CHAPTER–6 Development and Validation of a assay by HPLC...

Chapter-6 88

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


, efficiency and optimization of standard concentration. In validation section,

this chapter explains the all validation parameters like system suitability, system


(analyst to analyst, system to system and column to column variability, bench top /


temperature variation). At the same time the method explains

the forced degradation study.

Clopidogrel bisulfate is an inhibitor of adenosine diphosphate (ADP)


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


validation section,

all validation parameters like system suitability, system


ability, bench top /


temperature variation). At the same time the method explains

denosine diphosphate (ADP)-induced


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:


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:


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


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


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


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


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


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.



Preparation-2: Pipette out 2.5mL of the solution into 10ml volumetric flask


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.



Preparation-2: Pipette out 2.5mL of the solution into 10mL volumetric flask


Observation: Clopidogrel bisulphate was degraded to under peroxide solution.

F) Water hydrolysis: Weighed 200.20mg of Clopidogrel bisulphate sample to be


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



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


Sample solution preparation: Transferred 50 mg of sample into 100 mL


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 7

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:


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:


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-

HPLC method, year: 2008, volume-70, issue-5 ,Page :667-669.

2. Suhas Gurav, R. Venkata mahesh. Development and validation of derivative

UV spectropotometric methods for quantitative estimation of Clopidogrel in

bulk and pharmaceutical dosage form. International journal of chem tech

research CODEN( USA): IJCRGG ISSN : 0974-4290 Vol.4, No.2, pp 497-501.

3. Dr. U.C.Mashelkar , Sanjay D.Renapurkar. A LCMS compatible stability-

Indicating HPLC, assay method for Clopidogrelbisulphate, international

journal of chem tech research coden( USA): IJCRGG ISSN : 0974-4290. Vol.2,

No.2, pp 822-829, April-June 2010.

4. Journal of Pharmaceutical and Biomedical Analysis 61 (2012) 271– 276,

reversed phase liquid chromatographic method with UV detection for the

simultaneous determination of clopidogrel and acetylsalicylic acid and their

related substances in combined oral formulations was developed and

validated.

5. Dharmaraju.Ch , Sudhakarbabu. K , Shanthi. P , Lalitha. P , Hiranmayi. P ,

RosalineMishra, Himavani. K.V , Dr. Srinivas. J. S. HPLC method development

for determination of Para formaldehyde content in clopidogrelbisulphate

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