Download Syeda Bushra Yunus

Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.
Research Article
ISSN: 2319 – 9563
International Journal of Research
in
Pharmaceutical and Nano Sciences
Journal homepage: www.ijrpns.com
METHOD DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC
METHOD FOR THE ESTIMATION OF CLOPIDOGREL BISULPHATE IN PURE
AND TABLET DOSAGE FORM
Syeda Bushra Yunus*1, Karuppasamy. C1, Syeda Sheeba Ahamadi1, P. Dilip Kumar1,
Madhu Kumar. G1, Nagaraju. C1
*1
Department of Pharmaceutical Analysis, Creative Educational Society’s College of Pharmacy, Kurnool,
Andhra Pradesh, India.
ABSTRACT
A simple, accurate, precise and sensitive Spectrophotometric method was developed for the estimation of
Clopidogrel bisulphate in pure and tablet dosage forms. The estimation of Clopidogrel bisulphate was carried out
at maximum absorbance of 220 nm. The method was found to be linear and obeys Beer’s law in the
concentration range of 1-14 mcg / ml with a correlation coefficient 0.998. The developed method was validated
as per ICH guidelines and was found to be accurate and precise. Thus the proposed method can be successfully
applied for the estimation of Clopidogrel bisulphate in pure and tablet dosage forms.
KEYWORDS
Spectrophotometric, Clopidogrel bisulphate, Validation and ICH guidelines.
Author for correspondence:
Syeda Bushra Yunus,
Department of Pharmaceutical Analysis,
Creative Educational Society’s College of Pharmacy,
Kurnool, Andhra Pradesh, India.
Email: [email protected].
Available online: www.ijrpns.com
INTRODUCTION
Clopidogrel bisulphate is a potent oral antiplatelet
gent used in treatment of coronary artery disease.
Chemically it is Methyl (2s)-2-(2-chlorophenyl)-2(6, 7-dihydro-4H-thieno[3,2-c]pyridine-5-yl)acetate.
It has a molecular formula of C16H16 ClNO2S and a
molecular weight of 419.9g / mol and its structure
was given in (Figure No.1).
Literature survey revealed, few analytical methods
which include A Validated stability indicating
HPTLC method for determination of Clopidogrel
bisulphate in combined dosage form1, Determination
May - June
365
Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.
of Clopidogrel bisulphate by Chromatography2,
Stability indicating HPTLC Determination of
Clopidogrel
bisulphate
in
formulation3,
Determination of Clopidogrel bisulphate and its
impurity by TLC Method4, Validated HPLC Method
for the Estimation of Clopidogrel bisulphate and
Aspirin in combined tablet Formulation5,
Simultaneous Determination on of Clopidogrel
bisulphate and Aspirin in tablet formulation by
HPLC and HPTLC6. The present work deals with
estimation of Clopidogrel bisulphate in pure API and
tablets by UV-Spectrophotometry.
MATERIALS AND METHOD
Authentic drug sample of Clopidogrel bisulphate
was given as a gift sample by Aurobindo Pharma
Ltd, Hyderabad. Tablets of Clopidogrel bisulphate
were procured from local pharmacy. Distilled water
was used for the preparation of solutions.
Instrument
Labindia - 3000+ UV / Vis double beam
Spectrophotometer with a fixed slit width (2 nm)
and 10 millimeter quartz cell was used to obtain
spectrum and absorbance measurement.
Preparation of stock solution
100 mg of standard Clopidogrel bisulphate drug was
weighed, transferred to a 100 ml volumetric flask
and dissolved in distilled water. The flask was
shaken and volume was made up to the mark with
distilled water to give a solution containing 1000 µg
/ ml.
Determination of Absorbance Maxima
From above stock solution 10ml is taken in 100ml
volumetric flask and made upto 100ml to give
100µg/ml concentration and further dilution done
and was subjected for scanning between 200-400nm.
Effect of dilution on the absorbance maxima was
studied by diluting above solution to 1-14µg/ml and
scanned from 200-400 nm, shown in Figure No.2.
For the standard solution analytical concentration
range was found to be 1-14µg / ml and those values
were reported in Table No.1.
Appropriate volume of aliquots from standard
Clopidogrel bisulphate stock solutions were
transferred to different volumetric flasks of 10 ml
Available online: www.ijrpns.com
capacity. The volume was adjusted to the mark with
distilled water to obtain concentrations of
1,2,3,4,5,6,8,10,12,14µg / ml. Absorbance spectra of
each solution against distilled water as blank were
measured at 220 nm and the graphs of absorbance
against concentration was plotted and are shown in
Figure No.2. The regression equation and correlation
coefficient was determined.
ANALYSIS OF MARKETED FORMULATION
Accurately weigh 20 tablets of Clopidogrel
bisulphate and finely powdered. The powder
equivalent to 100mg of Clopidogrel bisulphate was
transferred into 100ml volumetric flask and 20 ml of
distilled water added and sonicated for 15 min to
dissolve the Clopidogrel bisulphate in it and made
volume to mark with distilled water. 10 ml of this
diluted with distilled water. The concentration of
Clopidogrel bisulphate in marketed formulation was
determined in Table No.2. To examine the absence
of either positive or negative interference of
excipients used in formulation, recovery studies were
carried out.
Accuracy was determined by recovery studies. The
recovery studies were carried out by adding the
known amount of Standard Clopidogrel bisulphate
drug to the sample solution of the tablets. Precision
for assay were determined by repeatability, interday,
intraday precision for drug (each in three replicate).
Limit of Detection (LOD) and limit of
Quantitation (LOQ)
The LOD and LOQ of Clopidogrel bisulphate are
determined by using calibration standards. Value of
LOD is determined by using the formula: 3.3ơ/S
And the value of LOQ is determined by: 10ơ/S,
where ‘ơ ‘ is the standard deviation of the y intercept
of the regression equation and ‘S’ is the slope of
calibration curve.
RESULTS AND DISCUSSION
The absorption spectral analysis shows the λmax of
Clopidogrel bisulphate at 220 nm (Figure No.3). The
calibration curve was obtained for a series of
concentration in the range of 1-14mcg/ml. It was
found to be linear and hence, suitable for the
estimation of the drug. The slope, intercept,
May - June
366
Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.
correlation coefficient and optical characteristics are
summarized in Table No.2. Regression analysis of
Beer’s law plot revealed a good correlation. The
effects of various excipients generally present in the
tablet dosage form of Clopidogrel bisulphate were
investigated. The results indicated that they did not
interfere in the assay in amounts far in excess of
their normal occurrence in it. The proposed method
was validated as per the ICH guidelines7,8. The
precision was measured in terms of repeatability,
which was determined by sufficient number of
aliquots of a homogenous sample. The % RSD was
found to be within the limits. This showed that the
precision of the method is satisfactory. The recovery
technique was performed to study the accuracy and
reproducibility of the proposed method. For this,
known quantities of the Clopidogrel bisulphate
solution was mixed with definite amounts of preanalyzed formulations and the mixtures were
analyzed. The total amount of Clopidogrel bisulphate
was determined by using the proposed methods and
the amount of added drug was calculated by the
difference. The % RSD was less than ± 2.0 (Table
No.3). This showed that the recoveries of
Clopidogrel bisulphate by the proposed methods are
satisfactory and the results are shown in Table No.4
and 5. Limit of detection (LOD) and Limit of
quantitation (LOQ) were determined by the proposed
method.
Table No.1: Results of Clopidogrel bisulphate calibration curve at 220 nm for by UV Spectroscopy
Concentration
Absorbance at
(mcg / ml)
220 nm
1
1
0.035
2
2
0.068
3
3
0.098
4
4
0.135
5
5
0.165
6
6
0.215
7
8
0.290
8
10
0.376
9
12
0.451
10
14
0.532
S.No
Available online: www.ijrpns.com
May - June
367
Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.
Table No.2: Shows Optical Characteristics of Clopidogrel bisulphate
S.No
Parameter
UV method
1
λmax (nm)
220
2
Beer’s law limits (mcg / ml)
1-14
3
Regression equation (Y*)
Y = 0.038x+0.015
4
Slope (m)
0.038
5
Intercept (c)
0.015
2
6
Correlation coefficient(r )
0.998
7
% RSD**
< 2%
8
Limit of detection (mcg / ml)
0.0151
9
Limit of quantitation (mcg / ml)
0.0457
*Y=mx+c where X is the concentration of Clopidogrel bisulphate in mcg/ml and
Y is the absorbance at the respective λmax.
**Average of six determinations.
Table No.3: Determination of Precision results for Clopidogrel bisulphate at 220 nm
S.No
1
2
3
Conc
Inter-day
mcg / ml
Absorbance Mean ± SD**
LQC
(3mcg/ml)
MQC
(6mcg/ml)
HQC
(12mcg/ml)
% RSD
Intra-day
%
Absorbance Mean ± SD**
RSD
0.162±0.000516
0.318
0.098±0.000516
0.526
0.099±0.0010
1.01
0.215±0.00154
0.716
0.455±0.0060
1.31
0.451±0.00206
0.456
**Average of six determinations.
Table No.4: Assay of Clopidogrel bisulphate formulation
Amount found (mg)
S.No
1
Formulation
Label claim
(n=3)
(mg/tab)
Mean ± SD
75
75.009±0.075
Clopilet
Available online: www.ijrpns.com
May - June
Assay
%RSD
98.73 %
1.002
368
Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.
Table No.5: Determination of Accuracy results for Clopidogrel bisulphate at 220 nm
Amount of sample
Amount of drug
Amount
% Recovery
(mcg / ml)
added (mcg / ml)
Recovered
± SD**
Clopilet
10
8
17.91
98.85±0.51
2
Clopilet
10
10
19.92
99.82±0.43
3
Clopilet
10
12
21.83
99.77±0.12
S.No
Brand name
1
Figure No.1: Structure of Clopidogrel Bisulphate
y = 0.038x + 0.015
R² = 0.998
0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
10
12
14
16
concentration ( mcg)
Figure No.2: Calibration curve of Clopidogrel bisulphate
Available online: www.ijrpns.com
May - June
369
Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.
Figure No.3: Spectra of Clopidogrel bisulphate
CONCLUSION
Thus it can be concluded that the methods developed
in the present investigation are simple, sensitive,
accurate, rapid and precise. Hence, the above said
method can be successfully applied for the
estimation of Clopidogrel bisulphate in tablet dosage
form.
ACKNOWLEDGEMENT
The authors are sincerely thanks to Creative
Educational Society’s College of Pharmacy,
Kurnool, Andhra Pradesh, India for providing the
facilities to complete this research work.
REFERENCES
1. Shinha P K, Mrinalini C D, Bothra K G. A
Validated stability indicating HPTLC Method for
determination of Aspirin and Clopidogrel
bisulphate in combined dosage form, Eur J
Chem, 4, 2009, 152-160.
2. Vocilkova L, Opatrilova R, Sramek V.
Determination of Clopidogrel bisulphate by
Chromatography, Cur Pharm Anal, 5, 2009, 424431.
Available online: www.ijrpns.com
3. Himani A, Neeraj K, Paradkar A R, Mahadik K
R. Stability indicating HPTLC Determination of
Clopidogrel bisulphate as Bulk drug and in
pharmaceutical dosage form, J Pharm and
Biomed Anal, 61, 2003, 581-589.
4. Anti D, Filipi S, Agbaba D. A simple and
sensitive TLC Method for determination of
Clopidogrel bisulphate and its impurity in
pharmaceutical product, Acta chromatogr, 18,
2007, 199-206.
5. Pankaj K, Kachhadia A S, Doshi H S. Validated
Column HPLC Method for Determination of
Aspirin and Clopidogrel bisulphate in combined
Tablets in the presence of Degradation Products
Formed under ICH Recommended Stress
Conditions, JAOAC Int, 92, 2009, 152-7.
6. Patel R B, Shankar M B, Patel M R, Bhatt K K.
Simultaneous Estimation of Acetylsalicylic Acid
and Clopidogrel bisulphate in Pure Powder and
Tablet Formulations by HPLC and HPTLC,
JAOAC International, 91, 2008, 750-755.
7. ICH/CGMP guidelines Q2A, Text on Validation
of Analytical Procedures, 1994.
8. ICH/CGMP guidelines Q2B, Validation of
Analytical Procedures-Methodology, 1996.
May - June
370