Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-HPLC in Bulk and Pharmaceutical Dosage Forms

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* Corresponding author:
E-mail address: mouni.568@gmail.com
IJPAR |Volume 1 | Issue 1 | Dec - 2012
Available Online at: www.ijpar.com
[Research article]
Method Development and Validation of Clopidogrel Bisulphate by Reverse
Phase-HPLC in Bulk and Pharmaceutical Dosage Forms
*A.Mounika, N.Sriram
Smt. Sarojini Ramulamma College of Pharmacy, shesadrinagar, mahabubnagar-509001.
Andrapradesh, India,.
ABSTRACT
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of
Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was
chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of
acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow
rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg
/ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra
and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was
99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for
routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation.
Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
INTRODUCTION
Clopidogrel is an oral, thienopyridine class
antiplatelet agent used to inhibit blood clots in
coronary artery disease, peripheral vascular
disease, and cerebrovascular disease. Clopidogrel is
a prodrug, the action of which may be related to an
ADP receptor on platelet cell membranes. The drug
specifically and irreversibly inhibits the P2Y12
subtype of ADP receptor, which is important in
activation of platelets and eventual cross-linking by
the protein fibrin. The blockade of this receptor
inhibits platelet aggregation by blocking activation
of the glycoprotein IIb/IIIa pathway. The IIb/IIIa
complex functions as a receptor, mainly for
fibrinogen and vitronectin but also for fibronectin
and von Willebrand factor. Activation of this
receptor complex is the "final common pathway"
for platelet aggregation and is important in the
cross-linking of platelets by fibrin.
Clopidogrel bisulphate is chemically (S)-(+)-
Methyl 2 - (2-chlorophenyl) -2- (6,7-dihydro-4H-
thieno [3,2-c] pyridin-5-yl) acetate hydrogen
sulfate[1].
The molecular formula of Clopidogrel
bisulphate is C16H16ClNO2S.H2SO4. The molecular
mass of is Clopidogrel bisulphate 419.03 g/mol. It
is an official drug in British Pharmacopoeia. It is
completely soluble in water, methyl alcohol, grain
alcohol and glacial acetic acid bit soluble in
acetone or chloroform[2].
The structure of
Clopidogrel was shown in fig 1.
Clopidogrel bisulphate was determined by HPLC
in normal mode [5]
and in reversed-phase ion pair
mode [15].
Clopidogrel bisulphate was also
determined in combination with other drugs like

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A.Mounica et al / Int. J. of Pharmacy and Analytical Research Vol-1(1) 2012 [1-7]
www.ijpar.com
aspirin [11]
and stability indicating HPLC of
Clopidogrel bisulphate [13]
and also by UV
spectrophotometric techniques [10,12,9],
and
impurities present in Clopidogrel[7,14]
and
potentiometric technique[6]
.
MATERIALS AND METHODS
Quantitative HPLC was performed on a isocratic
high pressure liquid chromatography (Waters
model 2695) Equipped with a photodiode array
detector capable of operating in the range of 190
nm to 400 nm Hypersil BDS C18 (150mm x
4.5mm, 5μm)
REAGENTS AND CHEMICALS
Sodium di hydrogen phosphate, ortho phosphoric
acid of AR grade, methanol of HPLC grade,
acetonitrile of HPLC grade and water HPLC grade
were obtained from Rankem Chemicals Ltd.,
Mumbai. Clopidogrel bisulphate was obtained as a
gift sample from Sun Pharma, India. The
commercially available Clopidogrel bisulphate
tablets were procured from the local market.
PREPARATION OF BUFFER SODIUM
DIHYDROGEN PHOSPHATE BUFFER (PH-
3.0)
Sodium dihydrogen phosphate buffer was prepared
by dissolving 158 gm of disodium hydrogen
Phosphate in 1000 ml of double distilled water and
the pH was adjusted to 3.0 with ortho-phosphoric
acid.
CHROMATOGRAPHIC CONDITIONS
The mobile phase consisting of acetonitrile and
sodium di hydrogen phosphate buffer (pH: 3.0) in
the ratio of 60:40 % v/v was filtered through 0.45μ
membrane filter before use, degassed and pumped
from the solvent reservoir into the column at a flow
rate of 1 ml/min. The detection was monitored at
224nm, and the run time was 20 minutes. The
volume of the injection loop was 10 μl and prior to
the injection of the drug solution; the column was
equilibrated for at least 30 minutes with the mobile
phase flowing through the system. The column and
the HPLC system were kept in 35°c temperature.
Stock standard solution of Clopidogrel bisulphate
was prepared by dissolving a quantity of
Clopidogrel bisulphate hydrochloride equivalent to
10.0 mg of Clopidogrel bisulphate in 10.0 mL of
diluent to obtain a solution having a known
concentration of 1.0 mg/mL Clopidogrel
bisulphate. Nominal (working) standard solution
was prepared by diluting 1 mL of stock standard
solution to 10 mL diluent to obtain a solution
having a known concentration of 100µg/mL
Clopidogrel bisulphate. Nominal solutions of the
formulated Clopidogrel bisulphate tablet solution
prepared by taking 75mg equivalent powder in 10
mL volumetric flask and dissolve with water from
that take 1.5 mL of solution transferred into 10 mL
volumetric flask and volume filled with water.
RESULTS
METHOD DEVELOPMENT
AC18 column (150mm x 4.5mm, 5μm) as a
stationary phase with a mobile phase of acetonitrile
and phosphate buffer pH3.0 (60:40, v/v) at a flow
rate of 1.0mL/min and a detection wavelength of
224 nm afforded the best separation of Clopidogrel
bisulphate. The standard solutions prepared as
above were injected into the 10 μl loop, and the
chromatogram was recorded as shown in fig 2. The
retention time of Clopidogrel was found to be
9.182 min. The calibration curve was constructed
by plotting concentration versus peak area ratio.
The amount of Clopidogrel present in the sample
was calculated through the standard calibration
curve.
ASSAY
Twenty tablets each containing 75 mg were
weighed accurately and powdered. A quantity
equivalent to10 mg of Clopidogrel was weighed
accurately and transferred to 10 ml volumetric flask
containing 3 ml of water. The contents were
sonicated for 20 min. and made up to the mark with
the water. The resulting solution is filtered through
13 mm × 0.45µm PVDF. 1.5mL of the above
solution was pipette into 10mL volumetric flask
and made up with water. The solution obtained was
diluted with the water to obtain a concentration in
the range of linearity previously determined for the
pure drug. The 10µl sample solution was injected
under the chromatographic conditions, and the
chromatogram was recorded. The amount of
Clopidogrel present in tablet formulation was
determined by comparing the peak area from the
standard. The results were furnished in Table 1.
METHOD VALIDATION
The linearity, precision, accuracy, limit of
detection, limit of quantitation, ruggedness and
robustness has been validated for the determination
of Clopidogrel. [3, 4]

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LINEARITY AND RANGE
The linearity experiment was carried out in
triplicate to ascertain accuracy and precision of the
method. The standard curve was obtained in the
concentration range of 50-150 μg /ml The peak
area ratios of the drug versus concentration were
found to be linear, and the results are furnished in
Table 2. The linearity was evaluated by linear
regression analysis using the least square method.
It was found that correlation coefficient and
regression analysis are within the limits. The
linearity graph was shown in fig 3.
ACCURACY
Accuracy of the method was performed by
preparing the placebo of the drug formulation
according to the formulation procedure. To the
required quantity of placebo, a known quantity of
Clopidogrel with the same proportion as in the
drug, formulation was added to get three
concentrations (50, 100, 150 µg/mL of
Clopidogrel). Results have shown that the recovery
of Clopidogrel is within 98.0–102%, and the RSD
is lower than 2.0%. The results are shown in Table
3.
PRECISION
Repeatability
Repeatability of the method was evaluated by
calculating the RSD of the peak areas of six
replicate injections for the standard concentration
(100%) of Clopidogrel, which was found to be
0.43%.The results are furnished in Table 4.
Intermediate precision (ruggedness)
The Intermediate precision method was also
evaluated by analyzing six samples of Clopidogrel
by two analysts in the same laboratory using
different HPLC systems. Results of this study
showed that the RSD of the percentage of
Clopidogrel in Clopidogrel tablets for the 12
samples (6 samples from each analyst) was 0.8%
and 0.4% indicating a good intermediate precision
of the method Table 5.
LIMIT OF DETECTION (LOD) AND LIMIT
OF QUANTITATION (LOQ)
The LOD and LOQ for Clopidogrel were predicted
basing on the parameters of standard error of
estimate and slope, calculated from linearity of the
response data of Clopidogrel Bisulphate. The
results were shown in Table 6.
ROBUSTNESS
The robustness was checked by changing the flow
rate to 0.8 and 1.2 ml/ min, the mobile phase pH
2.8 to 3.2, and column oven temperature 30°c to
40°c the method suits best, and the results are
shown in Table 7.
Fig 1: Structure of Clopidogrel bisulphate

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Fig 2.Chromatogram of Clopidogrel Bisulphate standard solution
Fig 3: Linearity graph for Clopidogrel Bisulphate
Table 1: Quantitative Estimation of Clopidogrel Bisulphate in tablet dosage form
Table 2: Linearity data of Clopidogrel bisulphate
S.
NO.
Tablet Sample
Label Claim in
mg/tablet
Peak Area
Amount found
mg/tablet
Percentage
content of DrugTest Standard
1
Clopidogrel
bisulphate
75 1229063 1515350 74.9 99.86
Concentration(µg/ml) Area
50 842089
75 1063739
100 1495425
125 1788165
150 2417255

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Table 3: Accuracy data of Clopidogrel Bisulphate
Sample Mean area counts Amt added
(µg/ml)
Amt recovered (µg/ml) %recovery Mean
50%-Rec-1 604982 50 49.9 99.8 99.86
50%-Rec-2 833740 50 50.1 100.2
50%-Rec-3 660589 50 49.8 99.6
100%-Rec-1 1518091 100 99.9 99.9 99.93
100%-Rec-2 1519697 100 99.8 99.8
100%-Rec-3 1525624 100 100.1 100.1
150%-Rec-1 2502382 150 149.8 99.8 99.6
150%-Rec-2 2517458 150 149.5 99.6
150%-Rec-3 2519699 150 149.2 99.4
Table 4: Repeatability data of Clopidogrel Bisulphate
Injection number Area of Clopidogrel bisulphate
1 1515163
2 1512512
3 1506778
4 1517826
5 1526474
6 1513348
Mean 1515350.17
SD 6564.30275
%RSD 0.433187186
Table 5: Ruggedness of Clopidogrel Bisulphate
S.No. System suitability
Observed value
Acceptance criteria
Analyst-1 Analyst-2
1
%RSD for Clopidogrel bisulphate in standard solution
0.4 0.8 NMT 2.0%
2 The Tailing factor 1.18 1.21 NMT 2.0
Table 6: LOD and LOQ data of Clopidogrel Bisulphate
S.NO Name LOD Value (µg/ml) LOQ Value (µg/ml)
1. Clopidogrel bisulphate 1.3 4.2

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Table 7: Robustness data of Clopidogrel bisulphate
CONCLUSION
The developed method is cheap, easy, and it gives
the sharp peak with high resolution. The developed
method is applied for the determination of
Clopidogrel bisulphate. The assay results are with
the label claim of the formulation. The developed
method is validated as per ICH guidelines using
parameters like Accuracy, Precision, Linearity, and
Range, Specificity, Ruggedness, LOD, LOQ and
Robustness. Hence the developed method is found
to be satisfactory, and it complies with all
validation parameters. So this developed method
can be used for the routine analysis of Clopidogrel
bisulphate in tablet dosage form.
REFERENCE
[1] United states pharmacopeia, National Formulary 25 volume 32, 2005 p.no 1802
[2] The merck index, twelfth edition p. no 406
[3] Validation of analytical procedure, methodology as per ICH harmonized tripartite guidelines 1996,
Q2A having reached step 4 of the ICH process at the ICH steering committee meeting on 27th
October ,
1994, pg.no.1-8.
[4] Ludwig Huber. Validation and Qualification in Analytical Laboratories, Second Edition, Germany,
2007 pg no 160-170
[5] G. Radha Krishna Reddy, Prof. V. Surya Narayana Rao, Jayam Naga Sateesh, Development and
validation of stability indicating related Substances method for clopidogrel bisulphate drug substance
by normal phase hplc, Journal of Global Trends in Pharmaceutical Sciences Vol.2, Issue 4, (2011) pp -
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[6] Amr lotfy saber, Mohmed alaa elmosallamy, Alaa alside ami and Hamada mohmed ahmed Killa,
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(2009) p no. 1-16,.
Para meter Tailing factor %RSD
Buffer pH
2.8 1.17 0.3
3 1.18 0.8
3.2 1.21 0.4
Flow rate (ml/min)
0.8 1.22 1.1
1 1.18 0.7
1.2 1.25 0.2
Temperature(°C)
30 1.23 0.2
35 1.21 0.4
40 1.2 0.1

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[10] Pravin B. Cholke, Raihan Ahmed, S. Z. Chemate1 and K. R. Jadhav, Development and Validation of
Spectrophotometric Method for Clopidogrel bisulfate in pure and in film coated tablet dosage form,
Scholars Research Library Archives of Applied Science Research, 4 (1)(2012), Pg.no 59-64.
[11] Getu Kahsay, Ann Van Schepdael, Erwin Adams, Development and validation of a liquid
chromatographic method for purity control of clopidogrel–acetylsalicylic acid in combined oral dosage
forms, Journal of Pharmaceutical and Biomedical Analysis, volume 61,( 2012) pg no 271– 276.
[12] Suhas Gurav, R. Venkatamahesh, Development and Validation of Derivative UVSpectropotometric
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International Journal of ChemTech Research, Vol.4, No.2, (2010), pp 497-50.
[13] Sanjay D.Renapurkar Dr. U.C.Mashelkar, A LCMS Compatible Stability-Indicating HPLC Assay
Method for Clopidogrel bisulphate, International Journal of ChemTech Research Vol.2, No.2 (2010),
pp 822-829.
[14] P. Aparnaa, Sumathi V. Raoa, K. M. Thomasa,K. Mukkantib and Kishore Deoc, Non chiral High
Performance Liquid Chromatography method for monitoring unknown impurities generated during
stability of Clopidogrel tablets, Scholars Research Library s2(4),(2010), pp 244-252.
[15] Sagar S.Panda, Ion-Pairing RP-HPLC Method for Simultaneous determination of Aspirin and
Clopidogrel bisulphate in Tablet and Capsule Dosage Form, International Journal of PharmTech
Research Vol.2, No.1, (2010) pp 269-273.
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Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-HPLC in Bulk and Pharmaceutical Dosage Forms

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Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-HPLC in Bulk and Pharmaceutical Dosage Forms