Beruflich Dokumente
Kultur Dokumente
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.)
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?
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)
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
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
DRUG REVIEW
Drug Chemical name : Clopidogrel
Chemical formula :
Molecular weight :
321.822
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
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
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
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
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% 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 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
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
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.
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
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
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-80%
Sample-90% Sample-100% Sample-110% Sample-120%
6
6 6 6 6
10
10 10 10 10
10
10 10 10 10
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.
4. System suitability
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