Two different spectrophotometric determinations of potential anticancer drug and its toxic metabolite

Article abstract of DOI:10.1016/j.saa.2015.03.009

Flutamide is a hormone therapy used for men with advanced prostate cancer. Flutamide is highly susceptible to hydrolysis with the production of 3-(trifluoromethyl)aniline, which is reported to be one of its toxic metabolites, impurities and related substances according to BP and USP. Flutamide was found to be stable when exposed to oxidation by 30% hydrogen peroxide and direct sunlight for up to 4 h. Two accurate and sensitive spectrophotometric methods were used for determination of flutamide in bulk and in pharmaceutical formulations. Method (I) is the area under curve (AUC) spectrophotometric method that depends on measuring the AUC in the wavelength ranges of 275-305 nm and 350-380 nm and using Cramer's rule. The linearity range was found to be 1-35 μg/mL and 0.5-16 μg/mL for the drug and the degradate, respectively. In method (II), combination of the isoabsorptive and dual wavelength spectrophotometric methods was used for resolving the binary mixture. The absorbance at 249.2 nm (λ_iso) was used for determination of total mixture concentration, while the difference in absorbance between 232 nm and 341.2 nm was used for measuring the drug concentration. By subtraction, the degradate concentration was obtained. Beer's law was obeyed in the range of 2-35 μg/mL and 0.5-20 μg/mL for the drug and its degradate, respectively. The two methods were validated according to USP guidelines and were applied for determination of the drug in its pharmaceutical dosage form. Moreover AUC method was used for the kinetic study of the hydrolytic degradation of flutamide. The kinetic degradation of flutamide was found to follow pseudo-first order kinetics and is pH and temperature dependent. Activation energy, kinetic rate constants and t_1/2 at different temperatures and pH values were calculated.

Full text of DOI:10.1016/j.saa.2015.03.009

Accepted Manuscript
Two Different Spectrophotometric Determinations of Potential Anticancer Drug
and its Toxic metabolite
Nehal F. Farid, Nada S. Abdelwahab
PII:
S1386-1425(15)00305-4
http://dx.doi.org/10.1016/j.saa.2015.03.009
SAA 13423
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Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
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23 February 2015
1 March 2015
Please cite this article as: N.F. Farid, N.S. Abdelwahab, Two Different Spectrophotometric Determinations of
Potential Anticancer Drug and its Toxic metabolite, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.03.009
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Two Different Spectrophotometric Determinations of Potential Anticancer
Drug and its Toxic metabolite
Nehal F. Farid*, Nada S. Abdelwahab
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Benisuef University, Benisuef, Egypt.
* Corresponding author
Name: Nehal Fayek Farid
Tel: +201277950994
Email: nehalfayek@gmail.com
1

ABSTRACT
Flutamide is a hormone therapy used for men with advanced prostate cancer. Flutamide is
highly susceptible to hydrolysis with the production of 3-(trifluoromethyl)aniline, which is
reported to be one of its toxic metabolites, impurities and related substances according to BP
and USP. Flutamide was found to be stable when exposed to oxidation by 30% hydrogen
peroxide and direct sunlight for up to four hours. Two accurate and sensitive
spectrophotometric methods were used for determination of flutamide in bulk and in
pharmaceutical formulations.
Method (I) is the area under curve (AUC) spectrophpotometric method that depends on
measuring the AUC in the wavelength ranges of 275-305 nm and 350-380 nm and using
Cramer's Rule. The linearity range was found to be 1-35 µg/mL and 0.5-16 µg/mL for the
drug and the degradate, respectively. In method (II), combination of the isoabsorpative and
dual wavelength spectrophotometric methods was used for resolving the binary mixture. The
absorbance at 249.2 nm (λiso) was used for determination of total mixture concentration,
while the difference in absorbance between 232 nm and 341.2 nm was used for measuring the
drug concentration. By subtraction, the degradate concentration was obtained. Beer's law was
obeyed in the range of 2-35 µg/mL and 0.5-20 µg/mL for the drug and its degradate,
respectively.
The two methods were validated according to USP guidelines and were applied for
determination of the drug in its pharmaceutical dosage form. Moreover AUC method was
used for the kinetic study of the hydrolytic degradation of flutamide. The kinetic degradation
of flutamide was found to follow pseudo-first order kinetics and is pH and temperature
dependent. Activation energy, kinetic rate constantsand t1/2 at different temperatures and pH
values were calculated.
Keywords: flutamide; area under curve; isoabsorpative; dual wavelength; kinetic study;
degradation.
2

1. INTRODUCTION
Flutamide is
chemically
known
as
2-methyl-N[4-nitro-3-
(trifluoromethyl)pheneyl]propamide [1]. It is a nonsteroidal component with anti-adrenergic
properties used for treatment of prostatic carcinoma [2]. Flutamide is susceptible to
hydrolytic degradation with the production of 3-(trifluoromethyl)aniline which is stated to be
one of the impurities and related substance of flutamide according to BP [3] and USP [4].The
resulted degradate was also reported to be one of the main toxic metabolites of flutamide [5].
Flutamide was determined in BP [3] by UV spectrophotometric method through
measuring the absorbance at 295 nm of methanolic solution. On the other hand USP [4]
determined the drug in pure form and in capsules by RP-HPLC method using acetonitrile:
water (1:1, v/v) as a mobile phase.
Several methods were reported for analysis of the studied drug including electrochemical
methods [6], different spectrophotometric methods [6-11] and chromatographic methods [6,
12-15].
Reaction kinetics is the study of chemical change and the way by which this change is
influenced by variable conditions such as concentration of reactant, the present chemicals and
temperature. It provides information that permits a rational approach to the stabilization of
drug products, prediction of shelf life and optimization of storage conditions [16-19]. Many
environmental conditions such as heat, light, hydrolysis or oxidation can take part in
pharmaceutical stability [19]. Hydrolysis is one of the most common degradation routes,
since water exists for most drugs and excipients as a solvent or moisture in the air [21].
Most reported spectrophotometric methods included the formation of colored products
that suffered from various drawbacks and tedious sample preparation. On the other hand,
none of the reported methods described the degradation behavior or mechanism of flutamide.
The work in this manuscript aimed to study the kinetic of the hydrolytic degradation of the
anticancer flutamide under acidic and alkaline conditions using different temperatures
through the development of stability indicating spectrophotometric methods. Characterization
of the produced degradate (flutamide toxic metabolite) was carried out by IR and mass
spectroscopic analyses. Other degradation pathways as oxidation and exposure to direct
3

sunlight were tried and the drug was found to be stable under these conditions. The developed
methods are easy to be applied, need simple sample preparation and data manipulation, hence
they can be used in quality control and stability studies of the proposed drug.
2. EXPERIMENTAL
Instruments
-A double beam UV-Visible spectrophotometer (SHIMADZU, Japan), model UV-1601 PC
with 1cm path length quartz cell is used and it is connected to IBM compatible computer. The
software was UVPC personal spectroscopy software version 3.7.
-UV lamp with short wavelength 254 nm (USA).
Materials
-Pure samples
-Flutamide sample was purchased from Sigma-Aldrishchemie GmbH., Germany, its purity
was found to be 99.25%.
-Pharmaceutical formulation
-Cytomed® tablets is labeled to contain 250 mg flutamide and manufactured by CIPLA LTD.
INDIA.
-Chemicals and reagents
All chemicals and reagents used throughout this work were of analytical grade and were used
without further purification.
Methanol and glacial acetic acid [(Sigma-Aldrich, Chromasolv®, Germany).
Sodium hydroxide (0.05 N,0.1 N and 0.2N aqueous solution), HCl (0.3N, 0.5N and
1Naqueous solution), hexane, ethylacetate (El-Nasr Pharmaceutical Chemicals Co., Abu-
Zabaal, Cairo, Egypt).
-Preparation of flutamide degradation product
0.2 g of flutamide was dissolved into 10mL methanol and then refluxed at 800C with 40mL
of either 1N HCl or 0.1N NaOH or 30% hydrogen peroxide for 3 hours. The drug solution
was also left for 4 hours in direct sunlight. The degradation process was followed by TLC
4

using a developing system consisting of hexane: ethyl acetate: glacial acetic acid (8: 3: 0.1,
by volume). After complete degradation, the produced yellow solution was extracted with
chloroform (3X10mL) and the extracted degradate was dried at 600C and then identified by
IR and mass spectroscopic analyses.
Solutions:
Stock solutions of flutamide and degradate(1mg/mL) were prepared by accurately weighing
0.1 gm flutamide and the prepared degradate in two separate 100-mL volumetric flasks and
dissolving in methanol.
Working solution of flutamide and degradate (0.1mg/mL) were prepared by transferring 10
mL from their respective stock solutions(1 mg/mL) into two separate 100-mL volumetric
flasks and then diluting with methanol.
For kinetic investigations, standard solutions of flutamide (2.9 x 10-3 and 4.3 x 10-3 M) were
prepared by dissolving the corresponding amount of flutamide in 10mL methanol and then
completing the volume to 25-mL with either HCl (0.3, 0.5 or 1N) or NaOH (0.05, 0.1 or
0.2N).
Preparation of laboratory prepared mixtures
Different aliquots of flutamide and its degradation product working solutions (0.1mg/mL)
were transferred to a set of 10-mL volumetric flasks and the volume was completed with
methanol to prepare laboratory prepared mixtures containing from 5% to 90% of the
degradation product.
Application to pharmaceutical formulation
Ten tablets of cytomed® were grinded well and then an accurately weighted amount was
transferred into 100-mL volumetric flask. 75 mL methanol was added and the solution was
ultra-sonicated for 30 minutes, filtered and then the volume was completed with the same
solvent to prepare sample stock solution (1mg/mL). Sample working solution (0.1mg/mL)
was then prepared.
Procedure
Spectral characteristics of flutamide and its degradation product
5

Zero order absorption spectra of 10µg/mL each of flutamide and its degradation product were
recorded in the range of 200-450 nm using methanol as a blank.
Construction of calibration curves
Concentrations of flutamide in the range of 1-35µg/mL and of the degradation product in the
range of 0.5-20µg/mL were prepared in methanol. The absorption spectra of the prepared
solutions were recorded in the range of 200-450nm.
For method (I) [area under curve method, AUC]: the area under the curve in the range of
275-305 nm (λ1-λ2) and 350-380nm (λ3-λ4) were recorded for the prepared solutions each of
flutamide and the degradation product. Then the absorptivity ‘Y’ values of each were
calculated where Y = the recorded area under curve of each component (from 275 to 305 nm
or 350 to 380 nm)/concentration of the component (in µg/mL). The concentrations of the
studied components in the prepared solutions were determined by applying Cramer’s rule and
matrices in the following equations:
A1= Yx1Cx+ Yz1Cz
A2=Yx2Cx+ Yz2Cz
(λ1-λ2)
(λ3-λ4)
Where A1, A2are the areas under curve in the range of 275-305 nm and 350-380nm,
respectively.Yx1, Yx2 are the absorptivity values of flutamide at (λ1-λ2) and (λ3-λ4),
respectively. Yz1and Yz2 are the absorptivity values of the degradate at (λ1-λ2) and (λ3-λ4),
respectively. Cx and Cz are the concentrations in µg/mL of flutamide and its degradate,
respectively.
For method (II)[dual wavelength and isoabsorbtivepoint methods]: The absorbance values at
232, 341.2 nm(for flutamide) and at 249.2 nm (for the degradate) were recorded.
Concentrations of flutamide were measured by plotting the absorbance difference at the
selected wavelengths (zero difference for the degradate) versus the corresponding
concentrations of flutamide from which the regression equation was computed. On the other
hand the absorbance at 249.2 nm (λiso) was used for construction of the calibration curve of
the degradation product.
Analysis of laboratory prepared mixtures
The absorption spectra of the previously prepared mixtures were recorded in the range of
200-450nm. For method (I), the area under curve in the range of 275-305nm (λ1-λ2) and 350-
380nm (λ3-λ4) were recorded then by substituting in the corresponding equations,
concentrations of the drug and the degradate could be obtained. For method (II), the
6

absorbance difference at 232nm and 341.2 nm was used for measuring flutamide
concentrations while the absorbance values at 249.2 nm were used for measuring the total
mixture concentration and by subtraction, concentrations of the degradation product could be
calculated.
Analysis of pharmaceutical formulation
Dilutions of the prepared sample working solution (0.1mg/mL) were performed to prepare
concentrations within the linearity range of the studied drug. Procure illustrated under
construction of the calibration curve for each method was followed. Moreover, recovery
studies were carried out at three levels (80, 100 and 120%) to access accuracy of the method.
Kinetic study
a- Effect of drug concentration
Two concentrations of flutamide (2.9 x 10-3 and 4.3 x 10-3 M) were prepared once in 0.5N
HCl (set-1) and the other in 0.1N NaOH (set-2). The prepared solutions were rapidly inserted
into thermostatic water baths set at 80 0C (for set-1) and 500C (for set-2).At the specified time
intervals, 0.25 mL of each sample was accurately transferred into 10-mL measuring flask,
0.25mL of either 0.5N NaOH (for set-1) or 0.1N HCl (for set-2) was added to stop the
reaction and then the volume was completed with methanol. Immediately the absorption
spectra of the prepared solutions were recorded and the remaining drug concentration was
determined by applying method (I). The kinetic parameters [order of the reaction, degradation
rate constant (K) and half-life of the reaction (t1/2)] were calculated by plotting the logarithm
of the remaining flutamide concentration versus time in minutes.
b- Effect of HCl and NaOH strength on the reaction rate
Solutions of 2.9 x 10-3M flutamide were prepared once in 0.3N, 0.5N and 1N HCl (set-1) and
the other in 0.05N, 0.1N and 0.2N NaOH (set-2). The solutions were then placed in
thermostated water baths set at 800C (for set-1) or at 500C (for set-2). The procedure
illustrated under (the effect of drug concentration) was then followed.
c- Effect of temperature on the reaction rate
Solutions of 2.9 x 10-3M flutamide were prepared one in 0.5N HCl (set-1) and the other in
0.1N NaOH (set-2). The solutions were then placed in thermostated water baths set at 700C,
800C and 900C (for set-1) or at 400C, 500C and 600C (for set-2). The procedure illustrated
under (the effect of drug concentration) was then followed. Temperature effect on drug
7

degradation rate was studied by plotting of Arrhenius plot (the logarithm of the reaction rate
constant (log K) versus the reciprocal of the absolute temperature (1/T) in kelvin) from which
the activation energy (Ea) was calculated (Slope= -Ea (KJ/mol)/R, R=8.314J/Kmol).
.
3- RESULTS AND DISCUSSION
Prostate cancer is one of the most common cancers in many industrialized nations and
among the leading causes of cancer death [22]. It may be less common in developing
countries, but its rate and mortality has been on the increase [23]. Flutamide is widely used to
treat advanced (metastatic) prostate cancer. Till now, no report has been found in the
literature for studying the stability and the degradation kinetic of the anticancer flutamide.
Moreover, the produced degradate is considered to be toxic metabolite, impurity and related
substance of the main drug. Due to the pharmaceutical importance of the studied drug, it is
important to develop sensitive, selective and accurate methods for its analysis and to study its
degradation behavior. Studying the reaction kinetics plays a vital role in providing
information about the chemical reaction rate, t1/2, thermodynamic effects as well as the effect
of various process variables.
-Degradation behavior of flutamide
Flutamideis an amide containing drug that is highly susceptible to alkaline and acidic
hydrolysis. Upon refluxing the cited drug with either 1N HCl or 0.1N NaOH at 800C for 3
hours, complete drug degradation was observed with the formation of a yellow degradate.
Structural elucidation of the resulted degradation product has been carried out by IR and mass
spectroscopic analyses.
The IR spectrum of flutamide, Fig. (1) showed a sharp peak at 1715 cm-1and a peak at
3360cm-1 corresponding to C=O and NH groups which disappeared in the IR spectrum of the
degradate and a new forked peak at 3371.92-3479.92 cm-1was appeared indicating the
cleavage of the amide linkage and the formation of a primary amine (NH2) containing
compound, Fig. (1).The electron impact of the degradate showed mass ion peak at 206m/z
corresponding to molecular weight of the expected degradation product, Fig. (1).
Flutamide was found to be stable to oxidation by 30% hydrogen peroxide and light for up
to four hours (no spots on TLC plate other than that of flutamide were observed).
8

-Methods development and optimization
The developed area under curve, dual wavelength and isoabsorptive point spectrophotometric
methods are sensitive, simple and selective with minimum sample preparation and data
manipulation comparing to all the reported spectrophotometric methods [3, 6-11] that are of
low selectivity and need tedious sample preparation steps.On the other hand the reported
chromatographic methods [6, 12-15] need expensive and sophisticated apparatus. Zero order
absorption spectra of flutamide and itsdegradate, Fig. 2 showed severe spectral overlap which
impeded their direct determinations. By applying the proposed methods, the drug and the
degradate spectral overlap could be successfully resolved. The developed area under curve
method (AUC)(method I) was used for simultaneous determination of the drug and the
degradate. In the second method (method II), dual wavelength method was applied for
determination flutamide while the isoabsorpitive method developed by Erram and Tipinis
[24-26] has been used measuring the total mixture concentration and by subtraction,
concentration of the degradate could be obtained.
In the area under curve method (AUC), selection of wavelength ranges is an important step
during method optimization hence it affects selectivity of the method. Different wavelength
ranges were tested where the wavelength ranges of 275-305nm and 350-380nm were the
most suitable ranges regarding selectivity for the drug and the degradate. On the other
hand,the developed dual wavelength method requires the careful selection of two
wavelengths where the interfering compound shows equal absorbance while the analyte of
interest shows significant difference in absorbance with concentration. Different wavelength
pairs were tested and good results were obtained when using the wavelength pair of 232nm
and 341.2 nm for selective determination of flutamide where the degradate showed zero
difference in absorbance.
-Methods validation
Method validation was carried out following USP[4] guideline.
- Linearity
a- Area under curve method (method I): In order to check the linearity of the developed area
under curve method, different concentrations of flutamide and the degradate in the range of
1-35 µg/mL and 0.5-20µg/mL were prepared. Then area under curve in the wavelength
9

ranges of 275-305nm and 350-380nm was recorded, Fig. 2. By using the absorptivity values
at the chosen ranges and applying cramer's rule and the following equations, concentrations
of the drug and itsdegradate could be calculated.
A1= 0.858Cdrug+ 0.366Cdeg
(λ1-λ2)
A2=0.201Cdrug + 1.270Cdeg(λ3-λ4)
Where A1, A2 are the areas under curve in the range of 275-305 nm and 350-380nm,
respectively.Cdrug and Cdeg are the concentrations in µg/mL of flutamide and itsdegradate,
respectively.
b- Dual wavelength and isoabsorptive point method(method II):the absorbance difference of
flutamide between 232nm and 341.2 nm was found to be proportional to its concentration in
the range of 2-35µg/mL. While the degradate showed linear relationship with the absorbance
at 249.2 (λiso) inthe range of 0.5-16µg/mL.Calibration curves were constructed from which
the following regression equations were computed:
A= 0.0245C+ 0.0098
241.2nm
r= 1
absorbance difference between 232nm&
A= 0.0174C+ 0.0028r=0.9999
For the total mixture at 249.2 nm (λ iso)
Where, A is the absorbance at the specified wavelengths, C is the concentration in µg/mL and
r is the correlation coefficient.
- Accuracy
Accuracy of the methods was tested by applying the methods for determination of different
three concentrations (n=3X3) of pure drug and degradate and then calculating the mean
percentage recoveries. The methods showed good accuracy, Table (1), where the obtained
percentage recoveries were found to be 100.03 and 101.46 (method I) for the drug and the
degradate, respectively and 100.10100.2646 (method II) for the drug and the degradate,
respectively. Accuracy was further confirmed by application of standard addition technique
where good results were obtained, Table (2) which confirmed that tablets excepients did not
interfere.
- Precision:
It measures the closeness between the measured values and was proved by testing method
repeatability and Intermediate precision. Repeatability was tested by analyzing three
concentrations of pure flutamide (10, 15 and 20µg/mL) and degradate (5,10 and 15µg/mL) in
10

triplicates on the same day. While intermediate precision was assessed by assaying the
prepared solutions of the above mentioned concentrations in triplicates in three successive
days. Mean recoveries and RSD% were then calculated for the two components and given in
Table (1).
- Specificity
It was tested by application of the proposed methods for determination of the dug and the
degradate in different laboratory prepared mixtures containing the degradate in the ratio from
5 to 90%. Good percentage recoveries were obtained, Table 3, confirming the selectivity of
the proposed methods.
- Limits of detection (LOD) and quantification (LOQ)
They were calculated by using the lower parts of the linear range of the calibration curves
and then applying in the following equations:
LOD = 3.3 × N/B
LOQ = 10 × N/B
Where N is the standard deviation of the intercept and B is the slope of the corresponding
calibration curve. The methods showed good sensitivities and results are presented in Table
(1).
-Application of the methods
The methods were applied for measuring flutamide concentration in the marketed
pharmaceutical preparation where no interference from tablets excepients were observed and
good results were obtained (100.86 1.751%), Table (2). Results of recovery study presented
in Table (2) confirmed the good accuracy of the developed methods Table (2).
-Statistical analysis
When the developed methods were compared with the official one [3] using F-value and
student's t-test, no significant difference was found between them, Table (2).
-Kinetic study
As previously mentioned, no kinetic spectrophotometric method has been reported in the
literature for analysis of flutamide. Hence, the developed area under curve method (method I)
was used for measuring the kinetic parameters of flutamide hydrolytic degradation.
11

Hydrolytic degradation is a bimolecular reaction containing water (one of the reactant) in
excess amount and concentration changes are negligible, hence this reaction follows pseudo
first order reaction. To test the order of the reaction, experiment at initial flutamide
concentration of 2.9X10-3mol/L, constant temperature and constant reagent (either HCl or
NaOH) normality was performed. By plotting the logarithm of the remaining concentration of
flutamide versus time (by minutes), straight line was obtained, Fig. (3)indicatingthat the
hydrolytic degradation of flutamide followed pseudo first order reaction. The reaction rate
constant (K) was calculated from the obtained curve (slope= -K/ 2.303) from which t1/2 was
calculated (t1/2= 0.693/K), Table (4).
Variables affecting the reaction rate such as flutamide concentration, reagent normality and
temperature have been tested.
a- Effect of drug concentration
When the experiment was repeated on two initial concentrations of flutamide(2.9 x 10-3 and
4.3 x 10-3 M) at constant temperature and constant NaOH (0.1N) and HCl (0.5N)
normality.On plotting the logarithm of the remaining flutamide concentration versus time (by
minutes), two parallel lines were obtained, Fig. (3), that have nearly the same slopesand
almost the same rate constant (K), hence t1/2 values near to each other. Results given in
Table (4) assessed that the reaction rate is not affected by the initial drug concentration and
hence followed pseudo first order reaction.
b- Effect of HCl and NaOH strength on the reaction rate
Flutamide degradation was found to be pH sensitive. As shown from Fig. (4)and results in
Table (4). K value is highly affected by normality of NaOH and HCl. Degradation with 0.2
NaOH possessed the highest K value and thehighest reaction speed.
c- Effect of temperature on the reaction rate
Hydrolysis of flutamide was significantly affected by temperature. As shown from the results
in Table (4), K values increased with the increasing in temperature either in alkaline or acidic
hydrolytic degradation indicating thatthe reaction rate (velocity) increases with increasing in
temperature. Moreover, the effect of temperature on the reaction rate constant was studied by
using Arrhenius plot, Fig. (5)from which activation energy was calculated and found to be
22097.38 KJ/Mole and 29032.49KJ/Mole for alkaline and acidic hydrolysis, respectively.
12

The values of activation energy indicated that flutamide acidic degradation reaction was
slower than its alkaline degradation reaction.
4- CONCLUSION
This work concerned with the development and validation of two stability indicating
spectrophotometric methods for determination of the anticancer, flutamide. The developed
methods were the first developed stability indicating spectrophotometric methods for
determination of the proposed drug. They were successfully applied for determination of
flutamide and its hydrolytic degradation product in raw materials and laboratory prepared
mixtures. Moreover, the developed area under curve method was utilized for kinetic study of
alkaline and acidic degradation of the drug and factors affecting reaction rate constant (K)
were studied. Flutamide was found to be highly degraded and its degradation was found to be
pH and temperature dependent.
REFERENCES
1- S. Budavari, "The Merck Index", An Encyclopedia of Chemicals, Drugs and Biologicals, 13th
Ed., Merck and Co.Inc., Whitehouse Station, NJ, (2002).
2- Martindale- Extra Pharmacopoeia, 34th Ed., "The Complete Drug References" The
pharmaceutical Press, London, UK, (2005).
3- "The British Pharmacopoeia", Her Majesty's, The Stationary Office, London (2007).
4- "The United States Pharmacopeia", National Formulary 35, United States Pharmacopeia
Convention Inc., 30th Ed., 2012.
5- R. Goda et al., Drug Metabolism and Disposition, 34 (2006) 828-835.
6- R. Sternal, N. Nugara; Analytical Profiles of Drug Substances and Excipients, volume 27,
2001, Academic Press, California, USA, pp. 116.
7- M. N. Reddy, T. K. Murthy, M. D. Reddy, D. G. Sankar, Asian J. Chem.13 (2001) 240-
243.
8- K. M. Reddy, K. Suvardhan, K. Suresh, S. Prabahar, P. Chiranjeevi,proceedings of the
third International Conference on Environmental and Health, Channai, India, 5-17
December. Chennai: Department of Geogaphy, University of Madras and Faculty of
Environmental Studies, York University (2003) 410-416
9- M. N. Reddy, T. K. Murthy, M. D. Reddy, D. G. Sankar, Asian J. Chem.13 (2001) 1261-
1262.
13

10-
11-
P. D. Trazanavaras, D. G. Themelis, J. Pharm. Biomed. Anal. 43 (2007) 1824-1830.
A. A. Smith, R. Manavalan, K. Kannan, N. Rajendiran, Oriental J. Chem. 24 (2008)
189-194.
12-
A. Miranda, I. Caramballo, M, Millaacuten, Drug Develop. Ind. Pharm. 28 (2002)
413-422.
13-
A. A. Smith, R. Manavalan, K. Kannan, N. Rajendrin, Int. J. PharmTech Res. 2
H. R. N. Salgado, M. Menezes, M. P. B. Storti,Acta Far. Bonaerense 24 (2005) 246-
I. Niopas, A. C. Daftsios. J. Chromatogr. B: Biomed. Sci. Appl. 759 (2001) 179-183.
(2009) 360-364.
14-
249.
15-
16- Y.X. Guo, Z.L. Xiu, D.J. Zahang, H. Wang, L.X. Wang, H.B. Xiao, J. Pharm. Biomed.
Anal. 43 (2007) 1249–1255.
17- S.M. Sabry, M. Abdel-Hady, M. Elsayed, O.T. Fahmy, H.M. Maher, J. Pharm. Biomed.
Anal. 32 (2003) 409–423.
18-H.E. Zaazaa, S.S. Abbas, M. Abdelkawy, M.M. Abdelrahman, Talanta 78 (2009) 874–
884.
19- H. E. Zaazaa , M. M. Abdelrahman, N. W. Ali, M. A. Magdy, M. Abdelkawy,
Spectrochim.Acta 132 (2014) 655–662
20-ICH Q1A (R2) Stability testing of new drug substances and products. Proceeding of the
International
Conference
on
Harmonization,
2003
[http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1A
_R2/Step4/Q1A_R2__Guideline.pdf] (accessed February 22, 2014).
21- K.C. Waterman etal., Pharm Dev. Technol. 7 (2002) 113-117.
22-A.Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C.Smigal, MJ. Thun, Cancer statistics,
2006, CA: a Cancer J.Clin., 56 (2006) 106–130.
[http://www.ncbi.nlm.nih.gov/pubmed/16514137] (Accessed September 29,2014)
23- B.N Deongchamps, A. Singh, G.P. Haas,CurrProbl Cancer 31 (2007 )226–236.
[http://www.ncbi.nlm.nih.gov/pubmed/17543950] (Accessed September 29,2014).
24- V. S. Erram and H. P.Tipinis,Indian Drugs 30 (1993) 555.
[Through Analytical Abstract Online search]
25- V. S. Erram and H. P. Tipinis; Indian Drugs, 30 (1993)462
14

[Through Anal.Abstr. Online search]
26- V. S. Erram and H. P. Tipinis; Indian Drugs, 31 (1993) 65.
[Through Anal.Abstr. Online search]
Figure captions:
Figure 1.IR and mass spectra of flutamide and itsdegradate
Figure 2: Zero order aborption spectra of 10µg/mL each of flutamide (
) and the
degradate (-----).
Figure 3: Kinetic plot of (A) alkaline, (B) acidic degradation of 2.9X 10-3mol/L and 4.3 X
10-3mol/L of flutamide.
Figure 4: Plot for the effect of sodium hydroxide (A) and HCl (B) concentration on the rate
of degradation of 2.9X 10-3mol/L flutamide.
Figure 5: Arrhenius plot for alkaline (A) and acidic (B) degradation of2.9X 10-3mol/L
flutamide.
Table captions:
Table 1: Assay and method validation parameters for the determination of pure samples of
flutamide and its degradate by the proposed methods.
Table 2: Determination of Flutamide in its pharmaceutical formulation by the proposed
methods and application of standard addition technique.
Table 3: Determination of Flutamide and itsdegradate in Laboratory prepared mixtures by the
proposed methods.
Table 4: Kinetic data of flutamide alkaline and acidic hydrolysis
15

Figures
A
IR spectrum of the flutamide
B
IR spectrum of the degradate
16

Mass spectrum of the degradate
Figure 1.IR and mass spectra of flutamide and its degradate
Figure 2: Zero order aborption spectra of 10µg/mL each of flutamide (
) and the
degradate (------).
17

A
Time in minutes
-2.1
0
15 30 45 60 75 90 105 120 135
-2.3
-2.5
-2.7
-2.9
-3.1
-3.3
-3.5
2.9 x 10-3M
4.3 x 10-3M
(A)
B
Time in minutes
-2.2
0
30
60
90
120
150
180
210
-2.4
-2.6
-2.8
-3
2.9 x 10-3M
4.3 x 10-3M
-3.2
-3.4
(B)
Figure 3: Kinetic plot of (A) alkaline, (B) acidic degradation of 2.9X 10-3mol/L and 4.3 X
10-3mol/L of flutamide.
18

Time in minutes
A
0
-0.5
-1
0
15 30 45 60 75 90 105 120 135
-1.5
-2
0.05 N NaOH
0.1 N NaOH
0.2 N NaOH
-2.5
-3
-3.5
-4
(A)
B
Time in minutes
0
-0.5
-1
0
30
60
90
120
150
180
-1.5
-2
0.3 N HCl
0.5 N HCl
1 N HCl
-2.5
-3
-3.5
-4
-4.5
(B)
Figure 4:Plot for the effect of sodium hydroxide (A) and HCl (B) concentration on the rate of
degradation of 2.9X 10-3mol/L flutamide.
19

A
1/T
0.0031
0
0.0029
0.003
0.0032
0.0033
-0.5
-1
-1.5
-2
y = -2,657.8521x + 6.3954
r = 0.9975
-2.5
(A)
B
1/T
0.0029
0
0.0027
0.0028
0.003
0.0031
-0.5
-1
y = -3492.x + 7.818
r= 0.9970
-1.5
-2
-2.5
-3
(B)
Figure 5: Arrhenius plot for alkaline (A) and acidic (B) degradation of2.9X 10-3mol/L
flutamide.
20

Tables
Table 1: Assay and method validation parameters for the determination of pure samples of
flutamide and its degradate by the proposed methods.
Flutamide
Difference
Degradate
Parameters
AUC
method
Isoabsorptive
point at 249.2 nm
AUC method
between
232 – 341.2 nm
Range ( µg/mL)
Accuracy
1 – 35
1 – 35
0.5-20
101.46
0.5 – 16
100.03
100.10
100.26
98.55
0.658
98.65
1.337
102.23 1.229
100 1.556
Selectivity
Precision (%RSD)
Repeatability a
Intermediate precision b
1.338
1.577
1.068
1.490
1.071
1.572
1.340
1.963
LOD ( µg/mL)
LOQ ( µg/mL)
0.31
0.95
0.30
0.90
0.15
0.45
0.16
0.48
a The intraday (n = 3), average of three different concentrations repeated three times within day.
b The interday (n = 3), average of three different concentrations repeated three times in three
successive days.
21

Table 2: Determination of Flutamide in its pharmaceutical formulation by the proposed methods and application of standard addition technique.
Reference method
[3] a
AUC method
Dual wavelength method
Pharmaceutical
formulation
Component
Taken
(µg/mL)
Found a %
RSD%
Added
(µg/mL)
Recovery
%
Found a %
RSD%
Added
(µg/mL)
Recovery
%
10.00
15.00
20.00
97.20
100.47
100.10
10.00
15.00
20.00
99.80
98.64
1.473
100.86
15.00
103.00
103.40
1.751
100.43 1.253
99.26
1.791
102.07
1.973
Mean SD
1.951
Mean SD
F-Test
(6.388 c )
1.381
2.074
Student's t-test
(2.306 c )
0.424
a Average of 5 determinations.
b Average of 3 determinations.
c The values between parenthesis are corresponding to the theoretical values of t and F (P = 0.05).
Reference method: direct spectrophotometric determination of methanolic solution of flutamide and measuring the absorbance at 295 nm.
22

Table 3: Determination of Flutamide and its degradate in Laboratory prepared mixtures by the
proposed methods.
Found %**
Dual
wavelength
method
Isoabsorptive
point method
Taken (µg/mL)
AUC method
% degradate
Flutamide Degradate Flutamide Degradate
Flutamide
Degradate
98.8
102.67
30.00
20.00
25.00
15.00
10.00
10.00
1.50
4.00
10.00
9.00
8.00
9.00
98.07
98.10
99.20
97.80
98.70
99.40
100.67
99.50
102.85
99.22
98.63
99.11
5%
98.10
101.28
97.73
98.00
98.00
103.75
100.00
102.11
102.38
102.44
20%
40%
60%
80%
90%
Mean
RSD%
98.55
0.658
100
1.556
98.65 1.337 102.23 1.229
** Average of 3 determinations.
23

Table 4: Kinetic data of flutamide alkaline and acidic hydrolysis
Temperature
(oC)
Concentration
(M/L)
Strength
t1/2(min.)
K(min.-1)
Alkaline degradation
2.9 x 10-3
40
50
50
50
50
60
0.1 N NaOH
0.05N NaOH
0.1N NaOH
0.1N NaOH
0.2 N NaOH
0.1 N NaOH
83.59
115.73
42.38
41.79
25.719
27.11
0.00829
0.00599
0.01635
0.01658
0.02695
0.02556
2.9 x 10-3
2.9 x 10-3
4.3 x 10-3
2.9 x 10-3
2.9 x 10-3
Acidic degradation
2.9 x 10-3
70
80
80
80
80
90
0.5 N HCl
0.3N HCl
0.5N HCl
0.5N HCl
1N HCl
158.38
158.38
75.23
71.65
36.25
48.53
0.00438
0.00438
0.0092
0.0097
0.01912
0.0148
2.9 x 10-3
2.9 x 10-3
4.3 x 10-3
2.9 x 10-3
0.5N HCl
2.9 x 10-3
24

25

High lights
1- No kinetic spectrophotometric study was published for assay of flutamide.
2- Developed methods were the first developed stability indicating ones for flutamide.
3- Degradation identification by IR and mass spectra analyses.
4- The drug degradation followed pseudo first order kinetics.
5- Degradation rate was highly sensitive to temperature and pH
26