INTRODUCTION:

Amlodipine Besylate¹ is chemically 3-ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate benzene sulphonate (fig.1) belongs to the class of Calcium channel blocker, used as anti-anginal. Molecular Formula – C₂₆H₃₁ClN₂O₈S , Molecular Weight – 567.1.Solubility - Slightly soluble in water and in isopropyl alcohol, sparingly soluble in dehydrated alcohol, freely soluble in methanol.

Fig.No.1.Cheical structure of Amlodipine basylate

Valsartan is chemically N-(1-Oxopentyl)-N-[[2'-(1H-tetrazol- 5-yl) [1, 1'-biphenyl]-4-yl] methyl]-L-valine.Valsartan is potent Angiotensin II receptor blocker. It is mainly used as anti-hypertensive drug.Valsartan is official in USP. There were few methods reported for determination of valsartan individually. The valine molecule of the Sertraline has one chiral centre. The (S) enantiomer is essentially used. Molecular Formula –

C₂₄H₂₉N₅O₃and Soluble in Acetonitrile, practically insoluble in water. The aim of the present study was to develop accurate, precise and selective reverse phase HPLC assay procedure for the analysis of Valsartan and Amlodipine in bulk drug formulation. The validation of proposed method is done according to the ICH guideline validation was done according to ICH guidelines.

Fig.No.2.Chemical structure of Valsartan.

From the literature survey it was found that many methods are available for determination of Amlodipine Besylate and Valsartan individually and few methods in combination with other drugs. However, no stability indicating HPLC has been reported for simultaneous determination of Amlodipine Besylate, and Valsartan in combination.

In the proposed study an attempt will be made to develop a stability indicating HPLC method for simultaneous estimation of Amlodipine Besylate, and Valsartan in pharmaceutical formulation (tablet).

Pharmaceutical grade of Amlodipine, and Valsartan were kindly supplied as gift samples by Morpen Laboratories, New Delhi, India, certified to contain > 99% (w/w) on dried basis. Commercially available Amlopres (Cipla Formulation Pvt. Ltd., Patalganga, India) tablets claimed to contain 5 mg Amlodipine and 80 mg Valsartan have been utilized in the present work. All chemicals and reagents used were of HPLC grade and were purchased from Agenta Chemicals, Hyderabad, India.

Chromatographic system and conditions:

The HPLC system (Analytical Technologies Gujarat, India) consisted of pump. The Analytical column a cosmosil packed column 5C₁₈ MS-II(250mm x 4.6 i.d.)was operated at ambient temperature (20±1^oc) . Isocratic elution with Acetonitrile : methanol:phosphate buffer (20:50:30 v/v pH 3)was used at flow rate at 1.0 ml/min column (250×4.6 i.d). The mobile phase. Before analysis the mobile phase was filtered through a 0.2 μm membrane and degassed by ultrasonification. Detection was monitored at 239 nm and injection volume was 20 μl. All the experiments were performed at ambient temperature.

Pharmaceutical grade of Amlodipine and Valsartan were kindly supplied as gift samples by Morpen Pharmaceuticals, New Delhi, India, certified to contain > 99% (w/w) on dried basis. Commercially available Amlopres (Cipla Formulation Pvt. Ltd., Patalaganga, India), tablets claimed to contain 5 mg Amlodipine and 80 mg Valsartan have been utilized in the present work. All chemicals and reagents used were of HPLC grade and were purchased from Agenta Chemicals, India.

Standard solutions and calibration graphs for chromatographic measurement:

Stock standard solutions were prepared by dissolving separately 5 mg of Valsartan and Amlodipine in 10 ml mobile phase (1000 μg/ml). The standard calibration solutions were prepared by appropriate dilution of the stock solution with methanol to reach a concentration range of 10-60µg/ml for Amlodipine and 10-60 μg/ml for Valsartan. 20 μl injections were made for each concentration and chromatographed under the optimized conditions described above. The peak area was plotted against the corresponding concentrations to obtain the calibration graphs.

Sample preparation:

Twenty tablets contents were accurately weighed, their mean weight was determined and they were mixed and finely powdered. A portion equivalent to about one tablet was accurately weighed and transferred into a 100 ml volumetric flask containing 50 ml mobile phase, sonicated for 15 min and diluted to 100 ml with mobile phase. The resulting solution was centrifuged at 100 rpm for 15 min. Supernatant was taken and after suitable dilution the sample solution was then filtered using 0.45 μ filter (Millipore, Milford, MA). The original stock solution was further diluted to get sample solution of drug concentration of 50 μg/ml Valsartan and 25 μg/ml Amlodipine. A 20 μl volume of sample solution was injected into HPLC, six times. The peak areas for the drugs were measured at 239 nm and amounts of Valsartan and Amlodipine were determined using the related linear regression equations.

Method validation:

The developed method was validated according to the ICH guidelines. The system suitability was evaluated by six replicate analyses of Amlodipine and Valsartan mixture at a concentration of 50 μg/ml Valsartan and 25 μg/ml Amlodipine. The acceptance criteria were a R.S.D. of peak areas and retention times less than 2%, Theoretical plate numbers (N) at least 2500 for each peak and tailing factors (T) less than 1% for Valsartan and Amlodipine.

Standard calibration curves were prepared in the mobile phase with six concentrations ranging from 10-60µg/ml for Amlodipine and 10-60 μg/ml for Valsartan into the HPLC system keeping the injection volume constant. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. To study the reliability and suitability of the developed method, recovery experiments were carried out at three levels 50, 100 and 150%. Known concentrations of commercial tablets were spiked with known amounts of Valsartan and Amlodipine. At each level of the amount six determinations were performed and the results obtained were compared with expected results. Recovery for pharmaceutical formulations should be within the range 100±5%. The percent R.S.D. of individual measurements was also determined. Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day) for 2 consecutive days. Three different concentrations of Valsartan and Amlodipine were analyzed in six independent series in the same day (intra-day precision) and 3 consecutive days (inter-day precision). The repeatability of sample application and measurement of peak area for active compounds were expressed in terms of percent RSD.

All chromatograms were examined to determine if compounds of interest co-eluted with each other or with any additional excipients peaks. Marketed formulations were analyzed to determine the specificity of the optimized method in the presence of common tablet excipients. Limit of detection (LOD) and limit of quantitation (LOQ) were estimated from the signal-to-noise ratio. LOD and LOQ were calculated using 3.3σ/s and 10σ/s formulae, respectively, where, σ is the standard deviation of the peak areas and s is the slope of the corresponding calibration curve. To evaluate robustness of HPLC method a few parameters were deliberately varied. The parameters included variation of flow rate, percentage of buffer in the mobile phase, and pH of mobile phase.

RESULTS AND DISCUSSION:

During the optimization of HPLC method, columns (Cosmosil packed column 5c-18 ms-II 250 mm × 4.6 i.d), two organic solvents (acetonitrile, methanol and phosphate buffer), two buffers (acetate and phosphate) at two different pH values (3 and 4) were tested. Initially methanol:water, acetonitrile:water, acetonitrile: phosphate buffer, methanol: phosphate buffer were tried in different ratios at pH 3 and 4. Amlodipine eluted with the tried mobile phases, but Valsartan was retained. Then, with acetonitrile:methanol: phosphate buffer all the two drugs eluted. The mobile phase conditions were optimized so the peak from the first-eluting compound did not interfere with those from the solvent, excipients. Other criteria, viz. time required for analysis, appropriate k range (1<k<10) for eluted peaks, assay sensitivity, solvent noise were also considered. Finally a mobile phase consisting of a mixture of acetonitrile: methanol: phosphate buffer pH 3 adjusted with orthophosphoric acid in ratio 20:50:30 (v/v), was selected as mobile phase to achieve maximum separation and sensitivity. Flow rates between 0.5 to 1.2 ml/min were studied. A flow rate of 1.0 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a reversed phase C18 column, the retention times for Amlodipine and Valsartan were observed to be 4.915 and 8.056 min. respectively. Total time of analysis was less than 10 min. The chromatogram at 239 nm showed a complete resolution of all peaks (fig. 2).

Representative chromatograms of standard solutions (a) standard solution of Valsartan (50 μg/ml); (b) standard solution of Amlodipine (25 μg/ml) and (c) a standard solution containing 50 μg/ml Valsartan, 25 μg/ml Amlodipine.

Validity of the analytical procedure as well as the resolution between different peaks of interest is ensured by the system suitability test. All critical parameters tested met the acceptance criteria on all days. As shown in the chromatogram, all three analytes are eluted by forming symmetrical single peaks well separated from the solvent front.

Excellent linearity was obtained for all the two drugs in the range of 10-60 Amlodipine and 10-60 Valsartan. The correlation coefficients (r²) were found to be greater than 0.999 (n=6) in all instances. The results of calibration studies are summarized in Table 1. The proposed method afforded high recoveries for Valsartan and Amlodipine tablet. Results obtained from recovery studies presented in Table 2, indicate that this assay procedure can be used for routine quality control analysis of this ternary mixture in tablet. Precision of the analytical method was found to be reliable based on % RSD (< 2%) corresponding to the peak areas and retention times. The % RSD values were less than 2, for intra-day and inter-day precision. Hence, the method was found to be precise for all the two drugs.

The chromatograms were checked for the appearance of any extra peaks. It was observed that single peak for Amlodipine (R_t±SD, 4.915±0.01) and Valsartan (R_t±SD, 8.056±0.01) were obtained under optimized conditions, showing no interference from common tablet excipients and impurities. Also the peak areas were compared with the standard and % purity calculated was found to be within the limits. These results demonstrate the specificity of the method

TABLE 1-Linearity Parameters for the Simultaneous Estimation of Amlodipine and Valsartan(N=6)

Parameters	Amlodipine	Valsartan
l_max(nm)	239	239
Beers law limit (μg/ml)	10-60	10 – 60
Correlation coefficient (r)	0.9994	0.9993
Regression equation (y=mx+c)	y= 230808.9x + 366397	y=249058 x + 2721008
Slope (m)	230808.9	249058
Intercept (c)	366397	2721008
LOD (μg/ml)	0.1379	0.0677
LOQ (μg/ml)	0.4180	0.2051
Standard Error	134339.4	162471.7

TABLE -3-System Suitability Parameters for the Optimized Chromatogram by RP - HPLC

PARAMETERS	AMLODIPINE	VALSARTAN
Tailing factor	1.10	1.18
Asymmetrical factor	1.10	1.18
Theoretical plates	24	11187
Capacity factor	6.697	14.377
Theoretical plate /unit Length	206.19	242.05
Resolution	Between AMLO and VALS 4.04

Fig.No.3. Calibration Curve of Amlodipine Basylate.

Fig.No.4.Calibration Curve of Valsartan

Fig.No.5. Typical Chromatogram of Mixture of Amlodipine and Valsartan

LOD and LOQ were found to be 0.1379 μg/ml and 0.4180 μg/ml for Valsartan and 0.0677 μg/ml and 0.2051 μg/ml for Amlodipine. In all deliberately varied conditions, the SD of retention times of Valsartan and Amlodipine were found to be well within the acceptable limit. The tailing factor for all the two peaks was found to be < 1.5 (Table 3). The validated method was used in the analysis of marketed conventional tablet Amlopres with a label claim: 80 mg valsartan and 5 mg Amlodipine per tablet. Representative chromatogram is shown in (fig. 4). The results for the drugs assay show a good agreement with the label claims.

The developed HPLC method is simple, specific, accurate and precise for the simultaneous determination of Amlodipine and Valsartan from tablets. The developed method provides good resolution between Valsartan and Amlodipine. It was successfully validated in terms of system suitability, linearity, range, precision, accuracy, specificity, LOD, LOQ and robustness in accordance with ICH guidelines. Thus, the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs either as such or in combination.

ACKNOWLEDGEMENT:

The authors would like to Thanks to Morpen Laboratories, New Delhi, India for providing a samples of Amlodipine and Valsartan. The authors are also thankful to Principal and Management of Chilkur Balaji College of Pharmacy –Hyderabad for providing all necessary facilities.

REFERENCES:

1 M. Manoranjani IJSID,et al., Journal of Pharmacy Research (2011) reported “RP-HPLC Method for the Estimation of Valsartan in Pharmaceutical Dosage Forms. Available from:htpp//www.en.wikipedia.org/wiki/Amlodipine and valsartan

3 Available from:htpp//www..Pubmed. Pharmacol.com /Amlodipine and valsartan

4 Available from:htpp//www.en.Rxlist..com /Amlodipine and valsartan

5. ICH Q2A Validation of Analytical procedure: Methodology International Conference on Harmonization, Geneva October 1994

6 ICH Q2B Validation of Analytical procedure: Methodology International Conference on Harmonization, Geneva March 1996.

7 Kasture AV, Ramteke M. Simultaneous UV-spectrometric method for the estimation of Atenolol and Amlodipine Besylate in combined dosage form. Indian Journal of Pharmaceutical Sciences .2006; 68(3):394-396.

8 Narasimham Y.S., Barhate V.D. Development and validation of stability indicating UPLC method for the simultaneous determination of beta-blockers and diuretic drugs in pharmaceutical dosage forms, Journal of chemicalmetrology 2010; 4{1}:1.20

9 Willard, Meritt, Dean and Settle. Instrumental Methods of Analysis. 7^th Edn., CBS Publishers and Distributors, New Delhi, 1986, 1, 592, 622-628.

10. Anonymous. Remington. The Science and Practice of Pharmacy. 21^st Edn., Wolters Kluwer Health (India) Pvt. Ltd., New Delhi, 2007, (I), 633-642

11 Code Q2A, Text on Validation of Analytical Procedures. ICH Harmonized Tripartite Guidelines, Geneva, Switzerland, 27 October, 1994, 1 - 5.

12. Code Q2B, Validation of Analytical Procedures; Methodology. ICH Harmonized Tripartite Guidelines, Geneva, Switzerland, 6^th November, 1996, 1 - 8.

13 Richa Sah and Saahil Arora. Development and validation of a HPLC analytical assay method for amlodipine besylate tablets: A Potent Ca+2 channel blocker. Journal of Advance Pharmacy Education and Research. 2012; 2 (3): 93-100.

15 Shalini Pachauri et al., Development and Validation of HPLC Method for Analysis of Some Antihypertensive Agents in their Pharmaceutical Dosage Forms. 2010.

16 Permender Rathee et al., Simultaneous Estimation of Amlodipine Besylate and Atenolol as A.P.I. and in Tablet Dosage Forms by Vierodt’s Method using UV Spectrophotometry. 2010.

Received on 18.07.2014 Accepted on 18.08.2014

Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 103-107