International Journal of ChemTech Research

CODEN( USA): IJCRGG ISSN : 0974-4290

Vol.1, No.4, pp 1189-1193, Oct-Dec 2009
DETERMINATION AND VALIDATION OF
UV SPECTROPHOTOMETRIC METHOD FOR ESTIMATION
OF BICALUTAMIDE TABLET
M. Swamivelmanickam
1
*, A.R.Gomes
2
, R. Manavalan
1
, D.Satyanarayana
1
,
P. Gangi Reddy
1
.
1. Department Of Pharmacy, Annamalai University, Chidambaram-608002,
Tamil Nadu. India.
2. Cipla pvt ltd, Bangalore, Karnataka.,India.
Corresponding author: swamivel@yahoo.com
ABSTRACT: A sensitive and direct spectrophotometric method is developed which is free from extraction,
derivatization, evaporation and complexation for the determination of Bicalutamide in pharmaceutical formulation. The
optimum conditions for the analysis of the drug are established. The method permits the determination of Bicalutamide
over a concentration range of 5mg/ml to 15mg/ml. Detection and quantification limit was found to be that 0.0117mg/ml
and 0.0355mg/ml respectively. The attained results shows that good recoveries of 99.72%, with relative standard
deviation of 0.21. All the calibration curve shows a linear relation between the absorbance and concentration with
correlation coefficient higher than 0.999. Precision and Accuracy of the method was established with recovery studies.
The proposed method is applicable for the assay of Bicalutamide investigation in dosage form and the results are in good
agreement.
Keywords: Bicalutamide, Spectroscopy, Determination
1. INTRODUCTION
Bicalutamide (BCA) N-[4-cyano-3-
(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-
hydroxy-2-methylpropanamide
Bicalutamide is an non-steroidal anti-androgen. It
competitively inhibits the action of androgen by binding
to cytosl androgen receptors in the target tissue, prostatic
carcinoma is known to be androgen and or removes the
source of androgen [1-3]
The structural formula of BCA by AM 1 method is
illustrated below:
It is well absorbed in oral administration. Co-
administration of Bicalutamide with food has no
clinically significant effect on rate or extent of
absorption, highly protein bound (96%), undergoes stereo
specific metabolism. Medicinal chemistry is concerned
with the understanding of chemical and biological
mechanism by which the action of drug molecule can be
explained [4 - 6]. It also tries to establish relation
between chemical structure and biological activity and to
link the later to the physical properties of the drug
molecules. The discovery of a new and biologically
important active compound usually gives rise to an
extended search for closely related compounds of similar
more effective, more specific or even opposite activity
[7] The S-isomer (inactive) is metabolized primarily by
glucuronidation. The R-isomer (active) also undergoes
glucuronidation but is predominantly oxidized to an
inactive metabolite followed by glucuronidation. Both the
Bicalutamide drug
M. Swamivelmanickam et al /Int.J. ChemTech Res.2009,1(4) 1190
parent metabolite glucuronides are eliminated in the urine
and feces. The S-enantiomer accounting for about 99 %
of total steady state plasma levels.
It is administered at dosages of 50 mg tablet daily. The
adverse effect of Bicalutamide includes hot flashes,
breast tenderness or pain and gynaecomastia. The
literature reveals that various methods for the
determination of BIC in biological fluids and
pharmaceutical formulations. Among these methods are
UV visible Spectrophotometry [13], HPLC method
using the uv detector [16], Rp-HPLC method [18-20], LC
method [15,22] in plasma were reported. The objective
of the present study is to develop a simple, precise,
accurate and economic analytical method with a better
detection range for the estimation of BIC in bulk drugs
and in pharmaceutical formulations. No extraction,
derivatization, or evaporation step, no complexation
agent, and no baleful chemicals are involved in the
proposed method, thereby decreasing the time and the
error in the quantization. This paper describes a simple,
reliable method for assaying bicalutamide by
Spectrophotometer which has been used to analyze the
formulation of BCA.
2. EXPERIMENTAL
2.1 Apparatus
A double-beam spectrophotometer Shimadzu UV 1601
PC model was used.
2.2 Chemicals and reagents
Determination and Validation of Bicalutamide was
carried at M/s Cipla Ltd, India. Methanol used was
spectro grade from S.D fine chemicals Ltd, India.
2.3 Standard solutions
Stock Solution
A methanolic primary stock solution of Bicalutamide (50
mg) was prepared in methanol. All the measurements
were performed at room temperature. The standard
solutions were prepared by the proper dilution of the
primary stock solution with methanol to obtain working
standard. For linearity study, serial dilutions were made
for Bicalutamide in the range of 5 to 15 g/ml
concentrations were prepared by diluting the stock
solution with methanol. The absorbances of these
solutions were fitted in the calibration curve to calculate
the accuracy and precision of the method.
2.4 For Formulation
The average weight of the tablets were determined by
weigh 20 tablets and powdered. Tablet powder equivalent
to 50mg of BCA was weighed and transferred to a 100 ml
volumetric flask. About 60 ml of methanol was added
and sonicated for 15 minutes complete dissolution of
drugs, made up to the volume with methanol and filtered
through whatman no 41 filter paper. Dilutions were made
with methanol to attain a concentration of 10 g/ml and
spectra was recorded. Six replicates of analysis were
carried out with sample weighed individually.
3. METHOD VALIDATION
3.1 Linearity
The method was validated according to ICH Q2B
guidelines [8] for validation of analytical procedures in
order to determine the Linearity, sensitivity, precision,
and accuracy of the analyte [8 - 10]. For BIC five point
calibration curves were generated with the appropriate
volumes of the working standard solutions for UV
methods. The linearity was evaluated by the least-squares
regression method using unweighted data.
3.2 Precision and Accuracy
Precision is the degree of repeatability of an analytical
method under normal operational conditions. The
precision and accuracy were determined with standard
quality control samples (in addition to calibration
standards) prepared in triplicate at different concentration
levels covering the entire linearity range[12]. The
precision of the assay was determined by repeatability
(intraday) and intermediate precision (inter-day) and
reported as RSD % for a statistically significant number
of replicate measurements [11]. The intermediate
precision was studied by comparing the assays on three
different days and the results are documented as the
standard deviation and RSD %. Accuracy is the percent
of analyte recovered by assay from a known added
amount. Data from nine determinations over three
concentration levels covering the specified range were
obtained.
3.3 LOD and LOQ
The limit of detection (LOD) is defied as the lowest
concentration of an analyte that an analytical process can
reliably differentiate from back-ground levels. In this
study, LOD and LOQ were based on the standard
deviation of the response and the slope of the
corresponding curve using the following equations
LOD = 3s/m; LOQ = 10s/m
Where s, the noise of estimate, is the standard deviation
of the absorbance of the sample and m is the slope of the
related calibrations graphs [14].
The limit of quantification (LOQ) is defined as the lowest
concentration of the standard curve that can be measured
with an acceptable accuracy, precision, and variability.
The values of LOD and LOQ were given in Table 1.
3.4 Recovery study
Recovery of the analyte of interest from a given matrix
can be used as a measure of the accuracy or the bias of
the method. The same range of concentrations as
employed in the linearity studies was used. To study the
accuracy, precision, and reproducibility of the proposed
method and dosage forms, recovery experiments were
carried out using the standard addition method [17].
These studies were performed by the addition of known
amounts of pure BIC to the pre-analyzed tablet
formulation and the mixtures were analyzed using the
proposed techniques. After parallel analyses, the recovery
results were calculated using the related calibration
equations.
M. Swamivelmanickam et al /Int.J. ChemTech Res.2009,1(4) 1191
4. RESULTS AND DISCUSSION
The development of a simple, rapid, sensitive, and
accurate analytical method for the routine quantitative
determination of samples will reduce unnecessary tedious
sample preparations and the cost of materials and labor.
BIC is a UV-absorbing molecule with specific
chromophores in the structure that absorb at a particular
wavelength and this fact was successfully employed for
their quantitative determinations using the UV
spectrophotometric method. The absorption spectrum of
BCA in methanolic solution is shown in Fig. 1.
4.1 Calibration curves
Calibration curve data was constructed in the range of the
expected concentrations of 5 to 15 g/ml. Beers law
was obeyed over this concentration range. The regression
equation was found to be y = 56.319x +0.0007. The
correlation coefficient (r) of the standard curve was found
to be greater than 0.9999. The stock solutions and
working standards were made in methanol. The
max
of
the drug for analysis was determined by taking scans of
the drug sample solutions in the entire UV region.
The characteristic of the calibration plot is presented in
Table 1 and the analytical characteristics and necessary
validation parameters for the UV techniques for BCA is
presented.
Performing replicate analyses of the standard solutions
was used to assess the accuracy, precision, and
reproducibility of the proposed methods. The selected
concentration within the calibration range was prepared
in methanol and analyzed with the relevant calibration
curves to determine the intra- and inter-day variability.
The intra- and inter-day precision were determined as the
RSD %. The precision, accuracy, and reproducibility of
the results given in table 1and 2 demonstrate a good
precision, accuracy, and reproducibility.
The proposed methods can be successfully applied for
assay in tablet dosage forms without any interference.
The assay showed the drug content of this product to be
in accordance with the labeled claim (Table 2). The
recovery of the analyte of interest from a given matrix
can be used as a measure of the accuracy of the method
(Table 2). In order to check the accuracy and precision of
the developed method and to prove the absence of
interference by excipients, recovery studies were carried
out after the addition of known amounts of the pure drug
to various pre-analyzed formulations of all drugs. The
application of this procedure is explained in the
experimental section. The obtained results demonstrate
the validity and accuracy of the proposed method for the
determination of all drugs in tablets (Table 2). These
results reveal that the developed method have an
adequate precision and accuracy, and consequently, can
be applied to the determination of BCA tablet in
pharmaceuticals without any interference from the
excipients.
5. CONCLUSIONS
A spectrophotometric method for quantifying
Bicalutamide in formulation samples has been developed
and validated. The assay is selective, precise, accurate
and linear over the concentration range studied. LOD was
approximately 0.0117mg/ml in formulation. In summary,
the proposed method can be used for the drug analysis in
routine quality control.
Table 1: Regression data of the calibration lines for quantitative determination of BCA by UV method.
Parameters Bicalutamide
Measured wavelength (
max
) 270
Linearity range, g/ml 5-15
Slope 56.319
Intercept 0.0007
Correlation coefficient (r) 1.0000
SE of slope 23.07
SE of intercept 1.387
LOD, g/ml 0.0117
LOQ, g/ml 0.0355
Repeatability of absorbance, RSD % 0.17
Repeatability of wavelength, RSD % 0.12
Reproducibility of absorbance, RSD % 0.39
Reproducibility of wavelength, RSD % 0.02
M. Swamivelmanickam et al /Int.J. ChemTech Res.2009,1(4) 1192
Table 2: Assay results from BCA tablets and mean recoveries in spiked tablets
*Mean of six determinations, ** three determinations
Figure 1: Absorbance
Parameters Bicalutamide
Labelled claim, mg 50
Amount found, mg* 49.1
RSD % 0.44
Added, % 150, 170, 190, 200
220, 250
Found, %** 99.31, 99.64, 99.80,
99.82, 99.85, 99.9
Recovery, % 99.72
RSD, % of recovery 0.21
M. Swamivelmanickam et al /Int.J. ChemTech Res.2009,1(4) 1193
A
b
s
o
r
b
a
n
c
e
Concentration (mg/ml)
Figure 2: Linearity of Concentration Vs Absorbance
Figure 2: Regression analysis of the calibration curve for Bicalutamide showed a linear relationship between the
intensity of absorbance and the concentration, with correlation coefficients higher than 0.9999 in all the curves
assayed.
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