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ANALYTICAL METHOD VALIDATION FOR ASSAY OF CLOPIDOGREL TABLETS BY HPLC METHOD

Supervisor Dr. Ajay Aggarwal (Assistant Professor) Presented byAman Thakur M.Pharm. 3rd Sem

Co- Supervisor Vijay Chauhan QC Executive Dr. Morepen Labs. Parwanoo (H.P.)

INSTITUTE OF PHARMACEUTICAL SCIENCES, 1 KURUKSHETRA UNIVERSITY, KURUKSHETRA

CONTENTS
Introduction

Review of Literature
Drug Review Plan of Work Material and Methods Current Status of Work

INTRODUCTION

METHOD VALIDATION

Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use.
In other words, validation is the process of establishing the performance characterstics and limitations of a analytical method and the identification of the influences which may change these characterstics and to what extent?

High Performance liquid Chromatography (HPLC)

HPLC equipment of Agilent Technologies

HPLC is a chromatographic technique involving mass transfer between stationary and mobile phase. HPLC employs liquid mobile phase while stationary phase (immiscible packing material in column) can be solid or liquid phase. Forces which are involved in the interaction of solute with stationary and mobile phase include-

a) Hydrophobic interactions ( Reversed phase Chromatography) b) Dipole-dipole interactions ( Normal phase Chromatography)

FLOW CHART OF HPLC

LITERATURE REVIEW

Rao et al ; A novel stability-indicating normal phase liquid chromatographic (NP-LC) method was developed for the determination of purity of clopidogrel drug. Chromatographic conditions used in this methoda) Column - Chiralcel OJ-H (250mm4.6mm,5 micron) b) Mobile Phase - n-Hexane,ethanol and diethyl amine (95:5:0.5) c) Detector wavelength - 240 nm. J. Pharm. Biomed. Anal., 2010, 52, 160-165

Kahsay et al; A 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.


Chromatographic condition used in this methoda) Column- Luna C18 column (150 mm 4.6 mm, 3 m). b) Mobile phase- Methanol and acetonitrile (5:95). c) Detector wavelength 220 nm.

J. Pharm. Biomed. Anal., 2012, 61, 271-276

Nawal et al; describes a simple stability-indicating reversed-phase HPLC assay for clopidogrel bisulfate. Chromatographic conditions used in this methoda) Column- C-18 column

b) Mobile phase- Na2HPO4 (pH 4): acetonitrile:: 80:20


c) Detector Wavelength- 235 nm. J. Saud. Chem. Soc., 2012, 16, 23-30

DRUG REVIEW
Drug Chemical name : Clopidogrel

: methyl (2S)-2-(2-chlorophenyl)-2{4H,5H,6H,7H-thieno[3,2c]pyridin-5-yl}acetate C16H16ClNO2S

Chemical formula :

Molecular weight :

321.822

CHEMICAL STRUCTURE OF CLOPIDOGREL


O O CH3

Methyl (2S)-2-(2-chlorophenyl)-2-{4H,5H,6H,7H-thieno[3,2-c]pyridin5-yl}acetate

DRUG DESCRIPTION
Clopidogrel is an antiplatlet drug used to reduce the atherosclerotic conditions such as myocardial infarction, stroke and vascular death in patients. Absorption: Absorption is at least 50 % based on urinary excretion of the metabolites. Metabolism: Elimination: Protein binding: Half-life: Liver is the main site of metabolism of drug. 50% of the drug is eliminated in urine. 98% 8 Days

PLAN OF WORK

PARAMETERS TO BE VALIDATED FOR METHOD VALIDATION


Specificity Linearity

Limit of detection and limit of quantification


Accuracy Precision Ruggedness Robustness

Stability of analytical Solution


System suitability

Specificity It is the power of discrimination between the analyte and closely related substances by the method. Linearity Linearity is the ability of the method to elicit test results that are directly proportional to analyte concentration with in a given range. Accuracy is used to describe the measure of exactness of an analytical method when compared with the reference value. Precision is the measure of the degree of repeatability of an analytical method under normal operation. Limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be distinguished from a blank.

Accuracy

Precision Limit of detection

The limit of quantification (LOQ) is defined as the Limit of lowest concentration of an analyte in a sample that quantification can be determined with acceptable precision and accuracy under the stated operational conditions of the method.
Ruggedness Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions ( Analysts, Laboratory conditions, instruments, reagents etc.). Robustness is the capacity of a method to remain unaffected by small deliberate variations in method parameters

Robustness

MATERIAL & METHODS

INSTRUMENTS REQUIRED FOR VALIDATION

HPLC with PDA detectors (Waters)

pH meter ( Electronic India)


Analytical balance ( Mettler) Sonicator (Powersonic)

REAGENTS AND CHEMICALS REQUIRED DURING THE VALIDATION

Monobasic Potassium Phosphate Acetonitrile (HPLC grade) Methanol ( HPLC grade)

0.45 micron nylon membrane filter (Millipore)

WORKING STANDARD, PLACEBO, AND SAMPLE USED DURING THE VALIDATION

Working standard- Clopidogrel Bisulfate


Batch No.- BDCLPP/090001 Purity- 97.89% (On as such basis) Placebo batch no.- R&D CGP-010211 Sample batch no.- RD/CGT - 02102011

CHROMATOGRAPHIC CONDITIONS DURING VALIDATION


Column Wavelength Flow Rate Injection volume Reagents L57 ( 150 mm X 4.6 mm) 5 micron 220 nm 1.0 ml/min 10 micro litre HPLC grade Acetonitrile Monobasic Potassium Phsphate ( KH2PO4) buffer Dissolve 1.36 gm. Of monobasic potassium phosphate (KH2PO4) in 1000 ml with HPLC grade water.

Buffer preparation ( phosphate buffer)

Mobile phase preparation

Buffer : Acetonitrile :: 750 : 250

CURRENT STATUS OF THE WORK

Parameters which are validated till datea) Specificity b) Linearity c) Accuracy d) Precision e) Limit of detection f) Limits of quantification

SPECIFICITY

a) Placebo Preparation Weight 183.5 mg of placebo in 100 ml methanol and filter with .45 micro filter paper Further dilute 5 ml to this solution to 50 ml. with methanol.

b) Standard preparation Weight 98 mg of clopidogrel bisulfate working standard in 100 ml. methanol and filter with 0.45 micro filter paper. Further dilute 5 ml to this solution with 50 ml methanol.

c) Test preparation- Determine the average weight and crush the tablet and weigh accurately equivalent to 75 mg of clopidogrel ( approx. 282 mg) and dissolve it in 100 ml of methanol and further filter. further dilute 5 ml to this solution to 50 ml with methanol.
Sample No. of Injection Injection Volume(ml) Run time ( minutes)

Blank
Placebo solution Standard Sample preparation

1
2 6 2

10
10 10 10

10
10 10 10

Sample sequence for specificity

CHROMATOGRAM OF STANDARD

CHROMATOGRAM OF SAMPLE

CHROMATOGRAM OF PLACEBO PREPARATION

LINEARITY OF RESPONSE

A series of standard solution shall be prepared over a concentration range of 50% to 150% w/v from the working standard of clopidogrel bisulfate. Standard stock Solution- Dissolve 98 mg of working standard in 100 ml of methanol. 37.5 ppm ( 50%): 5 ml of stock sol mix with methanol to make 100 ml solution.

56.25 ppm (75%) - 15 ml of stock sol mix with methanol to make 200 ml solution. 75 ppm (100%)10 ml of stock sol mix with methanol to make 100 ml solution.

93.75 ppm ( 125%) - 25 ml of stock sol mix with methanol to make 200 ml solution.
112.5 ppm ( 150%) - 15 ml of stock sol mix with methanol to make 100 ml solution.

SAMPLE SEQUENCE FOR LINEARITY

Sample name Blank ( Diluent) Standard Linearity 37.5 ppm (50%) Linearity 56.25 ppm (75%) Linearity 75 ppm (100%) Linearity 93.75 ppm (125%) Linearity 112.5 ppm (150%)

No. of injection 1 6 2 2 2 2 2

Injection Volume (ml) 10 10 10 10 10 10 10

Run Time (min.) 10 10 10 10 10 10 10

LINEARITY GRAPH STUDY AT DIFFERENT CONCENTRATION

LIMIT OF DETECTION & QUANTIFICATION


LOD - Prepare of dilution containing 0.2 ppm, 2 ppm and 20 ppm clopidogrel bisulfate. Inject each dilution and results were tabulated in table.

No. of injection
1 2 3 4 5 6

Area(0 .2ppm)
8483 8258 8217 8442 8951 9541

Area (2 ppm)
62986 62367 62981 62693 62633 62956

Area ( 20 ppm)
641171 641052 640776 642245 640801 640707

Mean
Std. Dev. %RSD

8648.8
509.1 5.9

62769.3
250.2 0.4

641125.5
576.9 0.1

Limit of Quantification - LOQ is conducted at 2 times of LOD(2 x2 ppm). Prepared 4 ppm sol. of clopidogrel and injected 6 times. Injection No. Response Peak area

1 2 3 4 5 6 Mean Std. Dev. % RSD

1272547 1268875 1274526 1268741 1275984 1272584 1272209.5 642.8 0.3

ACCURACY

For Accuracy, Clopidogrel tablets are prepared at three different concentration levels, 50%, 100% and 150% w/v in triplicate are prepared and analyzed as described under methodology.

Percent of the drug recovered is calculated to measure the extent


of accuracy of the method.

SAMPLE SEQUENCE FOR ACCURACY


Sample Name Mobile Phase standard Recovery-50%-1 No. of Injection 1 6 2 Injection volume (ml) 10 10 10 Run time (min.) 10 10 10

Recovery-50%-2
Recovery-50%-3 Recovery-100%-1

2
2 2

10
10 10

10
10 10

Recovery-100%-2
Recovery-100%-3 Recovery-150%-1 Recovery-150%-2 Recovery-150%-3

2
2 2 2 2

10
10 10 10 10

10
10 10 10 10

DATA FOR THE ACCURACY OF THE METHOD


Recovery 50%-1 Amt. of drug added(mg) 51.1 Peak area of sample 3219493.9 Amt. recovered(mg) 51.146 Recovery (%) 100.09

50%-2
50%-3

51.1
51.2

3207153.1
3233471.3

50.950
51.369

99.71
100.33

100%-1
100%-2 100%-3 150%-1 150%-2 150%-3 Mean

100.8
96.1 96.3 146.2 153.2 148.8 -

6331702.7
6018109.8 6036203.6 9157850.0 9636653.7 9357009.2 -

100.590
95.610 95.90 145.48 153.09 148.65 -

99.79
99.49 99.58 99.51 99.93 99.90 99.70

Std. Dev.
%RSD

0.1805547
0.181098

CALCULATION FOR THE ACCURACY OF METHOD


Percent recovery of drug (%age)Area of recover sample Std. Wt. 5 100 50 X X X X X P X 100 Area of std. preparation 100 50 Amount of drug 5 Where, P = Purity of clopidogrel working standard in % on as is basis

Acceptance Criteria- Average recovery at each level should be with in 98.0% to 102.0% and RSD is not more than 2.0%.

PRECISION

Precision of the analytical method for the test of assay of clopidogrel bisulfate tablets shall be established by carry out 80%, 90%, 100%, 110% and 120% of target concentration and 6 replicates of each concentration.

SAMPLE SEQUENCE FOR STANDARD PRECISION


Sample name Mobile Phase Standard -80% Standard-90% Standard 100% Standard -110% Standard 120% No. of injection 1 6 6 6 6 6 Injection Volume (L) 10 10 10 10 10 10 Run Time (minutes) 10 10 10 10 10 10

SAMPLE SEQUENCE FOR STANDARD PRECISION


Sample name Mobile Phase No. of Injection 1 Injection volume (L) 10 Run Time ( Minutes) 10

Sample-80%
Sample-90% Sample-100% Sample-110% Sample-120%

6
6 6 6 6

10
10 10 10 10

10
10 10 10 10

RESULT IN GIVEN TABLE FOR STANDARD PRECISION


No. of Injection 1
2 3 4 5 6 Mean Std. Dev. %RSD

Area 80% 55251064


5256604 5295719 5270952 5326868 5326868 5290839.7 38156.5 0.7

Area 90% 5764726


5759904 5717423 5756370 5795412 5825506 5769890.2 36895.6 0.6

Area 100% 6465465


6492837 6478722 6511760 6538076 6633853 6520127.1 61267.8 0.9

Area 110% 7035026


7050142 7065533 7090866 7111167 7142279 7082502.2 40134.5 0.6

Area 120% 7251561


7210247 7216146 7281642 7245928 7190780 7242717.4 35980.7 0.5

RESULTS IN TABLE FOR SAMPLE PRECISION


No. of injection Area 80% Area 90% Area 100% Area 110% Area 120%

1 2
3 4 5 6 Mean Std. Dev. %RSD

5247287 5259083
5309480 5338267 5378762 5370786

5598284 5707477
5729800 5756091 5744476 5784842

6515146 6481062
6505140 6470829 6508176 6481062

6431984 6491860
6491860 6431984 6442111 6501904

7002279 6940183
6949754 7081406 7069955 7100222

5317277.4 5720161.6 6493569.2 6465284.5 7023966.5 55551.9 1.0 65074.6 1.1 18126.4 0.3 33191.0 0.5 69608.2 1.0

Data evaluationThe assay of six sample preparations shall be calculated. Mean value of the result should be reported. RSD value for the saple shall be calculated and reported.

Acceptance criteriaRSD should not be more than 2.0%.

WORK TO BE DONE IN THE FUTURE

FOLLOWING PARAMETERS ARE STILL TO BE VALIDATED FOR THE METHOD

1. Ruggedness 2. Robustness 3. Stability of analytical solution

4. System suitability

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