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: S1386-1425(13)00808-1

DOI: http://dx.doi.org/10.1016/j.saa.2013.07.060

Reference: SAA 10804

To appear in: Spectrochimica Acta Part A: Molecular and Biomo‐

lecular Spectroscopy

Received Date: 2 March 2013

Revised Date: 23 June 2013

Accepted Date: 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

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1  

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

: mail-ECorresponding Author com.yahoo@jchaichi

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.

<Fig. 1>

2  

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

3  

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 Britton-

Robinson 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.

4  

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

5  

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].

<Table S1>

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).

<Table2>

<Table S2>

The function representing the relationship among pH, luminol concentrations and ratio of

Au/Ag alloy NPs/H2O2 and the chosen response is:

Y= 14.19 + 3.98X1 + 0.847X2 + 1.07X3-0.53X12 – 0.59X2

2 – 1.36X32 – 0.447X1X2 +0.81X1X3- 1.05 X2X3

According to analysis of variance (Table 3), it was shown that the predictability of the

model is at 95% confidence interval. The ANOVA of these responses demonstrated that the

model is highly significant as is evident from the value of Fstatistic (the ratio of mean square

due to regression to mean square to real error; Fmodel=25.74) and a very low probability value

(P= 0.001). The value of probability P>0.05 indicates that the model is considered

statistically significant [37]. The non-significant lack-of-fit (more than 0.05) showed that the

quadratic model is valid for present study. Non-significant lack-of-fit is good for data fitness

in the model.

<Table 3>

Therefore, the three factors were simultaneously optimized. Considering the results

obtained from the established response surface (Fig. 2), the following optimum values were

found: a 1×10-4 M luminal concentration, a ratio 2:1 of Au/Ag alloy NPs/H2O2 (both

corresponding to the highest level tested), and a pH 9.5. However, concentrations of 10-4 M of

luminol and, the ratio 2:1 of Au/Ag alloy NPs/H2O2 (10-4M) were chosen to be suitable for

6  

the purposes of the study, because higher concentrations of luminol and Au/Ag alloy

NPs/H2O2 could produce the saturation of the detector due to the high CL intensity of the

blank signal obtained.

<Fig. 2 (.a, b, c)>

3.3. Analytical performance

The ratio of the initial CL intensity I0 of luminaol- buffer- Au/Ag alloy NPs/H2O2

system to the CL intensity I at a given concentration of flutamide, I0/I, was proportional to the

concentration of flutamide. The flutamide concentration dependence of the CL intensity was

coincident to the fluorescence quenching described by a Stern- Volmer equation (Eq.2.):

I0/I=1+Ksv [Q] (2)

Ksv was found to be 22429 (mol-1 L) and this large value provided a sensitive CL

detection of flutamide. The Stern–Volmer plots for flutamide is shown in insert Fig. 3.

<Fig. 3>

Under the optimized experimental conditions, the relative CL intensity decreased

linearly in the concentration range of 1.0×10-4-5.0×10-7 mol L−1 for flutamide (R2 = 0.9981)

with detection limit of 1.8×10−9 mol L−1 at S/N ratio of 3. The resulting intensity–time plots

are shown in Fig. 3. (Table S3 lists the measured results of the content of flutamide in tablet).

<Table S3>

3.4. Mechanism discussion

The nanoparticles were primarily characterized by UV–Visible spectroscopy, which

has proved to be a very useful technique for the analysis of nanoparticles. In UV–Visible

spectrum a strong, broad peak, located between 420 and 440 nm, was observed for Ag/Au

alloy NPs prepared using the culture supernatant (Fig.4). The transmission electron

micrographs of Ag/Au alloy NPs are represented in Fig. 5. The particle size was found to a

range from 12 to 16 nm. The effects of Ag/Au alloy NPs on the chemiluminescent system

were investigated. As shown in insert Fig. 4, the CL signal was enhanced by Ag/Au alloy

NPs.

<Fig. 4>

<Fig. 5>

7  

The luminol based CL reaction is a well-known method for the detection of reactive

oxygen species, such as O2-, 1O2, and H2O2, because these species react quickly with luminol

in alkaline solution to emit light, as the hydroperoxide intermediate of luminol decomposes

into aminophthalate. The results (insert Fig.4) showed that the CL signal was enhanced by

Ag/Au alloy NPs, indicating that the luminophor in the both CL reactions between luminol

and hydrogen peroxide with and without Au/Ag alloy NPs is the same species, which is the

oxidation product 3-aminophthalate (3-APA*) of luminol [32].A schematic proposed reaction

process could be shown as Scheme 1.

Scheme 1><

3.5 Application

It has been reported that the reducing groups such as OH, NH2, or SH reacted readily

with the oxygen-containing intermediate radicals. In the luminol–buffer–Au/Ag alloy

NPs/H2O2 system, some intermediate radicals such as HO• and O2•− were formed during the

reaction. The reducing groups are likely to compete with luminol for active oxygen

intermediates (covered the surface of NPs) leading to a decrease in CL intensity. Therefore,

these compounds may interact with Au/Ag alloy NPs to interrupt the formation of luminal

radicals (L•−) and superoxide radical anion (O2•−) taking place on the surface of Au/Ag alloy

NPs, resulting in a decrease in CL intensity. The results demonstrate that the luminol–buffer–

Au/Ag alloy NPs/H2O2 CL system has a wide application for the determination of such

compounds via quenching phenomena [32].

Therefore the effects of flutamide on the CL system were investigated. As expected,

existence groups NHCO in flutamide as reducing functional groups inhibited the CL signal of

luminol–buffer–Au/Ag alloy NPs/H2O2 system. Also the functional groups of NO2 and CF3

are intense electron with drawing groups. Therefore N-H bond is become acidic and the

proton may be released easily. Protonation of the excited 3-APA led to quenching of the CL

system. Recovery tests were performed to evaluate the accuracy of this method. Results for

the contents and recoveries were summarized in Table 4. The recoveries ranged from 98% to

101%, with RSDs of <5.0%.

<Table 4>

8  

3.6. Interferences

It is well know that the chemiluminescence reaction is highly sensitive to the external

species. Under optimal experimental conditions, the interferences from selected metal ions

and organic compounds were evaluated. Most of the candidate compounds had no significant

influence on the determination of flutamide concentration. The tolerable molar concentration

ratios of foreign species to flutamide were >100 for Na+, K+, Ca+2, Mg+2, NO3- , Cl-, glucose,

starch and uric acid ;10 for Fe+2, Cu+2, SO4-2. However, ascorbic acid was found to be strong

interfering agents. Ascorbic acid in presence of oxidizing agents led to the quenching effect

on luminol CL reaction [38].

4. Conclusions

In conclusion, it was found that Au/Ag alloy NPs could strongly enhance the CL of

the luminol–H2O2 system. The mechanism of luminol–H2O2–Au/Ag alloy NPs CL reaction

was discussed. The proposed CL system has good linearity, high sensitivity and lower

detection limit and the method has been successfully applied to the determination of

flutamide in tablet.

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Figure captions

Scheme 1 Possible mechanism for the luminol–Au–Ag alloy NPs– H2O2 colloids CL system

Fig. 1. Chemical structure of flutamide

Fig. 2.a) Estimated response surface for [pH] =0, b) Estimated response surface for

[luminol] =0, c) Estimated response surface for [Au/Ag alloy NPs/H2O2] =0.

Fig. 3. Chemiluminescence emission intensity as a function of time for the luminal- buffer-

Au/Ag alloy NPs/H2O2 system with constant concentration of luminal (10-4M), buffer

(pH=9.5) and ratio 2:1 Au/Ag alloy NPs/H2O2 (10-4M) system, and varying concentrations of

flutamide: (1) 0.0 (2) 5×10-6 (3) 1.0×10-6 (4) 5×10-5 (5) 1.0× 10-5 (6) 5×10-4

Fig. 4 UV-Vis absorption spectra of gold and gold-silver alloy nanoparticles synthesized by

PEG stabilized metal. The inset shows the CL signal was enhanced by Ag/Au alloy NPs (a)

luminal- buffer- Au/Ag alloy NPs/H2O2 (b) luminal- buffer-H2O2 systems

Fig. 5 TEM image of gold-silver alloy nanoparticles, Particle size distribution of Au/Ag alloy

NPs 14.0 ± 2.0nm

Table captions

11  

Table1. Analytical parameters of the previous CL methods for the determination of flutamide

drugs

Table 2 Box-Benhken matrix for optimization of chemical factors

Table 3 ANOVA results for quadratic equation for CL intensity of flutamide

Table 4 Results of flutamide determination and recoveries in tablet sample (n=3)

Supporting information

Table S1 Experimental design level of chosen variable

Table S2 Test of significance for regression coefficients

Table S3 Determination of flutamide in pharmaceutical sample by the proposed method

12  

NH2CO2

CO2

_

_ *

O

O

NHNH

NH2O

O

NNH

NH2

_

O

NN

O2O

NH2

__

H2O2 OH HO2_

+

+ hv

+

+

Au-Ag alloy nanoparticle

+ OH +

H2O2Catalyst

Au-AgOH.

. OH

HO2

_ O2.

+

O

O

NNH

NH2

_

_

N

O

O

N

NH2

.

NH2CO2

CO2

_

_

Au-Ag

N2

H2O_

_H2O

Scheme 1 Possible mechanism for the luminol–Au–Ag alloy NPs– H2O2 colloids CL system

Fig. 1 Chemical structure of flutamide

13  

a

b

c

Fig. 2.a) Estimated response surface for [pH] =0, b) Estimated response surface for [luminol]

=0, c) Estimated response surface for [Au/Ag alloy NPs/H2O2] =0.

14  

Fig. 3. Chemiluminescence emission intensity as a function of time for the luminol- buffer-

Au/Ag alloy NPs/H2O2 system with constant concentration of luminol (10-4M), buffer

(pH=9.5) and ratio 2:1 Au/Ag alloy NPs/H2O2 (10-4M) system, and varying concentrations of

flutamide: (1) 0.0 (2) 5×10-6 (3) 1.0×10-6 (4) 5×10-5 (5) 1.0×10-5 (6) 5×10-4

Fig. 4. UV-Vis absorption spectra of gold and gold-silver alloy nanoparticles synthesized by

PEG stabilized metal. The inset shows the CL signal was enhanced by Ag/Au alloy NPs (a)

luminol- buffer- Au/Ag alloy NPs/H2O2 (b) luminol- buffer-H2O2 systems

15  

Fig. 5 TEM image of gold-silver alloy nanoparticles, Particle size distribution of Au/Ag alloy

NPs 14.0 ± 2.0nm

16  

Table1. Analytical parameters of the previous CL methods for the determination of flutamide drugs

Method Linear range

(mol L-1)

DL

(mol L-1)

Ref.

HPLC with UV detection

Flow- Injection method

spectrophotometric method

spectrophotometric method

differential pulse polarogrphy

voltammetry

This work

3.6×10-7- 2.1×10-6

3.6×10-4-1.4×10-3

2.8×10-6- 3.6×10-5

2.9×10-6- 5.1×10-5

2.0×10-9- 1.5×10-5

7.2×10-5- 5.7×10-4

5.0×10−7-1.0×10−4

3.1×10-8

4.3×10-7

4.7×10-7

4.6×10-7

1.7×10-9

1.8×10-7

1.2×10−8

[8]

[48]

[8]

[9]

[5]

[6]

-

Table 2. Box-Benhken matrix for optimization of chemical factors

SE expected Expected Experimental NPs/ H2O2 pH Luminol Experiment

0.477 14.199 13.658 0 0 0 1

0.715 17.452 16.700 0 + + 2

0.715 7.7940 8.5460 0 - - 3

0.715 8.0650 7.7340 - 0 - 4

0.477 14.199 14.923 0 0 0 5

0.715 10.382 10.571 0 + - 6

0.715 14.408 15.018 - 0 + 7

0.477 14.199 14.017 0 0 0 8

0.715 9.2700 8.8490 - - 0 9

0.715 8.5860 7.9760 + 0 - 10

0.715 13.517 13.375 + - 0 11

0.715 13.104 13.525 + + 0 12

0.715 18.170 18.501 + 0 + 13

0.715 16.652 16.463 0 - + 14

0.715 13.070 13.212 - + 0 15

17  

Table 3 ANOVA results for quadratic equation for CL intensity of flutamide

Prob>F P F-ratio Mean square Sum of square DF Source

Significant 0.001 25.74 17.547 157.925 9 Regression

0.6817 3.409 5 Residual error

Not Significant 0.350 2.01 0.8525 2.559 3 Lack-of-fit

0.4250 0.850 2 Pure error

161.334 14 Total

Table 4 Results of flutamide determination and recoveries in tablet sample (n=3)

Sample Added (10-5 Mol L−1)Found (10-5 Mol L−1) Recovery (%) R.S.D (n=3) (%)

1 0 1.04 ± 0.03 - -

2 1 2.03 ± 0.25 99 1.2

3 5 6.10 ± 0.51 101 4.8

4 10 10.84 ± 0.047 98 3.2

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A novel luminol chemiluminescent method catalyzed by silver/gold alloy

nanoparticles for determination of anticancer drug flutamide

Mohammad Javad Chaichi*, Seyed NaserAzizi and Maryam Heidarpour

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Highlight

A silver/gold alloy nanoparticle is used as catalysis for luminal CL system.

Box–Behnken experimental design is used in the CL measurement parameters.

The high sensitivity method is developed for determination of flutamide.

There is a suitable LOD and LDR for detection of flutamide in tablets.