Amrish Sharma*, Mukul Tailang, Bhaskar Gupta and Ashish Acharya
People’s Institute of Pharmacy and Research Centre, Bhanpur, Bhopal- 462 037(M.P.)
*Corresponding Author E-mail: amrish_sharma21@rediffmail.com
ABSTRACT:
A new and simple reverse phase HPLC method was developed for the determination of amlodipine in its pharmaceutical dosage form. The mobile phase used acetonitrile, methanol, KH2PO4 was in the ratio of 250: 250: 500. Buffer solution was prepared by dissolving 0.05m KH2PO4 + 0.1 % H3PO4 adjunct with triethylamine. The separation was achieved on hypersil C-18 column, phenomenex Gemini (250´4.6mm) and 5m particle size with rheodyne injector. The flow rate was 1ml/min and UV detection at 238nm. The retention time for amlodipine was 9 min. The linearity coefficient of Amlodipine was 0.99% and the percentage recoveries for amlopdipine was 100.02% respectively. The proposed method was accurate, precise and linear within the desired range. This method can be successfully employed for the quantitative analysis of amlodipine.
KEYWORDS: RP HPLC, Validation, Atenolol.
INTRODUCTION:
Amlodipine (C20H25ClN2O5 and Molecular weight is 408.88) (fig-1) is 2-[(2-Aminoethoxy) methyl]-4-(2-chlorophenyl)-1, 4-dihydro-6-methyl-3,5pyridinedi carboxylic acid, 3-ethyl, 5-methyl ester1 is a prototype second generation dihydropyridine calcium channel blocker2. It produces less reduction of myocardial contractility and indicated in treatment of Hypertension and angina pectoris. It can be safely combined with thiazides, beta-blocker, Ace inhibitors and nitrates. A survey of literature reveals that amlodipine is estimated by HPLC3, HPTLC4, Colorimetry5, Spectrophotometry6.Amlodipine is also studied with some other drugs for HPLC7, GLC8 and Spectrophotometry9.
MATERIAL AND METHODS:
Reagents and Chemicals:
Acetonitrile HPLC grade, Methanol HPLC grade, KH2PO4, Triethylamine.
Instruments:
Shimadzu HPLC (V.P Series), Spd-20 ATvp With UV-visible detector.
Hypersil C-18 column, PhenomenexGemini, 250´4.6mm, 5m particle size, Injector Rheodyne.
Pump LC- 20 AT, Mode of operation -Isocratic, Temperature-Ambient, Flow rate -1ml / min and UV detection at 238nm.
Standardization of method:
Acetonitrile, Methanol, KH2po4 are mixed in the ratio of (250: 250: 500) and used as a mobile phase.
Buffer solution was prepared by dissolving 0.05m KH2PO4+0.1% H3PO4 adjunct with Triethylamine.
An accurately weighed quantity 5mg of Amlodipine was dissolved in100 ml mobile phase this gave a concentration range of 500mg/ml of Amlodipine. From this solution 4, 4.5, 5, 5.5, 6, ml of the solution were taken and volume was made upto 25ml with the mobile phase to get concentration ranges of 80mg/ml, 90mg/ml, 100mg/ml, 110mg/ml, 120mg/ml which represents the range of 80%, 90%, 100%, 110%, 120%. These concentration ranges were used for further work of the analysis.
About 10 tablets were weighed (each tablet contains 5mg Amlodipine). Powder equivalent to average weight of 10 tablets was taken and dissolved in 100ml of the mobile phase and solution was filtered through what’s man filter paper No: 40. This gave a concentration of 500mg/ml of Amlodipine, from this solution 4, 4.5, 5, 5.5 and 6 ml of of solution were taken and the volume was made up to 25 ml with the mobile phase to get concentration ranges from 80mg/ml to 120mg/ml which represents the range of 80%, 90%, 100%, 110%, 120%. 20ml of the standard and sample solutions were injected respectively and the scans were recorded. Each solution was run five times at an interval of 15 min, to ensure elution of earlier injection.
Figure-1: Chemical structure of Amlodipine
The method is found to be specific because it doesn’t shows any interference with placebo solutions.
Varying quantities of the mixed standard stock solution was diluted with the mobile phase to give concentration of 80, 90, 100, 110 and 120 % of the Amlodipine. The injections were made at an interval of 15 min and peak area was determined. A calibration curve was determined by plotting the peak areas obtained against concentrations. There exists a linear relationship showing concentrations ranging from for Amlodipine 80mg/ml to 120mg/ml. From the data obtained, correlation coefficient for the Amlodipine was found 0.99. (Fig-3)
Precision:
Repeatability expresses the precision under the same operating conditions. Responses to the repeated injection of the same standard solution should have R.S.D< 1.0%. It is also termed as intra-assay precision. Injection of sample from each solution should have R.S.D< 2.0%. Our method precision is found R.S.D 0.98 and for system precision it is 0.80 for Amlodipine this indicates that our method is precise and accurate.
The number of theoretical plates 7371, tailing factor is 1.113 and retention time is 9min for the developed reverse phase HPLC method. The number of the theoretical plates was high indicating the efficient performance of the column (Fig-2).
Recovery Studies:
Determination of accuracy by direct comparison to reference standard is a preferred technique. Recovery studies were performed by spiking the blank matrix of the sample at different levels (80, 100, and 120%) of the known level in the sample. Average recovery of the analyte should be in the range of 99.0-102% at different levels of spiking.
RESULTS AND DISCUSSION:
The developed reverse phase HPLC method utilizes Acetonitrile, methanol and KH2PO4 in the ratio of (250:250:500) adjunct with Triethylamine as a mobile phase and column-Phenomenex Gemini C18 (5µ-250×4.6mm) as a stationary phase. The method precision and system precision are performed and found to be within the limits (table-1). The recovery study reveals the accuracy and precision of the method employed for the present studies.
|
S. No |
Parameter |
Acceptance criteria |
Results obtained |
|
|
1 |
Specificity |
Should not interfere with the placebo |
Passes |
|
|
2 |
Linearity |
Correlation coefficient not less than 0.99 |
0.99 |
|
|
3 |
Precision |
R.S.D for<1.0% system precision |
0.80 |
|
|
R.S.D<2.0% for method precision |
0.98 |
|||
|
4 |
Accuracy |
R.S.D for % recovery at each accuracy level not more than 2.0% |
Accuracy with concentration |
R.S.D |
|
80% |
0.40% |
|||
|
100% |
0.3% |
|||
|
120% |
0.29% |
|||
|
Recovery of the spiked drug (98-102%) |
Concentration |
% Recovery |
||
|
80% |
98.79% |
|||
|
100% |
101.02% |
|||
|
120% |
100.65% |
|||
CONCLUSION:
It is clear from the present study that the prescribed method of analysis is simple, accurate, Specific and precise in operation and can be employed for routine batch analysis of Amlodipine tablets.
Authors are thankful to Cadila Pharmaceuticals ltd. Ahmadabad for providing gift samples of the pure drug Amlodipine.
REFERENCES:
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3. Bereford, A.P; Macrae, P.V; Stopher, D.A;wood, B.A; J. Chromgr, 1987, PP: 178, 420.
4. llango, k; Kumar, P.B; Vijayaprasad, V.R; Ind.J.pharm. science, 1997,59(6), PP: 336-337.
5. Singhvi,I; Chaturvedi, S.C; Ind. J. pharm.Sci, 1998,60(5), PP: 309-310.
6. Meyyanathan, S.N; Joel.J, S; Suresh. B; Indian Drugs 1988, 35(5), PP: 296-297.
7. Jain, R; Jain, C.L; Analytical Abstract, 1993, vol-55, PP: 5G38.
8. Erram, S.V; Tipnis, H.P; Indian Drugs, 1992, 29(7), PP: 436-438.
9. Rao,G.R; Avadhanulum A.B; Giridhar,R; Pantulu, A.r Kokate, C.K; Analytical abstract, 1991, vol, 53, PP: 8G85.
Received on 26.09.2009 Modified on 19.11.2009
Accepted on 17.12.2009 © AJRC All right reserved
Asian J. Research Chem. 3(1):Jan.-Mar. 2010 page 129-131