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 Aligent 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. HPLC is a dynamic adsorption process. As the liquid passes through the column it gets separated into its components under high pressure. Forces which are involved in the interaction of solute with stationary and mobile phase includea) Hydrophobic interactions ( Reversed phase Chromatography) b) Dipole-dipole interactions ( Normal phase Chromatography)

LITERATURE REVIEW

 Rao D. (2010); A novel stability-indicating normal phase liquid chromatographic (NP-LC) method was developed for the determination of purity of clopidogrel drug substance and drug products in bulk samples and pharmaceutical dosage forms in the presence of its impurities and degradation products. This method can be also be used for the estimation of assay of clopidogrel in drug substance as well as in drug product. The method was developed using Chiralcel OJ-H (250mm4.6mm, 5m) column. nHexane, ethanol and diethyl amine in 95:5:0.05 (v/v/v) ratio was used as a mobile phase. The eluted compounds were monitored at 240 nm. Clopidogrel bisulfate was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation.

Nawal A. (2010); describes a simple stability-indicating reversedphase HPLC assay for antiplatelet drug, clopidogrel bisulfate. Separation of the drug and the degradation products, under stress conditions was successfully achieved on a C-18 column utilizing 0.01 M Na2HPO4 (pH 4): acetonitrile in the ratio 80:20 v/v, pumped at a flow rate of 0.5 ml min1 with UV detection at 235 nm. The retention time of clopidogrel was 6.84 min. The method was satisfactorily validated with respect to linearity, precision, accuracy, selectivity, sensitivity and ruggedness. The response was linear in the range of 0.23.5 lg ml1 with detection limit 0.079 lg ml1. The proposed method was successfully applied to the content uniformity testing of tablets and for determination of clopidogrel in presence of its coadministered drug, acetyl salicylic acid.

 Kahsay G. (2012); 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. Good separation was achieved on a Luna C18 column (150 mm 4.6 mm, 3 m) using gradient elution at a flow rate of 1 mL/min and a column temperature of 35 C. UV detection was performed at 220 nm. The method proved to be specific, sensitive (LOQ = 0.975 g/mL and 0.0384 g/mL for clopidogrel and acetylsalicylic acid, respectively), linear in the concentration range from LOQ to 325 g/mL for clopidogrel and from LOQ to 650 g/mL for acetylsalicylic acid, precise (RSD values for intermediate precision <1%) and accurate with mean recovery values of 100.7% and 100.2% for clopidogrel and acetylsalicylic acid, respectively.

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

System suitability Specificity

Linearity
Limit of detection and limit of quantification Accuracy Precision Ruggedness Robustness Stability of analytical Solution

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.

Accuracy

Precision

Limit of detection

Limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be distinguished from a blank.

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 L57 ( 150 mm X 4.6 mm) 5 micron

Wavelength
Flow Rate Injection volume Reagents

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 : Acetonitrile :: 750 : 250

Buffer preparation ( phosphate buffer) Mobile phase preparation

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 Blank Placebo solution Standard No. of Injection 1 2 6 Injection Volume(ml) 10 10 10 Run time ( minutes) 10 10 10

Sample preparation

10

10

Sample sequence for specificity

CHROMATOGRAM OF PLACEBO PREPARATION

CHROMATOGRAM OF STANDARD

CHROMATOGRAM OF SAMPLE

LINEARITY OF RESPONSE

A series of standard solution shall be prepared over a concentration range of 50% to 150% 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%)

No. of injection 1 6 2 2

Injection Volume (ml) 10 10 10 10

Run Time 10 10 10 10

Linearity 75 ppm (100%) Linearity 93.75 ppm (125%) Linearity 112.5 ppm (150%)

2 2 2

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

Area(0 .2ppm)
8483 8258 8217

Area (2 ppm)
62986 62367 62981

Area ( 20 ppm)
641171 641052 640776

4
5 6

8442
8951 9541

62693
62633 62956

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.

PREPARATION OF DIFFERENT CONCENTRATION OF CLOPIDOGREL BISULFATE TABLETS

Sample Name Amount of clopidogrel bisulfate drug(mg) 49.0 49.0 Volumetric flask to be used (ml) 100 100

Recovery-50%-1 Recovery-50%-2

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

49.0
98.0 98.0

100
100 100

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

98.0
147.0 147.0 147.0

100
100 100 100

Sample Name Mobile Phase

No. of Injection 1

Injection volume (ml) 10

Run time (min.) 10

standard
Recovery-50%-1 Recovery-50%-2

6
2 2

10
10 10

10
10 10

Recovery-50%-3
Recovery-100%-1 Recovery-100%-2 Recovery-100%-3 Recovery-150%-1 Recovery-150%-2 Recovery-150%-3

2
2 2 2 2 2 2

10
10 10 10 10 10 10

10
10 10 10 10 10 10

DATA FOR THE ACCURACY OF THE METHOD

Recovery
50%-1 50%-2

Amt. of drug added(mg)

51.1 51.1

Peak area of sample

3219493.9 3207153.1

Amt. recovered(mg)
51.146 50.950

Recovery (%)
100.09 99.71

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

51.2
100.8 96.1

3233471.3
6331702.7 6018109.8

51.369
100.590 95.610

100.33
99.79 99.49

100%-3
150%-1 150%-2 150%-3 Mean Std. Dev. %RSD

96.3
146.2 153.2 148.8 -

6036203.6
9157850.0 9636653.7 9357009.2 -

95.90
145.48 153.09 148.65 -

99.58
99.51 99.93 99.90 99.70 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% No. of injection 1 6 Injection Volume (L) 10 10 Run Time (minutes) 10 10

Standard-90% Standard 100%

Standard -110% Standard 120%

6 6
6 6

10 10
10 10

10 10
10 10

SAMPLE SEQUENCE FOR STANDARD PRECISION

Sample name Mobile Phase Sample-80% Sample-90% Sample-100% Sample-110% Sample-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

RESULT IN GIVEN TABLE FOR STANDARD PRECISION

No. of Injection 1 2 3 Area 80% 55251064 5256604 5295719 Area 90% 5764726 5759904 5717423 Area 100% 6465465 6492837 6478722 Area 110% 7035026 7050142 7065533 Area 120% 7251561 7210247 7216146

4
5 6

5270952
5326868 5326868

5756370
5795412 5825506

6511760
6538076 6633853

7090866
7111167 7142279

7281642
7245928 7190780

Mean
Std. Dev. %RSD

5290839.7
38156.5 0.7

5769890.2
36895.6 0.6

6520127.1
61267.8 0.9

7082502.2
40134.5 0.6

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

THANK YOU