Isocratic Reverse Phase High Performance Liquid Chromatographic Estimation of Ramipril and Amlodipine in Pharmaceutical Dosage Form

A new simple, rapid, specific, accurate, precise and novel reverse phase High Performance Liquid Chromatography (RP-HPLC) method has been developed for the simultaneous estimation of Ramipril and amlodipine besylate in the combined pharmaceutical dosage form. The chromatographic separation for ramipril and amlodipine besylate were achieved with mobile phase containing mixed phosphate buffer (0.02 M potassium di-hydrogen orthophosphate and 0.002 M di-potassium hydrogen phosphate anhydrous, pH 6.8) and acetonitrile (60:40 % v/v), phenomenex C18 column (150 × 4.6mm i.d, particle size of 5μ) at 50 C and UV detection at 237 nm. The compounds were eluted in the isocratic mode at a flow rate of 1 ml min-1. The retention times of Ramipril 2.13 ± 0.09 min and amlodipine besylate at 5.18 ± 0.12 min. The above method was validated in terms of linearity, accuracy, precision, LOD, LOQ etc. in accordance with ICH guidelines. Keyword: Ramipril, Amlodipine Besylate, RP-HPLC, Validation. INTRODUCTION Ramipril, chemically (2-[N-[(S)-1-(ethoxycarbonyl)-3phenylpropyl)] L-alanyl]-(1S, 3S, 5S)-2-azabicyclo [3-3-0] octane carboxylic acid (Fig.1), is an angiotensin-converting enzyme (ACE) inhibitor [1]. It acts on the renin–angiotensin aldosterone system by inhibiting the conversion of the inactive angiotensin I to the highly potent vasoconstrictor, angiotensin II, and also reduces the degradation of bradykinin. Amlodipine besylate (AMLO), chemically, Journal of Advanced Pharmacy Education & Research 2 (3) 137-145 (2012) ISSN 2249-3379 138 (2-[(2aminoethoxy) methyl]-4-(2-chlorophenyl)-1, 4-dihydro6-methyl-3, 5pyridinedicarboxylic acid 3-ethyl, 5-methyl ester) [2] (Fig.2). It is an anti-hypertensive and an antianginal agent in the form of the besylate salt, Amlodipine besylate. It is not official in any Pharmacopoeia. Literature survey reveals that there are several analytical methods for the estimation of amlodipine besylate and ramipril individually or in combination with other drugs [3-10]. Although the combination use of amlodipine besylate and ramipril is continuously increasing, there is no simple and economical RP-HPLC method for the determination of these drugs in the combined pharmaceutical dosage form. The purpose of present study is to investigate a simple, precise, accurate and economic RP-HPLC method in simultaneous determination of amlodipine besylate and ramipril in the combined pharmaceutical dosage form. Fig. 1. Structure of Ramipril Fig. 2. Structure of Amlodipine MATERIALS AND METHODS Ramipril and Amlodipine besylate gift samples were obtained from Taj Pharmaceuticals Ltd. India. Acetonitrile (HPLC grade) and dipotassium hydrogen phosphate (AR grade) were purchased from Merck Ltd, India. Water for HPLC was obtained from Qualigen fine chemicals, Mumbai, India. Analytical reagent potassium dihydrogen orthophosphate (AR grade) was obtained from Rankem Pvt. Ltd, Mumbai. Chromatographic separation was performed on Waters® HPLC system equipped with Waters 2489 UV/Visible detector and Empower software. Phenomenex C18 Column (150 × 4.6 mm i.d, particle size of 5μ) and constant flow pump and Auto injector with 20 μL loop were used. The composition of the mobile phase was in the ratio of mixed phosphate buffer (0.02 M potassium di-hydrogen orthophosphate and 0.002 M di-potassium hydrogen phosphate anhydrous, pH 6.8) and acetonitrile (60:40 % v/v) and was delivered at a flow rate of 1.0 ml min-1. The mobile Journal of Advanced Pharmacy Education & Research 2 (3) 137-145 (2012) ISSN 2249-3379 139 phase was filtered through a 0.45 μ membrane filter and sonicated for 15 min. Analysis was performed at 50 C temperature. Preparation of standard solutions 5 mg of Ramipril and 5 mg of amlodipine besylate were accurately weighed and dissolved in 50 ml of mobile phase to obtain standard stock solution of 50 μg mL-1 and 100 μg mL-1 respectively. Further working standard solutions of Ramipril and amlodipine besylate, 1.25 -7.5 μg mL-1 and 2.5–15 μg mL-1 respectively, were prepared by suitable dilution of the stock solution with mobile phase. A typical chromatogram obtained from the analysis of drugs using the developed method was shown in Fig. 3. Fig.3: A typical chromatogram of standard Ramipril (2.5 μg ml-1) and amlodipine besylate (5 μg ml-1) measured at 237 nm Preparation of capsules for assay For analysis of commercial formulations, 20 capsules were weighed and average weight was calculated. Weight equivalent to 2.5 mg and 5 mg of Ramipril and amlodipine besylate was taken and transferred into 100 ml volumetric flask and dissolved with mobile phase, filtered through a whatman filter paper. This solution was further diluted stepwise with mobile phase to get the concentration within the linearity range. Journal of Advanced Pharmacy Education & Research 2 (3) 137-145 (2012) ISSN 2249-3379 140 RESULTS AND DISCUSSION Phenomenex c18 (150 × 4.6 mm i.d, particle size of 5μ), column maintained at ambient temperature 50 C was used for the separation and the method was validated for the estimation of Ramipril and amlodipine besylate in capsules. The composition, pH and the flow rate of the mobile phase were optimized. A mobile phase consisting of mixture of 0.02 M potassium dihydrogen phosphate and 0.002 M dipotassium hydrogen phosphate (pH 6.8): acetonitrile (60:40 % v/v) set at a flow rate of 1 ml min-1 was selected for use of further studies after several preliminary investigatory chromatographic runs. Under the described experimental conditions, all peaks were well defined and free from tailing. The effects of small deliberate changes in the mobile phase composition, and flow rate were evaluated as a part of testing for method robustness. Method validation: The proposed method was validated as per International Conference on Harmonization (ICH) guidelines. Linearity and Range Linearity was established by least squares linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 1.5-7.5 μg mL-1 for Ramipril, 2.5-15 μg mL-1 for amlodipine besylate. Peak areas were plotted versus respective concentrations and linear regression analysis was performed on the resultant curves. Correlation coefficient values were found to be 0.999 and 0.999 for Ramipril and Amlodipine besylate respectively, (Fig. 4 and Fig. 5). The results are given in Table 1. Fig. 4: Calibration curve of Ramipril Journal of Advanced Pharmacy Education & Research 2 (3) 137-145 (2012) ISSN 2249-3379 141 Fig. 5: Calibration curve of Amlodipine besylate