INTRODUCTION:

Ramipril is (2S, 3aS, 6aS)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}propanoyl]-octahydrocyclopenta[b]pyrrole-2-carboxylic acid, is a long acting ACE inhibitor and Amlodipine Besylate is 3-Ethyl 5-methyl (±)-2-[(2-aminoethoxy)methyl]-4-(o-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, monobenzenesulfonate, is a calcium channel antagonist.^1,2,3

There are many analytical methods like UV, HPLC, HPTLC available for the quantization of Ramipril and Amlodipine Besylate individually as well as with other combinations.^4-13 But HPLC method for combination of Ramipril And Amlodipine Besylate was not reported. The purpose of this study was the development of a simple isocratic RP-HPLC method with ultraviolet detection for Ramipril and Amlodipine Besylate assay in tablets and validates the same as per the ICH guidelines.

MATERIAL AND METHODS:

HPLC grade acetonitrile, methanol and reagent grade hydrochloric acid, ortho -phosphoric acid, sodium lauryl sulphate and triethyl amine were purchased from Merck Ltd. Distilled water obtained from Merck Ltd. was used to prepare the mobile phase and standard solutions. The tablets containing combination of Ramipril (10mg) and Amlodipine Besylate (10mg) were procured from market.

The development and validation of the assay was performed on a HPLC system consisting of thermo separation quaternary gradient with UV detector using Data Ace software.

The analytical column used to achieve chromatographic separation was Xterra C18 column, 5 μ, 150 mm x 4.6 mm id.

For mobile phase, Buffer was made using 1 gm sodium lauryl sulfate added to 1000ml ultrapure water. 7 ml of triethyl amine was added and pH was adjusted to 2.5 by ortho-phosphoric acid. The final composition of mobile phase was Buffer: Acetonitrile: Methanol (45:16.5:38.5) V/V.

Injection volume was 10µl with flow rate of 1.5 ml/minute by keeping column temperature ambient. Scans for the drugs were carried on the UV detector for the elucidation of the lambda max of the drug. Two wavelengths were selected for analysis at 210 and 237nm, which are, reported lambda max of Ramipril and Amlodipine Besylate. On overlay it was observed that 210nm is the ideal wavelength for the method.

For diluents, 200 ml of 0.1 N Hydrochloric acid was mixed with 800ml of methanol and sonicated for 10 minutes with degassing.

Fig.1: Structure of Ramipril

Fig.2: Structure of Amlodipine Besylate

Linearity curves were constructed by plotting Concentrations against Area and regression equations were computed for both the drugs. The plots for Ramipril and Amlodipine Besylate were found to be linear in the range of 70 ppm to 420 ppm for Amlodipine Besylate and 50 ppm to 300 ppm for Ramipril with correlation coefficient 1 for Ramipril as well as Amlodipine Besylate.

Ten tablets of 10 mg strength were weighed and taken into 500 ml volumetric flask. To this about 250 ml of diluent was added and flask were shaken to disintegrate the tablets and sonicated for 10 minutes and finally volume was made with diluent. This solution was centrifuged at 3000 RPM for 10 minutes. The above solution was then filtered through 0.45 μ PVDA filter.

Analysis of formulations:

Commercial formulation of Ramipril and Amlodipine Besylate (Ramipril 10mg & Amlodipine Besylate 10mg) were selected for analysis. Sample preparations were done as per the procedure given for pure samples of Ramipril and Amlodipine Besylate and analyzed. Analysis was done with six replicates and injected into system and data were observed.

Accuracy studies:

Accuracy of proposed method was established by recovery experiment. This study was performed by addition of known amount of standard drug solution of Ramipril and Amlodipine Besylate to preanalysed tablet solutions at three different concentration levels (100 μg/ml-300 μg/ml). Resulting solution was reanalyzed by proposed method. Result of recovery studies are Table 1 for Ramipril and table 2 for Amlodipine Besylate.

Table 1: Accuracy for Ramipril

Sr. No.	Amount of Drug added (µg/ml)	*%Recovery± SD**	%RSD
1	100	100.53 ± 0.78	0.78
2	200	99.82 ± 0.34	0.35
3	300	99.51 ± 0.62	0.63

*mean of three estimation at each level.

Table 2: Accuracy for Amlodipine Besylate

Sr. No.	Amount of Drug added (µg/ml)	*%Recovery± SD**	%RSD
1	100	99.71 ± 0.71	0.71
2	200	99.42 ± 0.12	0.12
3	300	99.51 ± 0.72	0.73

*mean of three estimation at each level.

System suitability

For chromatographic separation following system suitability tests were done: Tailing factor, resolution, asymmetry and theoretical plates. The system suitability method acceptance criteria set in each validation run were: Resolution > 2.0, tailing factor ≤ 2.0, asymmetry ≤ 2.0 and theoretical plates > 2000. In all the analyte peak area for two consecutive injections was < 2.0%.

Fig.3: Chromatogram of Standard Preparation

Fig 4: Chromatogram of Test Preparation

Table 3: Linearity of Amlodipine Besylate

Level No.	Concentration (ppm)	Peak Area	Coefficient of Regression	Constant of Regression	Correlation Coefficient
1	70	718220	1041	12479	1
2	140	1439758
3	210	2178358
4	280	2887774
5	350	3633717
6	420	4356294

Table 4: Linearity of Ramipril

Level No.	Concentration (ppm)	Peak Area	Coefficient of Regression	Constant of Regression	Correlation Coefficient
1	50	926435	18769	18136	1
2	100	1852166
3	150	2806386
4	200	3717446
5	250	4678056
6	300	5617687

Linearity and Range:

A study was conducted to demonstrate the linearity of detector response for Ramipril and Amlodipine Besylate with respect to concentrations. Six solutions were prepared from 25% to 150% of the target concentration. Linearity of Detector Response was established by plotting a graph of peak area versus concentration and correlation of co-efficient was determined.

For Ramipril, linearity curve was obtained using 50-300 µg/ml concentration and for Amlodipine Besylate, linearity curve was obtained using 70-420 µg/ml concentration.

Fig 3: Linearity Plot for Amlodipine Besylate

Fig 4: Linearity Plot for Ramiril

Precision:

Precision of test method is demonstrated by analyzing six samples prepared as per test method for 10/10 mg strength tablets. Assay values and RSD of assay results were calculated. The assay method precision acceptance criteria set in the validation was R.S.D ≤ 2.0%.

The results are summarized as follows:

Table 5: Precision

Preparation No.	% Assay
Preparation No.	Ramipril	Amlodipine Besylate
1	99.2	98.3
2	99.4	98.5
3	99.3	98.3
4	99.6	98.4
5	99.7	98.7
6	99.6	98.7
MEAN	99.4	98.4
% RSD	0.1	0.1

Robustness

Typical variations in high performance liquid chromatography conditions were used to evaluate the robustness of the assay method. In this study, the chromatographic parameters monitored were retention time, area, capacity factor, tailing factor and theoretical plates. The robustness acceptance criteria were found to be in range.

In Robustness, different parameters like filter validation, flow rate variation, pH variation and temperature variation were studied. During these studies all system suitability parameters were found to be in range.

RESULT AND DISCUSSION:

All the system suitability parameters were optimized to obtain best resolution between multi-component formulations. Xterra column (150mm X 4.60mm) having particle size 5 μ and bonded phase octadecylsilane (C-18) were optimized with following conditions.

Chromatographic Condition For assay method:

Table 6: Chromatographic Condition For assay method

Equipment	Liquid chromatograph equipped with UV detector
Column	Xterra C-18 (150x4.6mm, 5 µm)
Column temperature	Ambient
Flow rate	1.5 ml/minute
Detection Wavelength	210 nm
Injection volume	10 µl
Run time	5 minutes
Mobile Phase	Buffer: Acetonitrile: Methanol (45:16.5:38.5)
Approx. RT	Ramipril: 3.14 min., Amlodipine Besylate: 3.82 min.

Linearity was observed by linear regression equation method for Ramipril and Amlodipine Besylate in different concentration range. The correlation coefficient of these drugs was formed to be 1.0, indicating good linearity. The developed HPLC method was validated for simultaneous estimation of Ramipril and Amlodipine Besylate using linearity, range, accuracy and precision. The %RSD for all parameters was found to be less than two, which indicates the validity of method and assay results obtained by this method were in fair agreement. Analysis of the results shown that the results obtained by various methods do not differ significantly and are quite comparable to the label claim of the formulations.

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Received on 29.04.2011 Modified on 12.10.2011

Asian J. Research Chem. 4(12): Dec., 2011; Page 1829-1832