Keywords:
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| Valsartan, RP-HPLC, Pharmaceutical Formulations, Validation, Mobile Phase, Specific. |
Introduction
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| Valsartan is chemically (S)-3-methyl-2-(N-{[2'-(2H-1, 2, 3, 4-tetrazol-5-yl)biphenyl 4 yl] methyl} pentanamido) butanoic acid [figure 1]. Valsartan is an angiotensin II receptor antagonist (more commonly called an "ARB", or angiotensin receptor blocker), with particularly high affinity for the type I (AT1) angiotensin receptor. By blocking the action of angiotensin, valsartan dilates blood vessels and reduces blood pressure [1] . In U.S,valsartan is indicated for treatment of high blood pressure, congestive heart failure (CHF), or post-myocardial infarction (MI) in 2005[2]. A study released in 2010, based on 819,491 cases in U.S. Veteran's Administration database from 2002– 2006, demonstrated a significant reduction in the incidence and progression of Alzheimer's disease and dementia.[3] An earlier study released by the Journal of Clinical Investigation in 2007 found some efficacy in the use of valsartan in the treatment and prevention of Alzheimer's disease.In the present investigation methods such as HPLC [4-6], LC-MS [7-9], Protein precipitation [10], Capillary electrophoresis [11] and Simultaneous UVspectrophotometric methods [12,13] are reported for estimation of Valsartan alone or in combination with other agents. However, there were few methods reported for determination of valsartan individually.In the present study a rapid, precise, accurate, specific and sensitive HPLC method was developed for quantitative estimation of Valsartan in bulk drug and pharmaceutical formulations using UV detector. The present method was simple, since diluted samples are directly used without any preliminary chemical derivatization or purification steps. |
Experimental
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| Instrumentation: |
| Agilent 1120 compact LC system, with variable wavelength programmable UV detector and Rheodyne injector with 20 μl fixed loop was used for the chromatographic separation. Ezchrome software was used for data analysis. Chromatographic separation was carried out on a C18 column [Agilent ODS UG 5 column, 250mm x 4.5mm]. Ultra-sonic bath sonicator, degasser is used. |
| Preparation of standard stock solution |
| 25mg of pure drug was weighed accurately and transferred in to 25ml volumetric flask and dissolved in 10ml solvent and made up to the mark with solvent to obtain a final concentration of 1000μg/ml (standard stock solution A). From the standard stock solution 2.5ml of aliquot was pippeted in to 25ml volumetric flask and dissolved in 10ml solvent and made up to the mark with the solvent to obtain a final concentration of 100μg/ml (standard stock solution ‘B’). |
| Preparation of sample stock solution |
| Marketed tablet formulation (VALENT) containing 80mg of Valsartan was analyzed by this method. Twenty tablets were accurately weighed and their average weight determined. The tablets were then crushed to fine powder and powder equivalent to 10mg was taken in 100ml volumetric flask and dissolved in 50ml of mobile phase. The solution was kept for sonication for 15min. The solution was made up to the mark with the mobile phase and filtered through 0.45 μ membrane filter to get the concentration of 100μg/ml (sample stock solution A).1.0ml aliquot of the above solution was transferred to a 10ml volumetric flask and diluted to the mark with the mobile phase to obtain a concentration of 10μg/ml (sample stock solution ‘B’). |
| Chemicals and reagents |
| HPLC grade Acetonitrile, Phosphate Buffer, Ortho Phosphoric acid, were used as solvents throughout the experiment.Valsartan was kindly supplied by Dr.Reddys laboratories (Hyderabad, A.P and India). All the solvents used in HPLC method are of HPLC grade. Commercial pharmaceutical preparations of Valsartan from Lupin limited (Aurangabad, India). Which were claimed to contain 80 mg Valsartan was used in analysis. After systematic and detailed study of the various parameters involved, as described under results and Discussion in this chapter, the following procedure was recommended for the determination of valsartan in Bulk samples and pharmaceutical formulations. |
| Preparation of Buffer: |
| A solution of 13.60 gm of potassium bi phosphate was dissolved in 900 ml of HPLC grade water, mix well by using sonicator, and make up the volume to 1000 ml with water. The pH of the resulting solution was adjusted to 3.0 ± 0.5 with 10 % orthophosphoric acid. The above solution was filtered through a 0.45μ membrane filter and degassed by ultra sonicator. |
| Chromatographic conditions |
| Mode of operation : Isocratic |
| Column temperature : Ambient |
| Detector wavelength : 273nm |
| Injection volume : 20 μL |
| Flow rate : 1ml/min |
| Run time : 7min |
| According to USP XXIV (621), system suitability tests are an integral part of chromatographic method. They are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions. |
| The calibration curve was linear in the concentration range of 10-50μg/ml with correlation coefficient 0.999, that was shown in the following figure. |
Results and discussion
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| The objective of this study was to develop a rapid and sensitive HPLC method for the analysis of Valsartan in bulk drug and in its Pharmaceutical dosage forms using the most commonly employed C-18column with UV- detection. Mobile phase and flow rate selection was based on peak parameters (height, capacity, theoretical plates, tailing or symmetry factor), run time, resolution. |
| The system with Acetonitrile: Phosphate buffer (70:30 v/v) with 1.0 ml / min flow rate was quite robust. The optimum wavelength for detection was 273 nm at which better detector response for the drug was obtained. The run time was set at 7 min and the retention time for was 3.48min.chromatogram was shown in the following figure 2. |
| Linearity |
| To establish the linearity of proposed method, aliquots 1,2,3,4 and 5 of standard stock solution ‘B’ were transferred in a series of 10ml volumetric flasks and the volume was made up to the mark with mobile phase to obtain final concentration of 10,20,30,40 and 50μg/ml. Three replicates per each concentration were injected and Peak areas of the above solutions were reported. A calibration curve of concentration vs. peak area was established. Regression equations were established and the correlation coefficients were determined in the following table1. |
| Accuracy |
| Accuracy of the method was examined by performing recovery studies by standard addition method, by adding a known amount of standard solution of pure drug to a pre-analyzed sample solution, at three levels 80%, 100%, 120% of the label claim. Accuracy was evaluated using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range. The mean recoveries were found in the range of 99.47-101.1 %. The values were determined in the following table 2. |
| Precision |
| The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. The values were reported in the following table 3 and 4. |
| Limit of Detection (LOD) |
| It is the lowest concentration of analyte that can be detected, but not necessarily quantified, by the analytical method. In chromatography the detection limit is the injected amount that results in a peak with a height at least twice or three times as high as the baseline noise level (S/N ratio~ 3). |
| Limit of Quantitation (LOQ) |
| It is the lowest concentration of analyte that can be determined with acceptable accuracy and precision by the analytical method. LOD was expressed as concentration of analyte generating an instrument response equivalent to ten times the noise (S/N ratio~ 10). |
| Limit of detection (LOD) and limit of quantitation (LOQ) were manually calculated from the slope of the calibration curve and standard deviation. |
| The LOD and LOQ values were calculated using the formula given below |
| LOD = 3.3 x 4/s |
| LOQ = 10 x 4/s |
| Where, σ = standard deviation of intercepts of calibration curve |
| S = slope of the calibration curve. |
| The LOD and LOQ values were reported in following table 5. |
| Robustness |
| The robustness of a method is its ability to remain unaffected by the deliberate variations in method parameters such as column temperature, analytical wavelength and flow rate. |
| Detection wave length was initially changed to 271nm and then to 273nm and a series of three injections of 100% concentration were given at each detection wave length and the % RSD was reported separately for each variable. |
| Flow rate was changed to 0.8ml/min and then to 1.2ml/min and a series of three injections of 100% concentration were given at each detection wave length and the % RSD was reported separately for each variable. The values were reported in table 6 and 7. |
| Ruggedness |
| Ruggedness is a measure of reproducibility of the results under instrument to instrument and analyst to analyst variations. Six Injections of 100% concentration were given by the co-analyst and the peak areas were recorded and the %RSD was calculated statistically. The values were determined in the following table 8. |
CONCLUSION:
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| A simple RP-HPLC method has been developed for the determination of Valsartan. The proposed method is rapid, accurate and precise. Its chromatographic retention time of 5 min allows the analysis of a large number of samples in short period of time. Therefore, it is suitable for the routine analysis of Valsartan. The results of the study reveals that the proposed RP-HPLC method for the estimation of Valsartan is simple and accurate in bulk and pharmaceutical dosage forms. |
Tables at a glance
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Table 8 |
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Figures at a glance
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