RP- HPLC Method
Development and Validation of Bicalutamide and Atenolol in its Pure Dosage
Forms
Prem Kumar Bichala1*,
R. Suthakaran2, Ch. Shankar, Syed Ghouse, Jannet Beula, R. Naresh
1Associate
Professor, Department of Pharmaceutical Analysis, Vijaya College of Pharmacy,
Munaganoor – 501511,
Hyderabad, Telangana, India
2Professor and
Principal, Department of Pharmaceutical Chemistry, Vijaya College of Pharmacy,
Munaganoor – 501511, Hyderabad,
Telangana, India
3Associate
Professor, Department of Pharmaceutical Analysis, Vijaya College of Pharmacy,
Munaganoor – 501511,
Hyderabad, Telangana, India
*Corresponding Author E-mail: prembichala@gmail.com
ABSTRACT:
A new method was established
for simultaneous estimation of Bicalutamide and Atenolol by RP-HPLC
method. The chromatographic conditions were successfully developed for
the separation of Bicalutamide and Atenolol by using Agilent C18 5µm
(4.6*250mm) column, flow rate was 1ml/min, mobile phase ratio was Water: ACN
(70:30%v/v), detection wave length was 254nm.
KEYWORDS: Agilent C18,
Bicalutamide and Atenolol, RP-HPLC method.
INTRODUCTION:
Bicalutamide is a
Non-steroidal anti-androgen receptor It competitively inhibits the action of
androgens by binding to cytosol androgen receptors in the target tissue3
Structure:
Bicalutamide
IUPAC Name: N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorobenzene)sulfonyl]-2-hydroxy-2-methylpropanamide
Chemical formula: C18H14F4N2O4S
Molecular weight: 430.373gm/mol
Atenolol ATL is a cardio
selective β blocker It is reported to lack intrinsic sympathomimetic
activity and membrane stabilizing properties This drug is used to treat
numerous cardiovascular disorders for example hypertension angina pectoris
cardiac arrhythmias and myocardial infarction It is official in USP IP and BP”1&2
Structure:
Atenolol
IUPAC Name: 2-(4-{2-hydroxy-3-[(propan-2-yl)amino]propoxy}phenyl)acetamide
Chemical formula: C14H22N2O3
Molecular weight: 266.3361
MATERIALS AND
METHODS:
Materials:
Bicalutamide and Atenolol API
was a gift sample by Celon Pvt Ltd., Hyderabad
Instrument:
The separation was carried out
on HPLC system 2695 (Waters) with Waters Quaternary pump, PDA detector (2998),
Waters Empower software.
Preparation of the
individual Bicalutamide standard preparation:
10mg of
Bicalutamide working standard was accurately weighed and transferred into a
10ml clean dry volumetric flask and about 2ml of DMF is added. Then it is
sonicated to dissolve it completely and made volume upto the mark with the
diluent (Stock solution). Further 10.0 ml from the above stock solution
is pipette into a 100 ml volumetric flask and was diluted upto the mark with
diluent.3
Preparation of the
individual Atenolol standard preparation:
10mg of Atenolol working
standard was accurately weighed and transferred into a 10ml clean dry
volumetric flask and about 2ml of DMF is added. Then it is sonicated to
dissolve it completely and made volume upto the mark with the diluent (Stock
solution). Further 10.0 ml from the above stock solution is pipette into
a 100 ml volumetric flask and was diluted upto the mark with diluent.5
Preparation of
Sample Solution :(Tablet)
Accurately 10
tablets are weighed and crushed in mortar and pestle and weight equivalent to
10 mg of Atenolol and Bicalutamide (marketed formulation)
sample into a 10mL clean dry volumetric flask and about 7mL of
Diluents is added and sonicated to dissolve it completely and
made volume upto the mark with the same solvent. (Stock solution) Further
3 ml of above stock solution was pipetted into a10ml volumetric flask and
diluted up to the mark with diluent.4
METHOD DEVELOPMENT:
Optimized
Chromatographic conditions:
Column
: Agilent C.18 5µm (4.6*250mm)
Mobile
Phase
: Water: ACN (70:30%v/v)
Detection
wavelength : 254 nm
Flow
rate
: 1ml/min
Sample
size
: 10μl
Column
temperature : Ambient
Figure 1: Chromatogram for
Optimized method
Fig. 2: Overlay
spectrum of Bicalutamide and Atenolol
Table 1: Accuracy
results for Bicalutamide and Atenolol
|
%Concentration (at specification Level) |
Bicalutamide |
Atenolol |
||||
|
Amount Added (ppm) |
Amount Found (ppm) |
% Recovery |
Amount Added (ppm) |
Amount Found (ppm) |
% Recovery |
|
|
50 |
5 |
4.9 |
101.8% |
5 |
5.0 |
101.3% |
|
100 |
10 |
9.98 |
99.9% |
10 |
9.96 |
99.6% |
|
150 |
15 |
15.0 |
100.0% |
15 |
14.9 |
99.3% |
|
Mean % Recovery |
|
|
102.5% |
|
|
101.0% |
RESULTS AND DISCUSSIONS:
Wavelength
Detection:7
The detection
wavelength was chosen by dissolving the drug in mobile section to induce a
amount of 10μg/ml for individual and mixed standards. The ensuing
resolution was scanned in U.V vary from 200-400nm. The overlay spectrum
of Bicalutamide and beta-adrenergic blocking agent was obtained and also the
isobestic purpose of Bicalutamide and beta-adrenergic blocking agent showed
absorbance’s maxima at 254nm. The spectrums are shown in Fig. 2
Method Validation:
Accuracy:
The accuracy study was
performed for five hundredth, 100 percent and a hundred and fifty you look
after Bicalutamide and beta-adrenergic blocker. every level was
injected in triplicate into natural action system. the realm of every level was
used for calculation of twenty-two recovery.8
Acceptance
criteria:
The % recovery for
each level should be between 98.0 to 102.0 %
Linearity:
The one-dimensionality study
was performed for the concentration of 100ppm to 500ppm and 1ppm to 5ppm level.
Every level was injected into action system. the world of every level was used
for calculation of correlation.8
Table 2: Linearity
results for Bicalutamide and Atenolol
|
S. No |
Bicalutamide |
Atenolol |
||
|
Working conc. (µg/ ml) |
Peak area |
Working conc. (µg/ ml) |
Peak area |
|
|
1 |
20 |
501543 |
25 |
224573 |
|
2 |
40 |
626277 |
50 |
441895 |
|
3 |
60 |
754999 |
75 |
635379 |
|
4 |
80 |
901124 |
100 |
842226 |
|
5 |
100 |
1016139 |
125 |
1041381 |
|
Correlation Coefficient (r) |
0.999 |
0.999 |
||
Acceptance
Criteria:
Correlation
coefficient should be not less than 0.999
Calibration curve of
Bicalutamide
Calibration curve
of Atenolol
Precision:
Repeatability:
To check the Repeatability
variation of the method, standard concentration was subjected to the proposed
HPLC method of analysis. The precision of the proposed method i.e. the
Repeatability in the peak area of the drug solutions was calculated in terms of
percent RSD. A statistical evaluation revealed that the relative standard
deviation of drugs at different concentration levels for 6 injections was less than
2.0. The results for intra-day precision were presented in Table 3
respectively.8
Table: 3. Repeatability
data for Atenolol and Bicalutamide
|
Injection No. |
Atenolol |
Bicalutamide |
||
|
Retention time (min) |
Peak area |
Retention time (min) |
Peak area |
|
|
1 |
2.527 |
1718661 |
4.630 |
2550539 |
|
2 |
2.528 |
1728892 |
4.628 |
2574105 |
|
3 |
2.528 |
1743398 |
4.626 |
2590892 |
|
4 |
2.529 |
1723892 |
4.632 |
2560539 |
|
5 |
2.527 |
1735820 |
4.626 |
2594102 |
|
6 |
2.528 |
1742589 |
4.628 |
2563982 |
|
Mean |
|
1732209 |
|
2572360 |
|
SD |
|
9216.839 |
|
15839.18 |
|
%RSD |
|
0.5 |
|
0.61 |
Intermediate
precision:
To check the
intra-day and inter-day variation of the method, standard concentration was
subjected to the proposed HPLC method of analysis. The precision of the
proposed method i.e. the intra and inter-day variations in the peak area of the
drug solutions was calculated in terms of percent RSD. A statistical evaluation
revealed that the relative standard deviation of drugs at different concentration
levels for 6 injections was less than 2.0. The results for intra-day and
inter-day precision were presented in Table 4 respectively.8
Table: 4. Intermediate Precision data for
Atenolol and Bicalutamide
|
Injection No. |
Atenolol |
Bicalutamide |
||
|
Retention time (min) |
Peak area |
Retention time (min) |
Peak area |
|
|
1 |
2.524 |
1763951 |
4.629 |
2575632 |
|
2 |
2.524 |
1794350 |
4.629 |
2570930 |
|
3 |
2.527 |
1792044 |
4.630 |
2613729 |
|
4 |
2.526 |
1792044 |
4.631 |
2613729 |
|
5 |
2.524 |
1783951 |
4.629 |
2575632 |
|
6 |
2.524 |
1794350 |
4.629 |
2570930 |
|
Mean |
|
1786782 |
|
2586764 |
|
SD |
|
10795.03 |
|
19163.75 |
|
%RSD |
|
0.60416 |
|
0.740839 |
Limit of Detection and Limit
of Quantification:8
LOD and LOQ were determined by
using the formula based on the standard deviation of the response and the
slope. LOD and LOQ were calculated by using equations, LOD = 3.3 × σ /
s and LOQ=10×σ/S., The results were presented in Table 10.
Where
σ = Standard deviation of
the response
S = Slope of the calibration
curve
Table: 5. Data table of LOD and
LOQ for Bicalutamide and Atenolol
|
Drug |
LOD (µg/ml) |
LOQ (µg/ml |
|
Bicalutamide |
0.54µg/ml |
1.6µg/ml |
|
Atenolol |
2.4µg/ml |
7.3µg/ml |
CONCLUSION:
The proposed HPLC
method was found to be simple, precise, accurate and sensitive for the
determination of Bicalutamide and Atenolol in pharmaceutical dosage forms.
Hence, this method can easily and conveniently adopt for routine quality
control analysis of Bicalutamide and Atenolol in pure and its pharmaceutical
dosage forms.
REFERENCES:
6. LR. Snyder, JJ
Kirkland, LG. Joseph, Practical HPLC Method Development, Wiley Inter
Science, New York, 2nd Ed, 1997, p. 1-56.
7. Ranjith singh, HPLC
Method Development and Validation- an Overview, J Pharm. Educ. Res.4 (2013)
26-33.
8. ICH: Q2B, Analytical
Validation – Methodology (1996)
Received on 08.09.2019 Modified on 18.11.2019
Accepted on 31.12.2019 ©Asian Pharma Press
All Right Reserved
Asian J. Pharm. Ana. 2020; 10(1):15-18.
DOI: 10.5958/2231-5675.2020.00004.6