INTRODUCTION:

Ramipril (Fig. 1) is an ACE inhibitor, indicated for the treatment of hypertension¹. It may be used alone or in combination with other antihypertensive agents. Chemically it is (2S,3aS,6aS)-1[(S)-N-[(S)-1-Carboxy-3-phenylpropyl]alanyl]octahydrocyclopenta [b]pyrrole-2-carboxylic acid, 1-ethyl ester. Ramipril lowers blood pressure by inhibiting angiotensin-converting enzyme (ACE) and is believed to be primarily suppression of the renin-angiotensin-aldosterone system².

Felodipine (Fig. 2) is a calcium channel blocker, indicated for the treatment of hypertension and angina pectoris³. Chemically it is ± ethyl methyl 4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate. Felodipine reversibly competes for dihydropyridine binding sites, blocks voltage-dependent Ca⁺⁺ currents⁴.

Fig. 1: Molecular structure of Ramipril

Fig. 2: Molecular structure of Felodipine

The approach of combination therapy may be theoretically favored by the fact that multiple factors contribute to the hypertension and achieving control of BP with single agent that acts through one particular mechanism may be unrealistic. Combining the second agent may lead to better control, acting by complimentary mechanism⁵.

Literature survey reveals that few spectrophotometric⁶, spectrofluorimetric, HPLC⁷ and LC-MS⁸ methods were reported for simultaneous estimation of Ramipril and Felodipine in combination dosage form. Therefore, an attempt has been made to develop an accurate, rapid and reproducible RP-HPLC⁹ method for simultaneous determination of Ramipril and Felodipine in tablet dosage form and validate it, in accordance with ICH¹⁰ guidelines.

MATERIALS AND METHODS:

Chromatographic conditions:

Separation was performed with Waters HPLC equipped with a pump 2695, auto sampler and UV detector. Empower2 software was applied for data collecting and processing. The separation was achieved on a Hypersil BDS C18 column (150 x 4.6 mm, 5 µ). The mobile phase consisted of phosphate buffer pH 5.5: acetonitrile in the ratio of 40: 60 V/V. The flow rate was 1.0 mL/min and UV detection was performed at 243 nm. The mobile phase was shaken on an ultrasonic bath for 30 min. The resulting transparent mobile phase was filtered through a 0.45 µ membrane filter. The injection volume was 20 µL and all the experiments were performed at temperature 30^◦C. The run time was set at 10 min.

Chemicals and reagents:

Pharmaceutical grade of Ramipril and Felodipine were kindly supplied as gift samples by Chandra Labs, Hyderabad, India. Commercially available TRIASYN tablets were purchased from local market. Tablets claimed to contain 5 mg of Ramipril and 5 mg of Felodipine have been utilized in the present work. Potassium dihydrogen phosphate and orthophosphoric acid of AR Grade were obtained from S.D. Fine Chemicals Ltd., Mumbai, India. HPLC grade acetonitrile was purchased from E. Merck (India) Ltd., Mumbai, India. HPLC grade water obtained from Milli Q water purification system was used throughout the study.

Preparation of standard solution:

Accurately weigh and transfer 5 mg of Ramipril and 5 mg of Felodipine working standard into 25 mL volumetric flask, add about 15 mL of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent (stock solution), from this stock solution pipette out 10 mL into 100 mL volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 µ filter.

Preparation of sample solution:

Twenty tablets were accurately weighed, their mean weight was determined and they were mixed and finally powdered. Transfer the sample equivalent to 5 mg of Ramipril and 5 mg of Felodipine into a 25 mL volumetric flask. Add about 15 mL of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 µ filter, from this stock solution pipette out 10 mL into 100 mL volumetric flask and dilute upto the mark with diluent. Mix well and filter through 0.45 µ filter.

Method validation:

The method was validated in accordance with ICH guidelines. The parameters assessed were linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), precision, reproducibility and robustness etc.

Linearity:

Several aliquots of standard solutions of Ramipril and Felodipine were taken in different 10 mL volumetric flasks and diluted up to the mark with mobile phase such that the final concentrations were 5 to 30 µg/mL for both Ramipril and Felodipine. Evaluation of the two drugs was performed with UV detector at 243 nm, peak area was recorded for all the peaks. The correlation coefficient values were R²=1 for both Ramipril and Felodipine. The results show that an excellent correlation exists between peak area and concentration of drugs within the concentration range indicated.

Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed HPLC method. The LOD for Ramipril and Felodipine were found to be 2.86 µg/mL and 2.21 µg/mL respectively. The LOQ for Ramipril and Felodipine were found to be 8.66 µg/mL and 6.71 µg/mL respectively.

Accuracy:

The accuracy of the method was assessed by recovery studies of Ramipril and Felodipine in the dosage form at three concentration levels. A fixed amount of preanalyzed sample was taken and standard drug was added at 50%, 100% and 150% levels. Each level was repeated three times. The contents of Ramipril and Felodipine per tablet were calculated. The % mean recoveries of Ramipril and Felodipine were 99.11% and 98.69% that shows there is no interference from excipients and the lower values of RSD of assay indicate the method is accurate.

Precision:

The precision was determined for both the drugs Ramipril and Felodipine in terms of intra-day and inter-day precision. For intra-day precision evaluation, a standard solution of fixed concentration was injected at various time intervals and %RSD for Ramipril and Felodipine were 0.08% and 0.21% respectively (limit %RSD < 2.0%). In addition, the inter-day precision was studied by injecting the same concentration of standard solution on consecutive days and the %RSD for Ramipril and Felodipine were 0.43% and 0.33% respectively (limit %RSD < 2.0%).

Ruggedness:

The ruggedness of the method was determined by carrying out the experiment on different instruments by different operators using different columns of similar types, which demonstrated that the developed HPLC method is rugged.

Robustness:

Robustness of the method was determined by making slight changes in the chromatographic conditions like mobile phase composition and flow rate. It was observed that there were no marked changes in the chromatograms, which demonstrated that the HPLC method so developed is robust.

Specificity:

Specificity is the ability of the analytical method to measure the analyte response in the presence of interferences including degradation products and related substances. No interference from any of the excipients was found at retention times of the examined drugs. These results demonstrate the absence of interference from other materials in the pharmaceutical formulations and therefore confirm the specificity of the proposed method.

Assay:

20 µL of each standard and sample solution were injected and from the peak area of Ramipril and Felodipine, amount of each drug in samples were computed. The result of assay undertaken yielded 99.46% and 100.09% of label claim of Ramipril and Felodipine respectively.

Table 1: Optimized chromatographic conditions of Ramipril and Felodipine

Parameter	Condition
Mobile phase	Phosphate buffer: acetonitrile (40: 60, V/V)
pH	5.5
Diluent	Mobile phase
Column	Hypersil BDS C18 column (150 x4.6 mm, 5μ)
Column temperature	30⁰C
Wave length	243 nm
Injection volume	20 µL
Flow rate	1.0 mL/min
Run time	10 min

Table 2: Linearity results of Ramipril and Felodipine

Concentration of Ramipril (μg/mL)	Area	Concentration of Felodipine (μg/mL)	Area
5	1034737	5	1089724
10	2071491	10	2184028
15	3091704	15	3256058
20	4102799	20	4319164
25	5112916	25	5379753
30	6132429	30	6452500

RESULTS AND DISCUSSION:

The HPLC procedure was optimized with a view to develop an accurate assay method for simultaneous estimation of Ramipril and Felodipine in tablet dosage form using Hypersil BDS C18 column (150 x 4.6 mm, 5 μ) in isocratic mode with mobile phase composition of phosphate buffer pH 5.5: acetonitrile in the ratio of 40: 60 V/V.

Fig. 3: Linearity curve of Ramipril

Fig. 4: Linearity curve of Felodipine

The use of phosphate buffer pH 5.5: acetonitrile in the ratio of 40: 60 V/V resulted in peak with good shape and resolution. The flow rate was 1.0 mL/min and both the components were measured with UV detector at 243 nm. The results of optimized HPLC conditions were shown in Table 1. The method was linear in the range of 5 to 30 µg/mL for both Ramipril and Felodipine with correlation coefficient of 1 for both Ramipril and Felodipine. Linear regression data for Ramipril and Felodipine were given in Table 2 and the linearity curves for Ramipril and Felodipine were shown in Fig. 3 and Fig. 4.

The % mean recoveries were found to be 99.11% for Ramipril and 98.69% for Felodipine, which indicate the method is accurate. The accuracy results were shown in Table 3. The %RSD for intra-day precision and inter-day precision for Ramipril were found to be 0.08 and 0.21 and for Felodipine were found to be 0.43 and 0.33 respectively, which indicate the method is precise. The precision results were shown in Table 4 and Table 5.

Table 4: Intra-day precision data of proposed method

S. No.	Ramipril	Felodipine
1	4080549	4303452
2	4081216	4303036
3	4087670	4302876
4	4079065	4303132
5	4079768	4303767
6	4080690	4281387
Average	4081493	4299608
SD	3118.9361	8932.279
%RSD	0.08	0.21

Table 5: Inter-day precision data of proposed method

S. No.	Ramipril	Felodipine
1	4081926	4306246
2	4082119	4304373
3	4095831	4315609
4	4103087	4323783
5	4117591	4336791
6	4124223	4337159
Average	4100796.17	4320660
SD	17695.7819	14427.21
%RSD	0.43	0.33

The retention time of Ramipril and Felodipine was 2.844 min and 5.727 min respectively. The number of theoretical plates calculated was 2756 for Ramipril and 4232 for Felodipine and symmetry factor was 1.09 for Ramipril and 1.10 for Felodipine, which indicates efficient performance of the column. The LOD for Ramipril and Felodipine were found to be 2.86 µg/mL and 2.21 µg/mL respectively. The LOQ for Ramipril and Felodipine were found to be 8.66 µg/mL and 6.71 µg/mL respectively, which indicate the sensitivity of the method. The summary of system suitability parameters and validation parameters were shown in Table 6. Validated method was applied for the determination of Ramipril and Felodipine in commercial formulations. The % assay was found to be 99.46% and 100.09% for Ramipril and Felodipine respectively and the assay results were shown in Table 7.

Table 6: System suitability parameters of proposed method

Parameters	Ramipril	Felodipine
Linearity (µg/mL)	5-30	5-30
Theoretical plates	2756	4232
Symmetry factor	1.09	1.10
Resolution	0.00	10.09
Retention time (min)	2.844	5.727
LOD (µg/mL)	2.86	2.21
LOQ (µg/mL)	8.66	6.71

Typical chromatogram of standard showing the separation of the drugs Ramipril and Felodipine was shown in Fig. 5. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulations did not interfere with the estimation of the drug by the proposed HPLC method.

CONCLUSION:

The developed HPLC method is simple, specific, accurate and precise for the simultaneous estimation of Ramipril and Felodipine in tablet dosage form. The developed method provides good resolution between Ramipril and Felodipine. It was successfully validated in terms of linearity, accuracy, precision, specificity, robustness, LOD, LOQ and system suitability in accordance with ICH guidelines. Thus the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs in combinations.

REFERENCES:

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2. J.E. Frampton and D.H. Peters, Ramipril, an updated review of its therapeutic use in essential hypertension and heart failure, Drugs, 1995, 49(3), 440-466.

3. P.H. Dunselman and B. Edgar, Felodipine clinical pharmacokinetics, Clin. Pharmacokinet., 1991, 21(6), 418-430.

4. M.B. El-Hawary, M.T. Khayall and Z. Isaak, Hand Book of Pharmacology, The Scientific Book Center, S.O.P. Press, Cairo, 1985.

5. K. Sanjay, K. Bharti and A. Navneet, Combination therapy in hypertension: an update, Diabetol. Metab. Syndr., 2010, 2, 44.

6. A.E. Fawzy, E.M. Mohamad, H.M. Hussan, M.S. Ghassan and M.S. Nada, Comparative spectrophotometric, spectrofluorometric and high-performance liquid chromatographic study for the quantitative determination of the binary mixture Felodipine and Ramipril in pharmaceutical formulations, Anal. Lett., 2008, 41(4), 853-870.

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Received on 05.08.2013 Modified on 30.08.2013

Asian J. Research Chem. 6(11): November 2013; Page 1018-1022

Spiked level of drug	Amount added (mg)		Amount found (mg)		% Recovery
Spiked level of drug	Ramipril	Felodipine	Ramipril	Felodipine	Ramipril	Felodipine
50%	50	50	49.44	49.33	98.87	98.67
100%	100	100	99.03	99.09	99.03	99.09
150%	150	150	149.14	147.47	99.43	98.32