INTRODUCTION:

Amlodipine Besylate: chemically is 3-ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate benzene sulphonate, belongs to the class of Calcium channel blocker, used as anti-anginal. Molecular Formula – C₂₆H₃₁ClN₂O₈S and Molecular Weight – 567.1.

Solubility-Slightly soluble in water and in isopropyl alcohol, sparingly soluble in dehydrated alcohol, freely soluble in methanol.

Atenolol: chemically is (RS)-4-(2-hydroxy-3-Isopropylaminopropoxy) phenylacetamide, belongs to the class of β-adrenergic blocker, used as an antihypertensive drug.

Fig.No.1.Chemical structure of Amlodipine Besylate

Fig.No.2.Chemical structure of Atenolol

Aspirin: chemically is 2-(acetyloxy) benzoic acid. It is a Platelet Aggregation Inhibitors, Anti-Inflammatory Agents, Non-Steroidal dug

Fig.No.3.Chemical structure of Aspirin

From the literature survey it was found that many methods are available for determination of Amlodipine Besylate, Aspirin and Atenolol individually and few methods in combination with other drugs. However, no stability indicating HPLC has been reported for simultaneous determination of Amlodipine Besylate, Aspirin and Atenolol in combination. In the proposed study an attempt will be made to develop a stability indicating HPLC method for simultaneous estimation of Amlodipine Besylate, Aspirin and Atenolol in pharmaceutical formulation (capsules).

Pharmaceutical grade of Amlodipine, Aspirin and Atenolol were kindly supplied as gift samples by Morpen Laboratories, New Delhi, India, certified to contain > 99% (w/w) on dried basis. Commercially available Ato-Guard (Perk Formulation Pvt. Ltd., Merrut, India) capsules claimed to contain 100 mg aspirin; 5 mg Amlodipine and 50 mg Atenolol 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 C18-MS-II (4.6 I.D. x 250mm; 5µ particle size) was operated at ambient temperature (20 ±1^oc) . Isocratic elution with Acetonitrile: Acetate buffer (80:20 v/v pH 4.6) was used at flow rate at 0.8ml/min column (150×4.6 mm; 5 μm).

The mobile phase. Before analysis the mobile phase was filtered through a 0.2 μm membrane and degassed by ultrasonification. Detection was monitored at 233 nm and injection volume was 20 μl. All the experiments were performed at ambient temperature. Pharmaceutical grade of Aspirin, Amlodipine and Atenolol were kindly supplied as gift samples by Morpen Pharmaceuticals, New Delhi, India, certified to contain > 99% (w/w) on dried basis. Commercially available Ato-Guard (Perk Formulation Pvt. Ltd., Merrut, India), capsules claimed to contain 100 mg Aspirin; 5 mg Amlodipine and 50 mg Atenolol 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 50 mg of Aspirn, Atenolol and Amlodipine in 50 ml mobile phase (1000 μg/ml). The standard calibration solutions were prepared by appropriate dilution of the stock solution with mobile phase to reach a concentration range of 5-30 μg/ml for Aspirin, 1-6µg/ml for Amlodipine and 2.5-15 μg/ml for Atenolol. Triplicate 20 μl injections were made for each concentration and chromatographed under the optimized conditions described above. The peak area were plotted against the corresponding concentrations to obtain the calibration graphs.

Sample preparation:

Twenty capsule contents were accurately weighed, their mean weight was determined and they were mixed and finely powdered. A portion equivalent to about one capsule 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 100 μg/ml Asprin, 50 μg/ml Atenolol 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 233 nm and amounts of Aspirin, Atenolol and Amlodipine were determined using the related linear regression equations.

Method validation:

The developed method was validated according to the ICH guideline. The system suitability was evaluated by six replicate analyses of Aspirin, Atenolol and Amlodipine mixture at a concentration of 100 μg/ml Aspirin, 50 μg/ml Atenolol 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 Aspirin, Atenolol and Amlodipine.

Standard calibration curves were prepared in the mobile phase with six concentrations ranging from 5-30 μg/ml for ASP and 1-6µg/ml for Amlodipine and 2.5-15 μg/ml for Atenolol 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 capsules were spiked with known amounts of Aspirin, Atenolol 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 two consecutive days. Three different concentrations of Aspirin, Atenolol 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 capsule 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 C18-MS-II (4.6 I.D.x250mm;5µ particle size), two organic solvents (Acetonitrile and Methanol), two buffers (acetate and phosphate) at two different pH values (3 and 4) were tested. Initially methanol:water, acetonitrile:water, acetonitrile:acetate buffer, methanol:phosphate buffer were tried in different ratios at pH 3 and 4. Amlodipine and Atenolol eluted with the tried mobile phases, but Aspirin was retained. Then, with acetonitrile: Acetate buffer all the three 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: Acetate buffer pH 4.6 adjusted with 30% acetic acid in ratio 80:20 (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 0.8 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a reversed phase C18 column, the retention times for Aspirin, Amlodipine and Atenolol were observed to be 4.415, 3.688 and 3.001 min. min, respectively. Total time of analysis was less than 10 min. The chromatogram at 233 nm showed a complete resolution of all peaks (fig. 2)

Representative chromatograms of standard solutions (a) Standard solution of Aspirin (100 μg/ml); (b) standard solution of Atenolol (50 μg/ml); (c) standard solution of Amlodipine (25 μg/ml) and (d) a standard solution containing 100 μg/ml Aspirin, 50 μg/ml Atenolol, 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 three drugs in the range of 5-30 μg/ml for Aspirin and 2.5-15 Atenolol and 1-6 μg/ml Amlodipine. 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 Aspirin, Atenolol and Amlodipine capsules. 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 capsules. 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 three drugs.

The chromatograms were checked for the appearance of any extra peaks. It was observed that single peak for Aspirin (R_t±SD, 4.415±0.01), Atenolol (R_t±SD, 3.001±0.01) and Amlodipine (R_t±SD, 3.688±0.01) were obtained under optimized conditions, showing no interference from common capsule 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

TABLE1 Linearity Parameters for the Simultaneous Estimation of Aspirin, Atenolol and Amlodipine (N=6)

PARAMETERS	ATENOLOL	AMLODIPINE	ASPIRIN
l_max(nm)	233	233	233
Beers law limit (μg/ml)	2.5-15	1 - 6	5-30
Correlation coefficient (r)	0.999032	0.999072	0.999618
Regression equation (y=mx+c)	y= 0.772151x + 2424786	y=0.326938 x + 542963.5	y= 0.488661x + 465108.4
Slope (m)	0.772151	0.326938	0.488661
Intercept (c)	2424786	542963.5	465108.4
LOD (μg/ml)	0.1379	0.0677	0.0478
LOQ (μg/ml)	0.4180	0.2051	0.0145
Standard Error	0.001295	85052.77	0.002046

TABLE No. -2 Recovery Analysis of Formulation (Ato-Gaurd) By RP - HPLC

DRUG	PERCENTAGE	%RECOVERY	S.D	%RSD	S.E
ASPIRIN	50% 100% 150%	97.94 98.71 99.22	0.6444	0.6580	0.3720
AMLODIPINE	50% 100% 150%	100.10 99.75 99.16	0.4751	0.4746	0.2743
ATENOLOL	50% 100% 150%	100.66 99.44 101.06	1.1069	1.0996	0.6391

TABLE -3 System suitability parameters for the optimized chromatogram by RP - HPLC

PARAMETERS	ATENOLOL	AMLODIPINE		ASPIRIN
Tailing factor	1.24	1.21		1.17
Asymmetrical factor	1.23	1.34		1.22
Theoretical plates	3097	4166		7089
Capacity factor	21.507	26.660		32.112
Theoretical plate per unit length	206.19	242.05		324.70
Resolution	Between ATEN and AMLO 3.04		Between AMLO and ASPI 1.72

Figure-1 Calibration Curve of Atenolol

Figure-1 Calibration curve of Amlodipine

Figure-2 Calibration curve of Amlodipine

Figure- 3 Calibration Curve of Aspirin

Figure-4.Chromarogrm Mixture of Aspirin, Atenolol and Amlodipine .

LOD and LOQ were found to be 0.0478μg/ml and 0.0145 μg/ml for Aspirin, 0.1379 μg/ml and 0.4180 μg/ml for Atenolol and 0.0677 μg/ml and 0.2051 μg/ml for Amlodipine. In all deliberately varied conditions, the SD of retention times of Aspirin, Atenolol and Amlodipine were found to be well within the acceptable limit. The tailing factor for all the three peaks was found to be < 1.5 (Table 3). The validated method was used in the analysis of marketed conventional capsules Ato-guard with a label claim: 100 mg Aspirin, 50 mg Atenolol and 5 mg Amlodipine per capsule. 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 Aspirin, Atenolol and Amlodipine from capsules. The developed method provides good resolution between Aspirin, Atenolol 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, Indian for providing a samples of Atenolol, Aspirin and Amlodipine. The authors are also thankful to Principal and Management of Chilkur Balaji College of Pharmacy–Hyderabad for providing all necessary facilities

REFERENCES:

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Received on 05.07.2014 Accepted on 16.08.2014

Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 116-120