Chapter-6 Page 188
CHAPTER–6
Development and Validation of a assay by HPLC
method for Clopidogrel bisulphate
Chapter-6
This chapter describes the
method for Clopidogrel
of mobile phase, selection of diluent, optimization of wavelength, optimization of
column, efficiency and optimiz
this chapter explains the
precision, specificity, precision, accuracy, linearity of detector response, ruggedness
(analyst to analyst, system to system and column to column vari
refrigerator stability) and robustness (mobile phase composition variation,
variation in flow rate, temperature variation). At the same time the method explains
the forced degradation study
Clopidogrel bisulfate is an inhibitor of a
platelet aggregation acting by direct inhibition of ADP binding to its receptor and of
the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa
Clopidogrel splatelet inhibiting activity makes it an e
incidence ischemic strokes, heart
such as atherosclerosis. By inhibiting platelet aggregation, Clopidogrel reduces the
chance of arterial blockage,
Figure
6.1. INTRODUCTION
This chapter describes the development and validation of a
bisulphate(1-17) Development section it explains the selection
of mobile phase, selection of diluent, optimization of wavelength, optimization of
, efficiency and optimization of standard concentration. In validation section,
this chapter explains the all validation parameters like system suitability, system
precision, specificity, precision, accuracy, linearity of detector response, ruggedness
(analyst to analyst, system to system and column to column variability, bench top /
refrigerator stability) and robustness (mobile phase composition variation,
temperature variation). At the same time the method explains
the forced degradation study.
Clopidogrel bisulfate is an inhibitor of adenosine diphosphate (ADP)
platelet aggregation acting by direct inhibition of ADP binding to its receptor and of
mediated activation of the glycoprotein GPIIb/IIIa
splatelet inhibiting activity makes it an effective drug for reducing the
incidence ischemic strokes, heart attacks or claudication due to vascular diseases
atherosclerosis. By inhibiting platelet aggregation, Clopidogrel reduces the
chance of arterial blockage, thus preventing strokes and heart attacks
Figure- 6.1: Structure of Clopidogrel bisulphate.
Page 189
a assay by HPLC
evelopment section it explains the selection
of mobile phase, selection of diluent, optimization of wavelength, optimization of
validation section,
all validation parameters like system suitability, system
precision, specificity, precision, accuracy, linearity of detector response, ruggedness
ability, bench top /
refrigerator stability) and robustness (mobile phase composition variation,
temperature variation). At the same time the method explains
denosine diphosphate (ADP)-induced
platelet aggregation acting by direct inhibition of ADP binding to its receptor and of
mediated activation of the glycoprotein GPIIb/IIIa complex.
ffective drug for reducing the
attacks or claudication due to vascular diseases
atherosclerosis. By inhibiting platelet aggregation, Clopidogrel reduces the
heart attacks.
Chapter-6 Page 190
Chemical name
: (+)-(S)-α-(2-chlorophenyl)-6,7-
dihydrothieno[3,2-c]pyridine-5(4H)-acetate
sulfate(1:1)
Molecular formula : C16H16ClNO2S•H2SO4
CAS Registry Number
: 113665-84-2
Molecular weight
Therapeutic category
:
:
419.9
Antiplatelet drug
Table-6.1:Clopidogrel bisulphate details:
S. No Impurity structure
Chemical name Molecular
weight
Clopidogrel bisulphate
N
SCl
COOCH3
H2SO4
Clopidogrel bisulfate
(+)-(S)-α-(2-
chlorophenyl)-
6,7-
dihydrothieno[3,
2-c]pyridine-
5(4H)-acetate
sulfate(1:1).
419.9
6.2. REVIEW OF LITERATURE
A simple, rapid, precise RP-HPLC method was developed for simultaneous
estimation of aspirin and clopidogrel bisulphate in tablet dosage form used in the
treatment of cardiovascular diseases. To achieve the maximum resolution,
acetonitrile:50mM potassium dihydrogen phosphate buffer:methanol, solution pH
adjusted to 3, in the ratio 50:30:20; v/v was selected as mobile phase. This mixture
was found to be appropriate allowing good separation of both the components at a
Chapter-6 Page 191
flow rate of 1.5 mL/min and detection wavelength 240 nm. In these conditions
Clopidogrel bisulfate and aspirin were eluted at the 7.47 and 2.2 min. The linearity
was found in the concentration range 1.5-7.5 and 3.5-15.0 µg/mL, respectively. All
the analytical validation parameters were determined and found with in the limit as
per ICH guidelines, which indicates the validity of method. The HPLC system
consisted of a solvent delivery module LC-10ATvp Shimadzu liquid chromatograph
pump equipped with 20 mL loop and model SPD M10Avp Shimadzu UV/VIS diode
array detector. Integration was achieved by using the soft ware LC-10 . Separation
was carried out on a phenomenex, (250×4.6mm) Luna 5m C-18 (2) 100A column.
The chromatographic analysis was performed at ambient temperature. The mobile
phase consisted of the mixture of solvents, acetonitrile:50mM potassium dihydrogen
phosphate buffer : methanol, solution pH adjusted to 3.0 with ortho phosphoric acid,
in the ratio 50:30:20; v/v. The prepared mobile phase was filtered through a
Millipore 0.45 mm membrane filter and ultrasonically degassed prior to use. The
detection wavelength was set at 240 nm and the peak area was recorded using
chromatographic data system. The flow rate and run time was set to 1.5 mL/min and
10 min, respectively. All the system suitability parameters capacity factor, plate
number, tailing factor, retention time, and resolution were optimized by freshly
prepared standard solution of CPS (3.0 mg/mL) and ASP (15.0 mg/mL). To develop
a suitable method accurately weighed CPS and ASP were dissolved in mobile phase
(acetonitrile:buffer:methanol (5:3:2 v/v) to obtain solution of 1.5, 3.0, 4.5, 6.0 and
7.5 mg/mL for CSP and 3.0, 6.0, 9.0, 12.0 and 15.0 mg/mL for ASP. Before the HPLC
injection the solution were filter through 0.45µm HPLC filter. Peak area under the
curve of mixed standard were observed and plotted against respective concentration
and linearity was observed in the range of 0.0-7.5 mg/mL for CPS and 0.0-15.0
mg/mL for ASP at the 240 nm.
A simple, precise and economical first-order (Method A), area under curve
[AUC] (Method B) UV Spectro photometric methods have been developed and
validated for the estimation of Clopidogrel in bulk and its tablet formulation. The
solutions of standard and sample were prepared in 0.1 N HCl. Clopidogrel was
Chapter-6 Page 192
estimated at 219 nm for the first order UV-Spectro photometric method, while area
under the zero order spectrum of Clopidogrel was measured in between 221 nm to
231 nm for AUC method. Beer’s law was obeyed in the concentration range of 10 -
30 μg / mL with (r) value 0.999 for first order method. Similarly in AUC method,
Beer’s law was obeyed in the concentration range of 10 - 30 μg / mL with (r) value
0.999. These methods were tested and validated for various parameters according to
ICH guidelines. The precision expressed as relative standard deviation, which was
with in the range of 0.169 % to 0.519 % for the above two methods. The proposed
methods were successfully applied for the determination of Clopidogrel in tablet
formulations. In addition, the proposed methods are simple, easy to apply, low-cost,
and requires relatively in expensive instruments.
Clopidogrel bisulphate is an inhibitor of adenosine diphosphate (ADP)-
induced platelet aggregation which makes it an effective drug for reducing the
incidence ischemic strokes, heart attacks or claudication due to vascular diseases
such as atherosclerosis. A stability-indicating HPLC method for the quantitative
determination of Clopidogrel bisulphate is described. Separation was achieved on a
Inertsil C8 HPLC column using a mobile phase which consists of a mixture of 0.1 %
trifluoroacetic acid (Solvent A) and acetonitrile (Solvent B). Degradation studies
were performed on bulk samples of Clopidogrel bisulphate using acidic (0.5 N
hydrochloric acid), basic (0.1 N sodium hydroxide), neutral (water:acetonitrile
mixture 1:1), oxidative (6 % v/v hydrogen peroxide), thermal (105°C) and
photolytic (UV light -254 nm). Conditions, degradation was observed under acidic,
basic and neutral hydrolysis conditions to give Clopidogrel carboxylic acid. Two
additional degradation products were observed under the conditions of oxidative
degradation. The degradation products observed during forced degradation studies
were monitored using the HPLC method developed. Method developed was LCMS
compatible and the same was used to identify the degradation products. The mass
spectrum provides the identity of degradation products formed and proves the
specificity of the method unambiguously The method was validated with respect to
specificity, linearity, accuracy, precision and robustness. The output signal was
Chapter-6 Page 193
monitored and processed using Empower software (Waters) on a Pentium computer
(HP). The column used was a Inertsil C8 (250 X 4.6 mm I.D., 5.0μm) and the mobile
phase consisted of solvent A (1.0mL of trifluoroacetic acid in 1000 mL of HPLC
grade water) and solvent B (acetonitrile). The flow rate was 1.0 mL/ min. The HPLC
gradient was T/B (where T is time in minutes and B is % concentration of solvent B
in terms of volume by volume i.e. v/v ) :0 min / 20%, 6 min /20% , 40 min / 80% ,
45 min /80 % , 48 min / 20% and 50 min / 20% v/v. The column temperature was
maintained at 25°C and the analysis was carried out at wavelength λ = 225 nm. The
injection volume was 20μL. A mixture of water and acetonitrile (1:1) was used as
diluent.
6.3. OBJECTIVE
The complete literature survey reveals the chemical methods and
instrumental methods with high column cost, no reproducibility of analysis and
most of the HPLC columns are very sensitive. The main objective of this study is to
develop and simple, column, novel and development and validation of a assay by
HPLC method for Clopidogrel bisulphate.
6.4. MATERIALS AND METHODS
6.4.1. Reagents & Chemicals.
a. Water : Merck
b. Acetonitrile HPLC grade : Merck
c. Potassium hydroxide : Merck
d. Potassium hydrogen orthophosphate : Merck
6.4.2. Drug Substances:
Clopidogrel bisulphate sample were obtained from M/S Aurobindo pharma
Ltd, Hyderabad(A.P), India.
6.4.3. Instrument details:
The High Performance Liquid Chromatography using waters HPLC
instrument having quaternary pumps including auto injector. This HPLC connected
Chapter-6 Page 194
with PDA detector, make waters instrument. All the components are controlled with
Empower2 software .
6.4.4. Method development:
Development trials were performed with all neutral buffer salts and different
make HPLC columns but finally the chromatographic conditions were optimized
with the suitable salt, acetonitrile and methanol with simple isocratic method.
6.4.4.1.Wave length selection:
The UV spectrums were generated for Clopidogrel bisulphate and impurities
using with Photo diode array detector (PDA). Clopidogrel bisulphate and its
impurities were found to have varying absorption maxima over a range of
wavelength. But it was found that at about 210 nm, Clopidogrel bisulphate were
found to have optimum UV absorption. Therefore, 210 nm was selected for the
study and quantification of clopidogre bisulphate.
Figure- 6.2: UV Spectra of Clopidogrel bisulphate.
6.4.4.2. Selection of mobile phase and stationary phase:
Clopidogrelbisulphate was found that different functional groups, shows
different affinities with mobile phases and stationary phase. A different column with
different selectivity provides good separation for method development. Two
Chapter-6 Page 195
parameters were chosen to get required theoretical plates and separations and
symmetrical peaks for Clopidogrelbisulphate i.e., Selection of the mobile phase and
column.
6.4.4.3. Selection of mobile phase:
Clopidogrelbisulphate is a thieno pyridine class inhibitor of P2Y12 ADP
platelet receptors derivative and the impurities of Clopidogrelbisulphate were
having wide range of polarities and the separation of these impurities mainly
depends on the column stationary phase. An isocratic method was mobile phase of
buffer is 1.5g of potassium dihydrogen phoshate in water pH adjusted to 7.5 and
acetonitrile was suitable for the separation of Clopidogrel bisulphate and its related
substances. Mobile phase was degassed and filtered through 0.22µm millipore filter
paper.
6.4.4.4. Selection of stationary phase:
Separation was achieved with inertsil ODS-3V C18 250 x 4.6mm I.D., 5.0µm
column. Different stationary phases were studies for the separation of Clopidogrel
bisulphate such as C18, Phenyl, and ULTRON using the mobile phase specified.
The experimentation was started using ULTRON 150 X 4.6 mm I.D.,5.0µm
column.
Trail-1:
The complete experiment details are as follows.
Column : ULTRON 150 X 4.6 mm I.D., 5.0 μm column
Mobile phase
Sample preparation
:
:
Buffer :acetonitrile in the ratio of 70:30(v/v)
0.5mg/mL
Wavelength : 210 nm
Flow rate
: 1.0 mL/ min
Oven temperature
: Ambient
Diluent
: Mobile phase
Injection volume : 10 μL
Chapter-6 Page 196
Observation: Peak shape is good and resolution also good, but injection to injection
no consistency of peak shape and number of injections very less. Hence, ultron
column is not suitable for the Clopidogrel bisulphate assay method.
Trail-2:
The complete experiment details are as follows.
Column : Phenyl 150 X 4.6 mm I.D., 5.0μm column
Mobile Phase
Sample Preparation
:
:
Buffer : acetonitrile in the ratio of 70:30(v/v)
0.5 mg/mL
Wavelength : 210 nm
Flow rate
Injection volume
:
:
1.0 mL/ min
10μL
Oven temperature : Ambient
Diluent : Mobile phase
Observation: Peak shape is good, but injection to injection no consistency of peak
shape. Hence, phenyl column is not suitable for the Clopidogrel bisulphate assay
method.
Trail-3:
The complete experiment details are as follows.
Column : Inertsil ODS-3V 250 X 4.6 mm I.D., 5.0μm column
Mobile Phase : Buffer : acetonitrile in the ratio of 70:30(v/v)
Wavelength : 210 nm
Flow rate : 0.8 mL/ min
Oven temperature : Ambient
Diluent : Mobile phase
Observation: Clopidogrel bisulphate peak shape is very good and symmetrical peak
shape was observed with good theoretical plates. Hence, inertsil ODS-3V column is
suitable for the symmetrical peak of Clopidogrel bisulphate assay by HPLC method
and also this method is useful for related substances by HPLC also.
Chapter-6 Page 197
Conclusion:
Based on the above study on stationary phase, it was concluded that
Clopidogrel bisulphate assay method was well separated from each other in column
Inertsil ODS-3V C18 250 X 4.6 mm I.D., 5.0μm column.
6.4.5. Optimized method:
Based on the above study, the below mentioned assay by HPLC parameters
was chosen for the separation and quantification of Clopidogrel bisulphate.
Column : Inertsil ODS-3V C18 250 X 4.6mm I.D.,5.0μm column
Buffer preparation : 1.5g of potassium dihydrogen phoshate in 1000ml water
pH adjusted to 7.5 with potassium hydroxide solution
Mobile phase
on
:
:
Mix buffer and acetonitrile in the ratio of 300:700
Wavelength : 210 nm
Flow rate : 0.8 mL/Min
Oven temperature : 40oC
Diluent : Mobile phase
Elution : Isocratic
Injection volume : 10 μL
a) Standard solution preparation: Weighed accurately 50 mg of standard into
100 mL volumetric flask, dissolved and diluted to volume with diluent.
b) All impurities spiked solution: 0.15% impurity-A,B,C impurity solution and
transferred 50mg of Clopidogrel bisulphate into 100ml volumetric flask,
dissolved and diluted to volume with diluent.
c) Sample solution preparation: Weighed 50 mg of standard into 100 mL
volumetric flask, dissolved and diluted to volume with diluent.
Procedure: Injected all above standard solution for six times and calculated
the system suitability parameters.
System suitability criteria: The % RSD for six replicate injections not more than
1.5.
Tailing factor for Clopidogrel bisulphate is not more than 2.0.
Chapter-6 Page 198
Theoretical plates for Clopidogrel bisulphate peak from standard preparation should
not be less than 4000.
Table-6.2: Specification:
S. No Name of the product Specification
01 Clopidogrel bisulphate Between the 97% - 101.5%.
Calculation: Calculate the ASSAY using below formula
ASSAY: At x Wt x 100 x P
As x Ws x (100-L)
Where,
At: Area due to Clopidogrel bisulphatepeak in sample solution.
As: Area due Clopidogrel bisulphatepeak in standard solution.
Ws: Weight of the working standard taken in mg.
Wt: Weight of the sample taken in mg.
L:Loss on drying (%) sample.
P:Potency of Clopidogrel bisulphate standard.
Take the average of assay of preparation-I and II.
Average assay: Assay -I + Assay- II
Average
Figure- 6.3: A typical HPLC Chromatogram of diluent.
Chapter-6 Page 199
Figure- 6.4: A typical HPLC Chromatogram of impurities blend solution.
Figure- 6.5: A typical HPLC Chromatogram of Clopidogrel bisulphate sample.
6.5. RESULTS AND DISCUSSION
6.5.1. Method validation:
Analytical method validation was performed as per ICH and USFDA guidelines with
specificity, precision, accuracy, linearity, limit of detection, limit of quantification,
ruggedness and robustness.
6.5.1.1. Assay by HPLC:
6.5.1.2. System suitability:
a) Standard solution preparation: Weighed accurately 50 mg of standard into
100 mL volumetric flask, dissolved and diluted to volume with diluent.
Chapter-6 Page 200
B) All impurities spiked solution: 0.15% impurity-A,B,C solution and 50mg of
Clopidogrel bisulphate into 100mL volumetric flask, dissolved and diluted to
volume with diluent.
c) Sample solution preparation: Weighed about 50 mg of standard into a 100
mL volumetric flask, dissolved and diluted to volume with diluent.
Table-6.3: Clopidogrel bisulphate assay System suitability results:
S. No. Parameter Observation
1 %RSD 0.2
2 Tailing factor 1.04
3 Plate count 17792
6.5.1.3. Specificity:
A) Thermal degradation: Weighed about 1.0 gm of the Clopidogrel bisulphate
sample into a Petri dish and then kept the sample into oven at 105°C for 7days.
Weighed 50.0 mg of Clopidogrel bisulphate in to 100 mL volumetric flask,
dissolved and diluted to volume with diluent.
Preperation-1: Weighed 50.27 mg of Clopidogrel bisulphate sample into 100
mL volumetric flask, dissolved and diluted to volume with diluent.
Preparation-2: Transferred 50.28 mg of Clopidogrel bisulphate sample into
100 mL volumetric flask, dissolved and diluted to volume with diluent.
Analyze the samples by HPLC and determine the assay and peak purity of main
peak .
Observation: Clopidogrel bisulphate sample is stable under thermal condition.
B) Photo degradation: Weighed accurately 1 gm of sample is taken and kept in
UV chamber i.e., at 210 nm for 48 hours and sample collected after 48 hours
and then sample analyzed.
Observation: A Clopidogrel bisulphate sample is stable under photo condition.
C) Acid hydrolysis: Weighed 200.23 mg of Clopidogrel bisulphate sample to be
analyzed and transferred it into 100mL volumetric flask, dissolved with about
Chapter-6 Page 201
25mL of 0.5M HCl solution by sonication and made up to the mark with
diluent. Solution kept for 12 hrs with constant stirring at 70°C temperature.
Preperation-1: Pipette out 2.5mL of the solution into 10mL volumetric flask
,and diluted up to the mark with diluent.
Preparation-2: Pipette out 2.5mL of the solution into 10mL volumetric flask,
and diluted up to the mark with diluent.
Observation: Clopidogrel bisulphate sample is stable under acid hydrolysis.
D) Base hydrolysis: Weighed 50.0 mg of Clopidogrel bisulphate sample to be
analyzed and transferred it into 25mL volumetric flask, dissolved with about
15mL of 0.1N NaOH solution by sonication and made up to the mark with
diluent. Solution kept for 30 min with constant stirring at room temperature.
Preperation-1: Pipette out 2.5mL of the solution into 10mL volumetric flask
and diluted up to the mark with diluent.
Preparation-2: Pipette out 2.5mL of the solution into 10ml volumetric flask
and diluted up to the mark with diluent.
Observation: Clopidogrel bisulphate was degraded under base hydrolysis.
E) Oxidation degradation: Accurately weighed 200.83 mg of Clopidogrel
bisulphate sample to be analyzed and transferred it into 100mL volumetric
flask, dissolved with about 25mL of 3.0% peroxide solution by sonication and
made up to the mark with diluent. Solution kept for 24 hrs with constant
stirring at room temperature.
Preperation-1: Pipette out 2.5mL of the solution into 10mL volumetric flask
and diluted up to the mark with diluent.
Preparation-2: Pipette out 2.5mL of the solution into 10mL volumetric flask
and diluted up to the mark with diluent.
Observation: Clopidogrel bisulphate was degraded to under peroxide solution.
F) Water hydrolysis: Weighed 200.20mg of Clopidogrel bisulphate sample to be
analyzed and transferred it into 100mL volumetric flask, dissolved with about
25 mL of water by sonication and make up to the mark with diluent. Solution
kept for 12hrs with constant stirring at 70°C temperature.
Chapter-6 Page 202
Preperation-1: Pipette out 2.5mL of the solution into 10mL volumetric flask
and diluted up to the mark with diluent.
Preparation-2: Pipette out 2.5mL of the solution into 10mL volumetric flask
and diluted up to the mark with Diluent.
Conclusion:
Clopidogrel bisulphate samples are stable in thermal, photo degradation, acid
hydrolysis and water hydrolysis. Clopidogrel bisulphate was degraded in peroxide
solution and base hydrolysis. All samples are analysed and found that degradation
peaks are separated from known impurities and Clopidogrel bisulphate. Peak purity
were established with PDA detector and proved that Clopidogrel bisulphate peak is
pure in all above conditions. The studies are summarized in table-6.4
Table- 6.4: Clopidogrel bisulphate degradation Data :
Stressed condition Time (hrs) % Assay
Thermal degradation 7 x 24 98.69
Photo degradation 7 x 24 96.23
Acid hydrolysis 12 95.44
Base hydrolysis 0.5 90.53
Oxidation degradation 24 97.97
Water hydrolysis 12 94.62
Figure- 6.6: A typical HPLC Chromatogram of thermal degradation sample.
Chapter-6 Page 203
Figure- 6.7: A typical HPLC Chromatogram of photo degradation sample.
Figure- 6.8: A typical HPLC Chromatogram of acid degradation sample.
Figure- 6.9: A typical HPLC Chromatogram of base degradation sample.
Chapter-6 Page 204
Figure- 6.10: A typical HPLC Chromatogram of oxidation degradation sample.
Figure- 6.11: A typical HPLC Chromatogram of water degradation sample.
6.5.1.4. Linearity and accuracy:
a) 50% Linearity / accuracy solution: Accurately weighed 50 mg of sample into
100 mL volumetric flask, dissolved and diluted to volume with diluent. Taken 5
mL of this solution into 10 mL volumetric flask, dissolved and diluted to
volume with diluent.
b) 75% Linearity / accuracy solution preparation: Transferred 50 mg of sample
into 100 mL volumetric flask, dissolved and diluted to volume with diluent.
Taken 7.5 mL of this solution into 10 mL volumetric flask, dissolved and diluted
to volume with diluent.
Chapter-6 Page 205
c) 100% Linearity / accuracy solution preparation: Weighed 50 mg of sample into
100 mL volumetric flask, dissolved and diluted to volume with diluent. Three
solutions prepared as mentioned above.
d) 125% Linearity / accuracy solution preparation: Transferred 62.5 mg of
sample into 100 mL volumetric flask, dissolved and diluted to volume with
diluent.
e) 150% Linearity / accuracy solution preparation: Accurately weighed 75 mg of
sample into 100 mL volumetric flask, dissolved and diluted to volume with
diluent.
Injected all the above solutions each preparation once and calculated the
correlation coefficient, slope and Y-intercept for Clopidogrel bisulphate.
Conclusion:
The linearity and accuracy established a crossed the range from 0.25 mg/mL 0.375
mg/mL, 0.50 mg/mL, 0.625 mg/mL, 0.75 mg/mL for Clopidogrel bisulphate.
Correlation coefficient (r) obtained was greater than 0.999 and the recoveries are
98.3%, 99.3% and 99.9% the results are summarized in the table-6.5.
Table -6.5: Clopidogrelbisulphate linearity and accuracy data:
S. No Level
(%)
Average wt of the
Clopidogrel bisulphate
in mg/mL
Accuracy
%
recovery
(n=3)
Area of Clopidogrel
bisulphate
1 50 0.25 98.3 6348.77295
2 75 0.375 NA 9651.93164
3 100 0.500 99.3 12824.4
4 125 0.625 NA 16043.4
5 150 0.7503 99.9 19311.2
Correlation coefficient ( r ) 0.9999
Slope 25840.67
Y-Intercept -85.94
(%)Y-Intercept -0.67
Chapter-6 Page 206
Figure- 6.12: Clopidogrel bisulphate linearity graph.
6.5.1.5. Precision:
Standard solution preparation: Acurately weighed 50 mg of standard into 100 mL
volumetric flask, dissolved and diluted to volume with diluent.
Sample solution preparation: Transferred 50 mg of sample into 100 mL
volumetric flask, dissolved and diluted to volume with diluent.
Injected above standard solution once and sample solution each preparation
twice and calculated the assay content for each preparation.
Results and discussion: The results are summarized in the following table-6.6
Chapter-6 Page 207
Table - 6.6: Clopidogrel bisulphate precision data:
6.5.1.6. Robustness:
a) Flow variation:
Sample solution preparation: Weighed 10 mg of sample into 100 mL volumetric
flask ,dissolved and diluted to volume with diluent.
Injected the above sample solution at flow rates 0.6 mL/min and at 1.0 mL/min and
observed the system suitability parameters and results are compared with 0.8
mL/min results.
b) Temperature variation:
Sample solution preparation: Weighed 10 mg of sample into 50 mL volumetric
flask ,dissolved and diluted to volume with diluent.
Injected the above sample solution at , 35°C and at 45°C and observed the system
suitability parameters and results are compared with 40°C results.
Results and discussion:
In all above conditions there is no significant changes are observed in system
suitability results as well as in assay content. The results reveal that method is
robust.
Preparation Clopidogrel bisulphate Assay
(% w/w)
1 99.7
2 99.7
3 99.9
4 99.9
5 100.0
6 100.1
Average 99.9
Standard deviation 0.146
%RSD 0.15
Chapter-6 Page 208
Table- 6.7: Clopidogrel bisulphate robustness data:
Parameter 0.6 mL/min 1.0 mL/min At 35°C At 45°C As such
% RSD 0.2 0.2 0.3 0.2 0.1
Tailing factor 1.04 1.06 1.06 1.06 1.06
Plate count 17792 16538 17041 17287 17287
6.5.1.7. Solution stability and mobile phase stability:
Sample solution preparation: Weighed 10 mg of sample into 50 mL volumetric
flask, dissolved and diluted to volume with diluent.
The solution stability of Clopidogrel bisulphate in the assay method was
carried out by leaving both the test solutions of sample and reference standard in
tightly capped volumetric flasks at room temperature for two days. The same sample
solutions were performed for 0 hrs(Sample solution initial), 12hrs, 24hrs and 48
hrs. The mobile phase stability was also carried out by performing the freshly
prepared sample solutions against freshly prepared was kept constant during the
study period. The % RSD for the assay of Clopidogrel bisulphate was calculated
during mobile phase and solution stability experiments.
Conclusion: No significant changes were observed in the assay content of
Clopidogrel bisulphate during solution stability and mobile phase stability
experiments when performed using the assay method. The solution stability and
mobile phase stability experiment data confirms that the sample solutions and
mobile phases used during assay were stable for at least 48 hours. The results are
summarized in the table-6.8
Chapter-6 Page 209
Table- 6.8: Clopidogrel bisulphate solution stability and mobile phase
stability:
Batch analysis data:
6.6. SUMMARY AND CONCLUSION
The developed HPLC method was validated with respect to linearity,
accuracy, precision, specificity and robustness. The developed HPLC method to
determination of Clopidogrel assay is simple, comprehensive and can be used to
evaluate the quality of regular production samples. It can be also used to test the
stability samples of Clopidogrel. To the best of our knowledge, the validated stability
indicating RP-HPLC Assay method was not reported elsewhere.
Duration Solution stability % assay Mobile phase stability % assay
Initial 100.5 100.5
After 12 hrs 100.4 100.2
After 24 hrs 100.5 100.3
After 48 hrs 100.3 100.2
Lot Number % Assay
001 100.5
Chapter-6 Page 210
6.7. REFERENCES
1. PK Shrivastava, PK Basniwal, Deepti Jain, SK Shrivastava,Concurrent
estimation of clopidogrel bisulfate and aspirin in tablets by validated RP-
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