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gold alloy nanoparticles for determination of anticancer drug flutamide
Accepted Manuscript A novel luminol chemiluminescent method catalyzed by silver/gold alloy nano‐ particles for determination of anticancer drug flutamide Mohammad Javad Chaichi, Seyed Naser Azizi, Maryam Heidarpour PII: DOI: Reference:
S1386-1425(13)00808-1 http://dx.doi.org/10.1016/j.saa.2013.07.060 SAA 10804
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo‐ lecular Spectroscopy
Received Date: Revised Date: Accepted Date:
2 March 2013 23 June 2013 22 July 2013
Please cite this article as: M.J. Chaichi, S.N. Azizi, M. Heidarpour, A novel luminol chemiluminescent method catalyzed by silver/gold alloy nanoparticles for determination of anticancer drug flutamide, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2013), doi: http://dx.doi.org/10.1016/j.saa.2013.07.060
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel luminol chemiluminescent method catalyzed by silver/gold alloy nanoparticles for determination of anticancer drug flutamide Mohammad Javad Chaichi, Seyed Naser Azizi and Maryam Heidarpour Faculty of Chemistry, Mazandaran University, Babolsar, Postcode 47416-95447, Iran [email protected] Author E-mail:
Abstract It
was
found
that
silver/gold
alloy
nanoparticles
enhance
the
chemiluminescence (CL) of the luminol–H2O2 system in alkaline solution. The studies of UV–Vis spectra, CL spectra, effects of concentrations luminol, hydrogen peroxide and silver/gold alloy nanoparticles solutions were carried out to explore the CL enhancement mechanism. Flutamide was found to quench the CL signals of the luminol– H2O2 reaction catalyzed by silver/gold alloy nanoparticles, which made it applicable for the determination of flutamide. Under the optimum conditions, the CL intensity is proportional to the concentration of the flutamide in solution over the range 5.0×10−7 to 1.0×10−4mol L-1. Detection limit was obtained 1.2×10−8 mol L-1and the relative standard deviation (RSD)
5%. This work is introduced as a new method
for the determination of flutamide in commercial tablets. Box-Behnken experimental design is applied to investigate and validate the CL measurement parameters. Keywords: Flutamide, Silver/Gold alloy nanoparticles, Luminol, hydrogen peroxide, chemiluminescence
1. Introduction Flutamide, 4-nitro-3-trifluoromethyl-isobutilanilide, is a synthetic antiandrogenic agent devoid of hormonal agonist activity (Fig. 1).It seems to have antiandrogenic specificity only in genitalia organsandrogen-dependent, and it also shows therapeutic use in prostatic cancer [1, 2]. Flutamide is an unusual example of an antiandrogenic drug lacking with a steroidal structure.
1
This drug and its primary hydroxy metabolite decrease metabolism of C-19 steroids by the cytochrome P-450 system at the target cells in the secondary sex organ [3]. A survey of literature reveals that there are not many methods for the assay of the drug. The reported methods include polarography [4, 5], voltammetery [6] gas chromatography [7], UV spectrophotometric method [8, 9] and high performance liquid chromatography [10]. The analytical parameters of the previous reported CL methods for the determination of flutamide drugs were summarized in Table 1. < Table 1> Among luminescence techniques, chemiluminescence (CL) is considered as the most sensitive and versatile analytical technique that can be used in the determination of different compounds depending on their participation in different CL systems [11]. CL analysis has advantages such as high sensitivity, easy to use and simple instrumentation, being actively applied for detection of chemical species at ultra-trace levels [12-18]. Though CL has been investigated for years, study of CL was limited to some molecular systems. Recently, nanoparticles reveal remarkable catalytic qualities for a variety of chemical reactions, depending upon their high surface areas, good adsorption characteristics, high activity, and high selectivity [19–24]. For example, Cui et al [25–27] reported that noble metal nanoparticles can enhance the CL of the lucigenin–KI and luminol–H2O2 systems. In these systems, metal nanoparticles can participate in CL reactions as a catalyst, reductant, luminophor, and energy acceptor [28-31]. However, the application of bimetallic nanoparticles as catalysts in luminol chemiluminescence system is rarely used. In the present study, the function of Au/Ag alloy nanoparticles (NPs) in luminol–H2O2 weak CL system has been explored [32]. It was found that Au/Ag nanoparticles could enhance intensely the CL from the reaction between luminol and H2O2. The effects of the reaction conditions such as pH and reagents concentration on the CL intensity was investigated. The UV-Visible spectra, CL spectra were investigated and the CL mechanism has been proposed. Moreover, the influences of flutamide on the luminol– H2O2−Au/Ag alloy nanoparticles CL system were also explored in detail.
2. Experimental 2.1. Reagents and solutions A 10−4 molL−1 stock solution of luminol (3-aminophthalhydrazide) was prepared by dissolving luminol (Fluka,) in 0.1 mol L−1 sodium hydroxide solutions without purification. A stock solution of hydrogen peroxide (30%, v/v, commercially available) was prepared by 2
appropriate dilution of 30% solution with water. HAuCl4·4H2O (48% w/w), AgNO3, NaBH4, buffer phosphate and polyethylen glycol (PEG) were obtained from Merck (Schuchardt, Germany). All the chemicals and reagents were of analytical grade and used without further purification; the deionized and triple-distilled water was used throughout. Stock solutions of flutamide were prepared at a constant concentration of 2.5×10-3 M in ethanol. An aliquot of stock solution was taken and diluted with ethanol/ 0.04 M BrittonRobinson buffer mixture (20/80) to obtain a final working solution concentration of 1 ×10-4 M. Flutamide (100% Analytical grade) was obtained from Sigma Aldrich (USA) and the commercial tablets of Drogenil (250.0 mg flutamide per tablet) were obtained commercially.
2.2. Apparatus and software A 3030 Jenway pH meter (leeds, UK) was used for pH measurements. Transmission electron microscopy (TEM) images were recorded on a Philips CM10 transmission electron microscope
(Andover,
USA).
Absorption
spectra
were
recorded
using
UV–Vis
spectrophotometer Cecil, CE5501 (Cambridge, UK). The CL light intensity time curve was carried out by a Sirius-tube luminometer (Pforzheim, Germany) with a photomultiplier tube detector. Also, 15 experimental runs (formulation combinations) for optimization of factor levelswere generated and analyzed using MINITAB 13.
2.3. Synthesis of gold–silver alloy Gold–Silver alloy NPs were synthesized by the hydroborate reduction method. In this paper, the solutions of 0.1M PEG was prepared in 25ml of water. Then 2.5 ml of both HAuCl4 and AgNO3 (5mM) was added and sonicated for 30 min in order to homogeneously distribute the metal ions in the solution. Co-reduced by dropwise adding 20 mL of freshly prepare 3.33 mmol/L NaBH4 to the PEG stabilized metal ion solution. The formed gold/silver nanoparticles were stabilized by the PEG. The size and shape of the synthesized nanoparticles were characterized by transmission electron microscope (TEM). Statistical analysis of TEM data revealed that the average diameters of Au–Ag alloy (3: 2) nanoparticles [33, 34] were 14.0 ± 2.0nm.
3
2.4. Preparation of standard solutions A series of ten solutions was which prepared containing flutamide concentration ranging between 1×10-4-5×10-7 M ethanol / 0.04 M Britton-Robinson buffer mixture (20/80) at pH 8.0.
2.5. Sample preparation Ten solutions of one tablet (amount declared 250.0 mg flutamide per tablet) were prepared in ethanol, sonicated and diluted to 50 mL. Then, these solutions were centrifuged for 10 min at 4000 rpm. 0.3 mL aliquot of each solution taken and diluted to 50 mL with ethanol/0.04M Britton Robinson buffer mixture (20/80), pH 8.0. The 0.0156 mg flutamide in the sample solution were calculated from a prepared standard calibration plot.
2.6. General procedure Solution A was made by mixing 0.3 mL of luminol (various concentrations), 0.1 mL of phosphate buffer (pH=8.5-10.5) and 50 µl of flutamide (various concentrations). Solution B contained various volumes of hydrogen peroxide 10-4 M and various volumes of NPs 10-4 M (1:2, 2:1, 1:1). Solution A was transferred into glass cell and then 100 µL of solution B was injected in the glass cell, chemiluminescence light intensity- time spectrum was recorded soon after mixing of the solutions. As mentioned above, flutamide was found to quench the CL emission of quinoxaline. Therefore, the concentration of flutamide was determined on the basis of changing the chemiluminescence intensity (ΔIcL). ΔIcL is obtained from the ratio of CL intensities in the absence (I0) and presence (I) of flutamide i.e., ΔIcL= I0 / I
3. Results and discussion 3.1. Optimization of the method In the present study, the 3-level 3-factor Box–Behnken experimental design is applied to investigate and validate CL measurement parameters affecting the CL intensity of Luminol pH / Au/Ag alloy NPs/H2O2 system. The factor levels were coded as: −1 (low), 0 (central point or middle) and 1 (high) [35]. The variables and levels of the Box–Behnken design model are given in Table S1. The results were analyzed using the coefficient of determination (R2), Pareto analysis of variance (ANOVA) and statistical and response plots. A non-linear regression method was used to fit the second order polynomial (Eq. (1)) to the experimental
4
data and to identify the relevant model terms. Considering all the linear terms, square terms and linear by linear interaction items, the quadratic response model can be described as Y = β0 +Σβixi +Σβiix2ii +Σβijxixj + ε
(1)
where, βo is the offset term, βi is the slope or linear effect of the input factor xi, βii is the quadratic effect of input factor xi and βij is the linear by linear interaction effect between the input factor xi and xj [36]. 3.2. Optimization of experimental conditions A Box-Behnken design, a full factorial experimental design plus two centered points (Table 3), was used to optimize the pH and concentrations of luminol and ratio of Au/Ag alloy NPs/H2O2. This study showed that the luminol concentrations, ratio of Au/Ag alloy NPs/H2O2 and the quadratic of ratio of NPs/H2O2 were significant factors at 95% confidence interval, having important effects on the response when their values change in the selected region (Table S2).
S1386-1425(13)00808-1 http://dx.doi.org/10.1016/j.saa.2013.07.060 SAA 10804
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo‐ lecular Spectroscopy
Received Date: Revised Date: Accepted Date:
2 March 2013 23 June 2013 22 July 2013
Please cite this article as: M.J. Chaichi, S.N. Azizi, M. Heidarpour, A novel luminol chemiluminescent method catalyzed by silver/gold alloy nanoparticles for determination of anticancer drug flutamide, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2013), doi: http://dx.doi.org/10.1016/j.saa.2013.07.060
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel luminol chemiluminescent method catalyzed by silver/gold alloy nanoparticles for determination of anticancer drug flutamide Mohammad Javad Chaichi, Seyed Naser Azizi and Maryam Heidarpour Faculty of Chemistry, Mazandaran University, Babolsar, Postcode 47416-95447, Iran [email protected] Author E-mail:
Abstract It
was
found
that
silver/gold
alloy
nanoparticles
enhance
the
chemiluminescence (CL) of the luminol–H2O2 system in alkaline solution. The studies of UV–Vis spectra, CL spectra, effects of concentrations luminol, hydrogen peroxide and silver/gold alloy nanoparticles solutions were carried out to explore the CL enhancement mechanism. Flutamide was found to quench the CL signals of the luminol– H2O2 reaction catalyzed by silver/gold alloy nanoparticles, which made it applicable for the determination of flutamide. Under the optimum conditions, the CL intensity is proportional to the concentration of the flutamide in solution over the range 5.0×10−7 to 1.0×10−4mol L-1. Detection limit was obtained 1.2×10−8 mol L-1and the relative standard deviation (RSD)
5%. This work is introduced as a new method
for the determination of flutamide in commercial tablets. Box-Behnken experimental design is applied to investigate and validate the CL measurement parameters. Keywords: Flutamide, Silver/Gold alloy nanoparticles, Luminol, hydrogen peroxide, chemiluminescence
1. Introduction Flutamide, 4-nitro-3-trifluoromethyl-isobutilanilide, is a synthetic antiandrogenic agent devoid of hormonal agonist activity (Fig. 1).It seems to have antiandrogenic specificity only in genitalia organsandrogen-dependent, and it also shows therapeutic use in prostatic cancer [1, 2]. Flutamide is an unusual example of an antiandrogenic drug lacking with a steroidal structure.
1
This drug and its primary hydroxy metabolite decrease metabolism of C-19 steroids by the cytochrome P-450 system at the target cells in the secondary sex organ [3]. A survey of literature reveals that there are not many methods for the assay of the drug. The reported methods include polarography [4, 5], voltammetery [6] gas chromatography [7], UV spectrophotometric method [8, 9] and high performance liquid chromatography [10]. The analytical parameters of the previous reported CL methods for the determination of flutamide drugs were summarized in Table 1. < Table 1> Among luminescence techniques, chemiluminescence (CL) is considered as the most sensitive and versatile analytical technique that can be used in the determination of different compounds depending on their participation in different CL systems [11]. CL analysis has advantages such as high sensitivity, easy to use and simple instrumentation, being actively applied for detection of chemical species at ultra-trace levels [12-18]. Though CL has been investigated for years, study of CL was limited to some molecular systems. Recently, nanoparticles reveal remarkable catalytic qualities for a variety of chemical reactions, depending upon their high surface areas, good adsorption characteristics, high activity, and high selectivity [19–24]. For example, Cui et al [25–27] reported that noble metal nanoparticles can enhance the CL of the lucigenin–KI and luminol–H2O2 systems. In these systems, metal nanoparticles can participate in CL reactions as a catalyst, reductant, luminophor, and energy acceptor [28-31]. However, the application of bimetallic nanoparticles as catalysts in luminol chemiluminescence system is rarely used. In the present study, the function of Au/Ag alloy nanoparticles (NPs) in luminol–H2O2 weak CL system has been explored [32]. It was found that Au/Ag nanoparticles could enhance intensely the CL from the reaction between luminol and H2O2. The effects of the reaction conditions such as pH and reagents concentration on the CL intensity was investigated. The UV-Visible spectra, CL spectra were investigated and the CL mechanism has been proposed. Moreover, the influences of flutamide on the luminol– H2O2−Au/Ag alloy nanoparticles CL system were also explored in detail.
2. Experimental 2.1. Reagents and solutions A 10−4 molL−1 stock solution of luminol (3-aminophthalhydrazide) was prepared by dissolving luminol (Fluka,) in 0.1 mol L−1 sodium hydroxide solutions without purification. A stock solution of hydrogen peroxide (30%, v/v, commercially available) was prepared by 2
appropriate dilution of 30% solution with water. HAuCl4·4H2O (48% w/w), AgNO3, NaBH4, buffer phosphate and polyethylen glycol (PEG) were obtained from Merck (Schuchardt, Germany). All the chemicals and reagents were of analytical grade and used without further purification; the deionized and triple-distilled water was used throughout. Stock solutions of flutamide were prepared at a constant concentration of 2.5×10-3 M in ethanol. An aliquot of stock solution was taken and diluted with ethanol/ 0.04 M BrittonRobinson buffer mixture (20/80) to obtain a final working solution concentration of 1 ×10-4 M. Flutamide (100% Analytical grade) was obtained from Sigma Aldrich (USA) and the commercial tablets of Drogenil (250.0 mg flutamide per tablet) were obtained commercially.
2.2. Apparatus and software A 3030 Jenway pH meter (leeds, UK) was used for pH measurements. Transmission electron microscopy (TEM) images were recorded on a Philips CM10 transmission electron microscope
(Andover,
USA).
Absorption
spectra
were
recorded
using
UV–Vis
spectrophotometer Cecil, CE5501 (Cambridge, UK). The CL light intensity time curve was carried out by a Sirius-tube luminometer (Pforzheim, Germany) with a photomultiplier tube detector. Also, 15 experimental runs (formulation combinations) for optimization of factor levelswere generated and analyzed using MINITAB 13.
2.3. Synthesis of gold–silver alloy Gold–Silver alloy NPs were synthesized by the hydroborate reduction method. In this paper, the solutions of 0.1M PEG was prepared in 25ml of water. Then 2.5 ml of both HAuCl4 and AgNO3 (5mM) was added and sonicated for 30 min in order to homogeneously distribute the metal ions in the solution. Co-reduced by dropwise adding 20 mL of freshly prepare 3.33 mmol/L NaBH4 to the PEG stabilized metal ion solution. The formed gold/silver nanoparticles were stabilized by the PEG. The size and shape of the synthesized nanoparticles were characterized by transmission electron microscope (TEM). Statistical analysis of TEM data revealed that the average diameters of Au–Ag alloy (3: 2) nanoparticles [33, 34] were 14.0 ± 2.0nm.
3
2.4. Preparation of standard solutions A series of ten solutions was which prepared containing flutamide concentration ranging between 1×10-4-5×10-7 M ethanol / 0.04 M Britton-Robinson buffer mixture (20/80) at pH 8.0.
2.5. Sample preparation Ten solutions of one tablet (amount declared 250.0 mg flutamide per tablet) were prepared in ethanol, sonicated and diluted to 50 mL. Then, these solutions were centrifuged for 10 min at 4000 rpm. 0.3 mL aliquot of each solution taken and diluted to 50 mL with ethanol/0.04M Britton Robinson buffer mixture (20/80), pH 8.0. The 0.0156 mg flutamide in the sample solution were calculated from a prepared standard calibration plot.
2.6. General procedure Solution A was made by mixing 0.3 mL of luminol (various concentrations), 0.1 mL of phosphate buffer (pH=8.5-10.5) and 50 µl of flutamide (various concentrations). Solution B contained various volumes of hydrogen peroxide 10-4 M and various volumes of NPs 10-4 M (1:2, 2:1, 1:1). Solution A was transferred into glass cell and then 100 µL of solution B was injected in the glass cell, chemiluminescence light intensity- time spectrum was recorded soon after mixing of the solutions. As mentioned above, flutamide was found to quench the CL emission of quinoxaline. Therefore, the concentration of flutamide was determined on the basis of changing the chemiluminescence intensity (ΔIcL). ΔIcL is obtained from the ratio of CL intensities in the absence (I0) and presence (I) of flutamide i.e., ΔIcL= I0 / I
3. Results and discussion 3.1. Optimization of the method In the present study, the 3-level 3-factor Box–Behnken experimental design is applied to investigate and validate CL measurement parameters affecting the CL intensity of Luminol pH / Au/Ag alloy NPs/H2O2 system. The factor levels were coded as: −1 (low), 0 (central point or middle) and 1 (high) [35]. The variables and levels of the Box–Behnken design model are given in Table S1. The results were analyzed using the coefficient of determination (R2), Pareto analysis of variance (ANOVA) and statistical and response plots. A non-linear regression method was used to fit the second order polynomial (Eq. (1)) to the experimental
4
data and to identify the relevant model terms. Considering all the linear terms, square terms and linear by linear interaction items, the quadratic response model can be described as Y = β0 +Σβixi +Σβiix2ii +Σβijxixj + ε
(1)
where, βo is the offset term, βi is the slope or linear effect of the input factor xi, βii is the quadratic effect of input factor xi and βij is the linear by linear interaction effect between the input factor xi and xj [36].