with an Ag/AgCl double junction reference electrode, were immersed in the above mentioned test solutions and allowed to equilibrate while stirring. The potential was recorded after stabilizing to ±1 mV and the electromotive force was plotted as a function of the negative logarithm of FLU concentration. The sensors were washed in distilled water between measurements.

2.3.7 Effect of pH. The effect of pH on the response of the investigated electrodes was studied using 10⁻⁴ and 10⁻⁵ M solutions of FLU with pH ranging from 3 to 8 (while adjusting pH using citro-phosphate buffer).

2.3.8 Sensors selectivity. The potentiometric selectivity coefficients (K^pot_A.B) of the proposed sensors towards different substances were determined by a separate solution method using the following equation:¹⁵

−log(K^pot_A.B) = E₁ − E₂/(2.303RT/Z_AF) + (1 − Z_A/Z_B)log α_A (1)

where K^pot_A.B is the potentiometric selectivity coefficient, E₁ is the potential measured in 10⁻³ M FLU solution, E₂ is the potential measured in 10⁻⁴ M interferent solution, Z_A and Z_B are the charges of FLU and interfering ion, respectively, α_A is the activity of the drug and 2.303RT/Z_AF represents the slope of the investigated sensors (mV per concentration decade).

2.3.9 Determination of FLU in its pharmaceutical formulation. The content of one capsule of Treflucan® capsules was weighed. An accurately weighed amount of powder equivalent to 0.015 g FLU was transferred into 100 ml volumetric flask and filled to the mark with water to prepare 4.89 × 10⁻⁴ M stock solution. Then, a suitable dilution was made from the prepared stock to obtain 4.89 × 10⁻⁵ and 4.89 × 10⁻⁶ M samples of Treflucan®. The potentiometric measurements of the prepared samples were performed using the proposed sensors in conjunction with an Aldrich glass reference electrode, and the potential readings were recorded. Concentrations of FLU in the prepared solutions were calculated from the regression equation of each of the three electrodes.

2.3.10 Direct potentiometric determination of FLU in spiked plasma samples. Half milliliter of each of 5 × 10⁻³, 5 × 10⁻⁴ and 5 × 10⁻⁵ M standard drug solution were added separately into three 25 ml stoppered shaking tubes each containing 1 ml of plasma to prepare 1 × 10⁻⁴, 1 × 10⁻⁵ and 1 × 10⁻⁶ M FLU plasma solutions. The tubes were shaken for 1 min. The membrane sensors were immersed in conjunction with the reference electrode in the previously prepared spiked plasma solutions and then washed with water between measurements. The emf produced for each solution was measured by the proposed sensors, and the concentration of FLU was obtained from the corresponding regression equation.

2.3.11 Determination of FLU in spiked plasma samples after treatment. Half milliliter of each of 5 × 10⁻³, 5 × 10⁻⁴ and 5 × 10⁻⁵ M standard drug solution were added separately into three 20 ml stoppered shaking tubes each containing 1 ml of plasma and 2 ml methanol. The tubes were shaken for 1 min and then centrifuged for 50 min at 8000 rpm. Then the supernatant was transferred into three 25 ml volumetric flasks and completed to the mark with water to prepare 1 × 10⁻⁴, 1 × 10⁻⁵ and 1 × 10⁻⁶ M FLU plasma solutions. The membrane sensors were immersed in conjunction with the reference electrode in the previously prepared spiked plasma solutions and then washed with water between measurements. The emf produced for each solution was measured by the proposed sensors, and the concentration of FLU was obtained from the corresponding regression equation.

2.3.12 Direct potentiometric determination of FLU in spiked urine samples. One milliliter of each of 1 × 10⁻³, 1 × 10⁻⁴ and 1 × 10⁻⁵ M standard drug solution were added separately into three 20 ml stoppered shaking tubes each containing 9 ml of urine to prepare 1 × 10⁻⁴, 1 × 10⁻⁵ and 1 × 10⁻⁶ M FLU urine solutions. The tubes were shaken for 1 min. The membrane sensors were immersed in conjunction with the reference electrode in the previously prepared spiked urine solutions and then washed with water between measurements. The emf produced for each solution was measured by the proposed sensors, and the concentration of FLU was obtained from the corresponding regression equation.

2.3.13 Direct potentiometric determination of FLU in spiked milk samples. One milliliter of each of 1 × 10⁻³, 1 × 10⁻⁴ and 1 × 10⁻⁵ M standard drug solution were added separately into three 20 ml stoppered shaking tubes each containing 9 ml of milk to prepare 1 × 10⁻⁴, 1 × 10⁻⁵ and 1 × 10⁻⁶ M FLU milk solutions. The tubes were shaken for 1 min. The membrane sensors were immersed in conjunction with the reference electrode in the previously prepared spiked urine solutions and then washed with water between measurements. The emf produced for each solution was measured by the proposed sensors, and the concentration of FLU was obtained from the corresponding regression equation.

3 Results and discussion

Molecular recognition and inclusion complexation are of current interest in host–guest and supramolecular chemistry and offer a promising approach to chemical sensing.^16,17 The use of selective inclusion complexation and complementary ionic or hydrogen bonding are two main strategies for preparing synthetic host molecules, which recognize the structure of guest molecules.¹⁸

Modified cyclodextrins (CDs), either natural or synthetic, are viewed as molecular receptors, as is shown in Fig. 3(a). In the case of natural CD, cooperative binding with certain guest molecules was mostly attributed to intermolecular hydrogen bonding between the CD molecules, whereas intermolecular interactions between the host and guest molecules (hydrogen bonds, hydrophobic interactions and van der Waals forces) contributed to cooperative binding processes when synthetic CDs were used.¹⁹ Although the size and geometry of the guest mainly govern the binding strength, it is possible to influence the host–guest interactions by modifying three hydroxyl groups on each glucose unit. Indeed, the use of 2-hydroxypropyl β-cyclodextrin enhances the interaction properties between host and guest molecules.²⁰

Fig. 3 (a) Structure of HP-β-CD and (b) FLZ docked with β-CD (top-view).

Sensor 1 was based on electroactive membrane consisting of HP β-CD as an ionophore. The principle of the determination lies in the ability to selectively form inclusion complexes between the host (HP-β-CD) and the guest (FLU).

Inclusion complexes of FLU with β-CDs were investigated by applying NMR and molecular modelling methods. The 1 : 1 stoichiometry of the FLU : β-CD complex was determined by a continuous variation (Job's plot) method. The shielding of cavity protons of β-CD and the deshielding of aromatic protons of FLU in various ¹H-NMR experiments showed complexation between β-CD and FLU. Based on spectral data obtained from 2D ROESY, a reasonable geometry for the complex could be proposed implicating the insertion of the m-difluorophenyl ring of FLU into the wide end of the torus cavity of β-CD. Indeed, the best docked complex in terms of binding free energy supports the model proposed from NMR experiments and the m-difluorophenyl ring of FLU is observed to enter into the torus cavity of β-CD from the wider end, as shown in Fig. 3(b).²¹

In sensor 2 and 3, the ionophore was adsorbed on the magnetic nanoparticles. By adding the resulting magnetic fluids into the sample solution, the ionophore-functionalized magnetic nanoparticles could be dispersed in the solution swiftly and symmetrically.^22,23 Upon the application of a magnetic field, the magnetic nanoparticles were aggregated to the inner side of the polymeric membrane and the ionophore adsorbed on the nanoparticles could be dissolved on the surface of the membrane, which yielded a significant potentiometric response.

The present work evaluates the influence of magnetic iron oxide particles on the response of the three proposed sensors, the effect of its particle size and the relation between their presence and size to increasing the sensitivity of the proposed sensors.

3.1 Performance characteristics of FLU sensors

Table 1 shows electrochemical response characteristics of the three investigated FLU sensors.

Table 1 Electrochemical response characteristics of the three investigated FLU sensors

Parameter	Sensor 1	Sensor 2	Sensor 3
a Average of three determinations.b Limit of detection (measured by intersection of the extrapolated arms of calibration curve).
Slope (mV per decade)^a	58.90	59.14	60.42
Intercept (mV)	582.5	709.71	844.33
LOD (M)^b	9.60 × 10^−8	9.63 × 10^−10	9.14 × 10^−11
Response time (s)	60	40	25
Working pH range	4–6	4–6	4–6
Concentration range (M)	1 × 10^−7 to 1 × 10^−3	1 × 10^−9 to 1 × 10^−3	1 × 10^−10 to 1 × 10^−3
Stability (days)	51	55	48
Correlation coefficient	0.9998	0.9999	0.9999
Average recovery (%) ± S.D	99.52 ± 0.52	99.26 ± 1.05	99.53 ± 0.86
Repeatability (SD r)	0.72	0.65	0.41
Intermediate precision (SD int)	0.91	0.87	0.65

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Typical calibration plots are shown in Fig. 4. The slopes of the calibration plots are 58.9, 59.143 and 60.417 mV per concentration decade for sensors 1, 2 and 3, respectively. Deviation from the ideal Nernstian slope (60 mV) is due to the electrodes responding to the activity of the drug cation rather than its concentration. The sensors displayed constant potential readings for day to day measurements, and the calibration slopes did not change by more than ±2 mV per decade over a period of 51, 55 and 48 days for sensors 1, 2 and 3 respectively. The detection limits of the three sensors were estimated according to the IUPAC definition.¹⁵ Table 1 shows that sensor 3 could detect FLU in very dilute solutions down to 9.14 × 10⁻¹¹ M, sensor 2 could detect FLU down to 9.63 × 10⁻¹⁰ M, whereas sensor 1 could only detect FLU down to 9.60 × 10⁻⁸ M. As shown in Table 2, the selectivity of the proposed sensors in the presence of related substances that may be present in the dosage form with FLU, such as MgSO₄, CTAB, SLS and ZnSO₄, and in the presence of other anti-fungal drug such as Tinidazole is excellent.

Fig. 4 Profile of the potential in mV versus-log concentration of fluconazole in M obtained by using the sensors 1, 2 and 3.

Table 2 Potentiometric selectivity coefficients (K^pot_I) of the two proposed sensors using the separate solutions method (SSM)

Interferent^b	Selectivity coefficient^a
	Sensor 1	Sensor 2	Sensor 3
a Each value is the average of three determinations.b All interferents are in the form of 1 × 10^−4 M solution.
MgSO4	2.43 × 10^−3	3.28 × 10^−4	1.05 × 10^−6
Tinidazole	9.47 × 10^−5	7.79 × 10^−5	1.11 × 10^−6
Glycine	2.93 × 10^−5	2.62 × 10^−5	2.64 × 10^−6
L-Cysteine	8.79 × 10^−4	1.50 × 10^−3	2.07 × 10^−5
EDTA	6.69 × 10^−4	1.51 × 10^−5	8.95 × 10^−6
CTAB	3.73 × 10^−3	5.25 × 10^−4	3.27 × 10^−5
SLS	3.70 × 10^−4	1.97 × 10^−3	1.06 × 10^−6
ZnSO4	6.69 × 10^−4	2.62 × 10^−5	4.86 × 10^−6

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To examine the validity of the proposed sensors, the obtained results were compared to those of the reported method²⁴ and no significant difference was observed, as shown in Table 3.

Table 3 Statistical comparison for the results obtained the proposed electrodes and the official method (Basha 2011) for the analysis of fluconazole in pure powder form

Item	Sensor 1	Sensor 2	Sensor 3	Reported method^b
a Average of three determinations.b Spectrophotometric measurement at 260 nm.c The values in parentheses are the corresponding theoretical values for t and F at P = 0.05.
Mean^a	99.52	99.26	99.53	100.38
S.D^a	0.52	1.05	0.86	0.94
Variance	0.27	1.11	0.75	0.88
N	5	7	8	5
Student's t-test^c	1.80(2.45)	1.94(2.62)	1.65(2.31)	—
F value^c	3.26(6.39)	1.26(6.16)	1.17(4.12)	—

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3.2 Potentiometric determination of FLU in pharmaceutical formulation

The results obtained for the determination of FLU in pharmaceutical formulation show that a wide concentration range of the drug could be determined by the investigated sensors with high precision and accuracy. The results presented in Table 4 showed that sensors 2 and 3 are more sensitive than sensor 1.

Table 4 Determination of fluconazole in pharmaceutical formulation by the three proposed sensors

Sensor 1			Sensor 2			Sensor 3
Taken (M)	Found (M)	%Recovery^a	Taken (M)	Found (M)	%Recovery^a	Taken (M)	Found (M)	%Recovery^a
a Average of three determinations.
4.89 × 10^−4	4.92 × 10^−4	100.61	4.89 × 10^−4	4.91 × 10^−4	100.41	4.89 × 10^−4	4.83 × 10^−4	98.77
4.89 × 10^−5	4.90 × 10^−5	100.20	4.89 × 10^−5	4.90 × 10^−5	100.2	4.89 × 10^−5	4.86 × 10^−5	99.39
4.89 × 10^−6	4.86 × 10^−6	99.39	4.89 × 10^−6	4.92 × 10^−6	100.61	4.89 × 10^−6	4.86 × 10^−6	99.39
Mean ± S.D		100.01 ± 0.62	Mean ± S.D		100.41 ± 0.21	Mean ± S.D		99.18 ± 0.36

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3.3 Potentiometric determination of FLU in plasma, urine and milk

The results obtained for the determination of FLU in spiked human plasma show that a wide concentration range of the drug could be determined by the investigated sensors with high precision and accuracy. The results presented in Table 5 show that sensors 2 and 3 are more sensitive than sensor 1 in plasma samples.

Table 5 Determination of fluconazole in spiked human plasma (pretreated) by the proposed sensors

Added (M)	%Recovery ± S.D^a
	Sensor 1	Sensor 2	Sensor 3
a Average of three determinations.
10^−4	99.33 ± 1.70	98.80 ± 1.31	99.80 ± 1.31
10^−5	98.47 ± 1.55	98.83 ± 1.77	99.20 ± 1.72
10^−6	98.63 ± 0.95	98.10 ± 1.15	99.83 ± 1.11

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For the application of the sensors to urine and milk, it was found that the three sensors are reliable and give stable results with good accuracy and high percentage recovery, as shown in Tables 5 and 6.

Table 6 Determination of fluconazole in spiked human urine by the proposed sensors

Added (M)	%Recovery ± S.D^a
	Sensor 1	Sensor 2	Sensor 3
a Average of three determinations.
10^−4	98.63 ± 1.87	99.67 ± 1.53	99.8 ± 1.31
10^−5	97.60 ± 1.61	98.67 ± 1.46	99.10 ± 0.30
10^−6	98.30 ± 1.47	98.87 ± 1.33	99.83 ± 1.11

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The response times of the proposed sensors are rapid (within seconds), so the sensors were rapidly transferred back and forth between the biological samples and the bi-distilled water between measurements to protect the sensing component from adhering to the surface of some matrix components. It is concluded that the proposed sensors could be successfully applied to in vitro studies and for clinical use without the need for any pretreatment or preliminary extraction procedures from biological fluids. There was no significant difference between the results of FLU determinations in either pretreated or untreated plasma samples, as shown in Tables 7 and 8.

Table 7 Determination of fluconazole in spiked cow milk by the proposed sensors

Added (M)	%Recovery ± S.D^a
	Sensor 1	Sensor 2	Sensor 3
a Average of three determination.
10^−4	98.90 ± 1.01	99.00 ± 1.32	99.47 ± 0.92
10^−5	98.63 ± 1.58	98.40 ± 1.60	99.10 ± 1.90
10^−6	99.10 ± 1.23	99.33 ± 0.83	99.83 ± 1.11

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Table 8 Determination of fluconazole in spiked human plasma (untreated) by the proposed sensors

Added (M)	%Recovery ± S.D^a
	Sensor 1	Sensor 2	Sensor 3
a Average of three determinations.
10^−4	96.49 ± 1.27	97.53 ± 1.29	99.60 ± 1.31
10^−5	98.10 ± 1.90	98.87 ± 1.27	99.10 ± 1.90
10^−6	98.00 ± 1.97	99.40 ± 0.72	99.33 ± 1.60

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4 Conclusion

The described sensors are sufficiently simple and selective for the quantitative determination of FLU in pure form, pharmaceutical formulations, milk, plasma and urine. The proposed sensors offer the advantages of fast response and elimination of drug pre-treatment or separation steps. They could therefore be used for routine analysis of FLU in quality-control laboratories. It is also noticed that magnetic iron oxide particles play a role in increasing the sensitivity of sensor 2 more than 1, and of sensor 3 more than 2. Thus, decreasing the particle size of magnetic iron oxide particles offers this advantage of increasing sensitivity by significantly reducing the mass transfer distance.

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