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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