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ANALYTICAL METHOD VALIDATION FOR ASSAY OF CLOPIDOGREL TABLETS

BY HPLC METHOD

INSTITUTE OF PHARMACEUTICAL SCIENCES, KURUKSHETRA UNIVERSITY, KURUKSHETRA

SupervisorDr. Ajay Aggarwal(Assistant Professor)

Co- SupervisorVijay Chauhan QC ExecutiveDr. Morepen Labs.Parwanoo (H.P.)Presented by-

Aman ThakurM.Pharm. 3rd Sem

Introduction

Review of Literature

Drug Review

Plan of Work

Material and Methods

Current Status of Work

CONTENTS

INTRODUCTION

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?

METHOD VALIDATION

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 include-

a) 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 (250mm×4.6mm, 5m) column. n-Hexane, 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 reversed-phase 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 0.2–3.5 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 co-administered 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 : Clopidogrel

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

Chemical formula : C16H16ClNO2S

Molecular weight : 321.822

DRUG REVIEW

CHEMICAL STRUCTURE OF CLOPIDOGREL

N

O O

CH3

S

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

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: Liver is the main site of metabolism of drug.

Elimination: 50% of the drug is eliminated in urine.

Protein binding: 98%

Half-life: 8 Days

DRUG DESCRIPTION

PLAN OF WORK

System suitability

Specificity

Linearity

Limit of detection and limit of quantification

Accuracy

Precision

Ruggedness

Robustness

Stability of analytical Solution

PARAMETERS TO BE VALIDATED FOR METHOD VALIDATION

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 Accuracy is used to describe the measure of exactness of an analytical method when compared with the reference value.

Precision Precision is the measure of the degree of repeatability of an analytical method under normal operation.

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.

Limit of quantification

The limit of quantification (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method.

RuggednessRuggedness is the degree of reproducibility of the results obtained under a variety of conditions ( Analysts, Laboratory conditions, instruments, reagents etc.).

Robustness

Robustness is the capacity of a method to remain unaffected by small deliberate variations in method parameters

MATERIAL & METHODS

HPLC with PDA detectors (Waters)

pH meter ( Electronic India)

Analytical balance ( Mettler)

Sonicator (Powersonic)

INSTRUMENTS REQUIRED FOR VALIDATION

Monobasic Potassium Phosphate

Acetonitrile (HPLC grade)

Methanol ( HPLC grade)

0.45 micron nylon membrane filter (Millipore)

REAGENTS AND CHEMICALS REQUIRED 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

WORKING STANDARD, PLACEBO, AND SAMPLE USED DURING THE VALIDATION

Column L57 ( 150 mm X 4.6 mm) 5 micron

Wavelength 220 nm

Flow Rate 1.0 ml/min

Injection volume 10 micro litre

Reagents HPLC grade Acetonitrile Monobasic Potassium Phsphate ( KH2PO4) buffer

Buffer preparation ( phosphate buffer)

Dissolve 1.36 gm. Of monobasic potassium phosphate (KH2PO4) in 1000 ml with HPLC grade water.

Mobile phase preparation Buffer : Acetonitrile :: 750 : 250

CHROMATOGRAPHIC CONDITIONS DURING VALIDATION

Parameters which are validated till date-

a) Specificity

b) Linearity

c) Accuracy

d) Precision

e) Limit of detection

f) Limits of quantification

CURRENT STATUS OF THE WORK

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.

SPECIFICITY

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 1 10 10

Placebo solution 2 10 10

Standard 6 10 10

Sample preparation

2 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 name No. of injection

Injection Volume (ml)

Run Time

Blank ( Diluent) 1 10 10

Standard 6 10 10

Linearity 37.5 ppm (50%) 2 10 10

Linearity 56.25 ppm (75%) 2 10 10

Linearity 75 ppm (100%) 2 10 10

Linearity 93.75 ppm (125%) 2 10 10

Linearity 112.5 ppm (150%) 2 10 10

SAMPLE SEQUENCE FOR LINEARITY

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 Area(0 .2ppm) Area (2 ppm) Area ( 20 ppm)

1 8483 62986 641171

2 8258 62367 641052

3 8217 62981 640776

4 8442 62693 642245

5 8951 62633 640801

6 9541 62956 640707

Mean 8648.8 62769.3 641125.5

Std. Dev. 509.1 250.2 576.9

%RSD 5.9 0.4 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 1272547

2 1268875

3 1274526

4 1268741

5 1275984

6 1272584

Mean 1272209.5

Std. Dev. 642.8

% RSD 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 Name Amount of clopidogrel bisulfate

drug(mg)

Volumetric flask to be used (ml)

Recovery-50%-1 49.0 100

Recovery-50%-2 49.0 100

Recovery-50%-3 49.0 100

Recovery-100%-1 98.0 100

Recovery-100%-2 98.0 100

Recovery-100%-1 98.0 100

Recovery-150%-1 147.0 100

Recovery-150%-2 147.0 100

Recovery-150%-3 147.0 100

PREPARATION OF DIFFERENT CONCENTRATION OF CLOPIDOGREL

BISULFATE TABLETS

Sample Name No. of Injection Injection volume (ml)

Run time (min.)

Mobile Phase 1 10 10

standard 6 10 10

Recovery-50%-1 2 10 10

Recovery-50%-2 2 10 10

Recovery-50%-3 2 10 10

Recovery-100%-1 2 10 10

Recovery-100%-2 2 10 10

Recovery-100%-3 2 10 10

Recovery-150%-1 2 10 10

Recovery-150%-2 2 10 10

Recovery-150%-3 2 10 10

Recovery Amt. of drug added(mg)

Peak area of sample

Amt. recovered(mg)

Recovery (%)

50%-1 51.1 3219493.9 51.146 100.09

50%-2 51.1 3207153.1 50.950 99.71

50%-3 51.2 3233471.3 51.369 100.33

100%-1 100.8 6331702.7 100.590 99.79

100%-2 96.1 6018109.8 95.610 99.49

100%-3 96.3 6036203.6 95.90 99.58

150%-1 146.2 9157850.0 145.48 99.51

150%-2 153.2 9636653.7 153.09 99.93

150%-3 148.8 9357009.2 148.65 99.90

Mean - - - 99.70

Std. Dev. - - - 0.1805547

%RSD - - - 0.181098

DATA FOR THE ACCURACY OF THE METHOD

Percent recovery of drug (%age)-

Area of recover sample Std. Wt. 5 100 50 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%.

CALCULATION FOR THE ACCURACY OF METHOD

X X XX X P X 100

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.

PRECISION

SAMPLE SEQUENCE FOR STANDARD PRECISION

Sample name No. of injection

Injection Volume (µL)

Run Time (minutes)

Mobile Phase 1 10 10

Standard -80% 6 10 10

Standard-90% 6 10 10

Standard – 100% 6 10 10

Standard -110% 6 10 10

Standard – 120% 6 10 10

SAMPLE SEQUENCE FOR STANDARD PRECISION

Sample name No. of Injection

Injection volume (µL)

Run Time ( Minutes)

Mobile Phase 1 10 10

Sample-80% 6 10 10

Sample-90% 6 10 10

Sample-100% 6 10 10

Sample-110% 6 10 10

Sample-120% 6 10 10

No. of Injection

Area 80%

Area 90%

Area 100%

Area 110%

Area 120%

1 55251064 5764726 6465465 7035026 7251561

2 5256604 5759904 6492837 7050142 7210247

3 5295719 5717423 6478722 7065533 7216146

4 5270952 5756370 6511760 7090866 7281642

5 5326868 5795412 6538076 7111167 7245928

6 5326868 5825506 6633853 7142279 7190780

Mean 5290839.7 5769890.2 6520127.1 7082502.2 7242717.4

Std. Dev. 38156.5 36895.6 61267.8 40134.5 35980.7

%RSD 0.7 0.6 0.9 0.6 0.5

RESULT IN GIVEN TABLE FOR STANDARD PRECISION

No. of injection

Area 80%

Area 90%

Area 100%

Area 110%

Area 120%

1 5247287 5598284 6515146 6431984 7002279

2 5259083 5707477 6481062 6491860 6940183

3 5309480 5729800 6505140 6491860 6949754

4 5338267 5756091 6470829 6431984 7081406

5 5378762 5744476 6508176 6442111 7069955

6 5370786 5784842 6481062 6501904 7100222

Mean 5317277.4 5720161.6 6493569.2 6465284.5 7023966.5

Std. Dev. 55551.9 65074.6 18126.4 33191.0 69608.2

%RSD 1.0 1.1 0.3 0.5 1.0

RESULTS IN TABLE FOR SAMPLE PRECISION

Data evaluation-

The 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 criteria-

RSD 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