RP-HPLC method development and validation for simultaneous estimation of ramipril and felodipine European Journal of Chemistry 10 (2) (2019) 113-117 European Journal of Chemistry View Journal Online View Article Online RP-HPLC method development and validation for simultaneous estimation of ramipril and felodipine Elham Anwar Taha 1, Manal Mohammed Fouad 2,3,*, Ali Kamal Attia 1 and Zainab Mahmoud Yousef 1 1 National Organization for Drug Control and Research (NODCAR), Giza, 12553, Egypt dr_elhamtaha@hotmail.com (E.A.T.), alikamal1978@hotmail.com (A.K.A.), zeze.badawy@gmail.com (Z.M.Y.) 2 Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, 11651, Egypt manalfoad2000@yahoo.com (M.M.F) 3 Analytical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts, 6 October City, Giza, 12585, Egypt * Corresponding author at: Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, 11651, Egypt. Tel: +202.0122.3337669 Fax: +202.002.37603811 e-mail: manalfoad2000@yahoo.com (M.M. Fouad). 10.5155/eurjchem.10.2.113-117.1832 Received: 09 February 2019 Received in revised form: 20 April 2019 Accepted: 04 May 2019 Published online: 30 June 2019 Printed: 30 June 2019 A rapid and sensitive High Performance Liquid Chromatography (HPLC) method has been developed and validated as per ICH guideline for simultaneous determination of ramipril and felodipine binary mixture. Chromatographic separation was achieved on a Hyperchom C18 column (250 × 4.6 mm i.d., 5 μm) using an isocratic mobile phase of potassium di- hydrogen phosphate (pH = 3.4): methanol: acetonitrile in the ratio 15:15:70 (v:v:v). The flow rate was 1.5 mL/min, temperature of the column was maintained at 30 °C and detection was made at 210 nm. Linearity studies indicated that the drugs obey Beer’s law over the range of 10-80 μg/mL for ramipril and 5-80 μg/mL for felodipine. The proposed method is precise, accurate, linear and robust. The short retention time allows the analysis of a large number of samples in a short period of time and, therefore, considered to be cost-effective that can be used for routine analysis of both drugs in the pharmaceutical industry. Isocratic Ramipril Felodipine Routine analysis Pharmaceuticals Chromatography Cite this: Eur. J. Chem. 2019, 10(2), 113-117 Journal website: www.eurjchem.com 1. Introduction Today’s pharmaceutical industries are looking for new ways to cut cost and shorten time for the development of drugs while simultaneously improving the quality of their products. The high-performance liquid chromatography is a well-established reliable technique used in controlling the quality and consistency of active pharmaceutical ingredients and dosage forms, it is one of the most promising develop- ments in the area of fast chromatographic separations with its unique characteristics of high chromatographic resolution, speed, and sensitivity analysis [1]. In the present work, this technology has been applied to the method development and validation study of mixture of ramipril and felodipine. Ramipril (RAM) is (2S,3aS,6aS)-1-[(2S)-2[[(1S)-1-(ethoxy carbonyl)-3-phenylpropyl]amino]propanoyl] octahydro cyclo penta[b]pyrrole-2-carboxylic acid, used as angiotensin conver- ting enzyme inhibitor (ACE inhibitor) Figure 1 [2]. Angio- tensin-converting enzyme (ACE) inhibitors lower blood pressure by reducing peripheral vascular resistance without reflexively increasing cardiac output, heart rate, or contrac- tility [3]. These drugs block the enzyme ACE which cleaves angiotensin I to form the potent vasoconstrictor angiotensin II [4]. RAM is officially listed in British Pharmacopoeia, which describes a potentiometric titration with 0.1 M sodium hydroxide for its assay in bulk [2]. Felodipine (FLD), chemically known as ethylmethyl(4RS)- 4-(2, 3-dichlorophenyl)-2, 6-dimethyl-1,4dihydropyridine-3, dicarboxylate, used as calcium channel blocker, Figure 1 [2]. Felodipine was classified as dihydropyridines, this class of calcium channel blockers have the advantage of showing little interaction with other cardiovascular drugs, such as digoxin or warfarin, which are often used concomitantly with calcium channel blockers [4]. The drug is officially listed in British Pharmacopoeia, which describes a titration with 0.1 M cerium sulphate for its assay in bulk [2]. Ramipril in combination with felodipine are used in several antihypertension preparations. RMP was determined individually by spectrophotometric methods [5-7], HPLC [6,8- 10], HPTLC [11,12] and electrochemical methods [13-16]. On ABSTRACT RESEARCH ARTICLE KEYWORDS European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2019 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.10.2.113-117.1832 http://dx.doi.org/10.5155/eurjchem.10.2.113-117.1832 https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.2.113-117.1832&domain=pdf&date_stamp=2019-06-30 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.10.2.113-117.1832 mailto:dr_elhamtaha@hotmail.com mailto:alikamal1978@hotmail.com mailto:zeze.badawy@gmail.com mailto:manalfoad2000@yahoo.com mailto:manalfoad2000@yahoo.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.10.2.113-117.1832&domain=pdf&date_stamp=2019-06-30� 114 Taha et al. / European Journal of Chemistry 10 (2) (2019) 113-117 the other hand, FLD was determined individually by UV spectroscopy [17-20], HPLC [18,21-23], spectrofluorimetric [24], gas chromatography [25] and electrochemical methods [26,27]. Meanwhile, some spectrophotometric [28,29], HPLC [28-31] and HPTLC [32] methods were reported for the simultaneous determination of both drugs. NH O O N O O OHH H RAM N Cl Cl O O O O FLD Figure 1. Chemical structures of ramipril and felodipine. 2. Experimental 2.1. Instrumentation Chromatographic separation was performed on an Agilent 1100 HPLC (USA) with UV detector. Analysis was performed on a Hyperchom C 18 column (250 mm, 4.6 mm i.d., 5 μm) at 30 °C. Data acquisition and processing was performed using ChemStation software. An Elma S100 ultrasonic processor (Germany) was used for the degassing of the mobile phase. 2.2. Materials and reagents 2.2.1. Pure samples Ramipril (99.96%) [2] was obtained from Kahira Pharmaceuticals & Chemical Industries Co. and felodipine (98.9%) [2] was kindly supplied from Egyptian Group for Pharmaceutical Industries (EGPi Co., Egypt). 2.2.2. Market samples Triacor® (Aventis Pharma, Deutschland GmbH - Germany) labelled to contain 5 mg RMP and 5 mg FEL, purchased from local pharmacy. 2.2.3. Solvents Ortho-phosphoric acid (Sigma-Aldrich, Germany), potassium dihydrogen phosphate (ADWIC Chemicals, Egypt), methanol and acetonitrile HPLC grade (Fisher, UK). Bi-distilled water was prepared and used throughout the procedure. 2.3. Solution preparation 2.3.1. Preparation of mobile phase A mixture of phosphate buffer (pH = 3.4), methanol and acetonitrile in their appropriate ratio was prepared, degased in ultrasonic water bath for 5 minutes and filtered through 0.45 μm filter under vacuum filtration. 2.3.2. Stock solution preparation 25 mg of each of RAM and FLD were accurately transferred into separate 25 mL volumetric flasks to which about 10 mL of methanol was added, sonicated then diluted to the mark with the same solvent to get a standard solution of 1 mg/mL. 2.3.3. Tablet solution preparation Twenty Triacor® tablets were weighed separately and powdered. Amount of powdered tablet equivalent to 25 mg RMP and 25 mg FLD were weighed, transferred into 25 mL volumetric flask and treated with 10 mL methanol. Solution was sonicated for 15 min, adjusted to volume with methanol and filtered through 0.45-micron membrane filter paper. The resulting solution was further diluted with methanol to get a final concentration of 1 mg/mL RMP and FLD. 2.4. Chromatographic conditions The analysis was achieved on a Hyperchom C18 column (250 mm, 4.6 mm i.d., 5 μm). Isocratic elution was performed using a mobile phase of potassium dihydrogen phosphate (adjusted to pH = 3.4 using orthophosphoric acid): methanol: acetonitrile in the ratio 70:15:15 (v:v:v) at a flow rate of 1.5 mL/min. The detection was monitored at the wavelength of 210 nm. Analysis was performed at 30 °C column temperature and the injection volume was 10.0 μL. 2.5. Method validation The method was validated in accordance with ICH guidelines [33]. 2.5.1. Linearity Linearity of the method was studied by injecting five concentrations of the drugs in triplicate having concentration ranges from 10-80 μg/mL for RMP and 5-80 μg/mL for FLD into the HPLC system. Linear graphs were plotted by using the peak areas against concentration in μg/mL from which the correlation coefficients, slopes and y-intercepts of the calibration curves were determined. 2.5.2. Accuracy Five different concentrations (15, 30, 50, 70 and 75 μg/mL) covering the linearity range of both RAM and FLD were analysed for accuracy. The chromatograms were recor- ded and the %Recovery±S.D. was calculated from regression equations of the calibration curves. 2.5.3. Precision Three different concentrations (20, 40 and 60 µg/mL) of both RAM and FLD were analysed in triplicates within the same day for intraday and for three successive days for interday precision. The chromatograms were recorded and the percentage relative standard deviation (RSD%) was calculated from regression equations of the calibration curves. 2.5.4. Limit of quantification The limit of quantitation (LOQ) of the method was deter- mined by standard deviation of response and slope method. 2.5.5. Specificity In the present work, specificity was checked by analysing RMP with FLD in their laboratory prepared mixtures containing different ratios of the cited drugs within the linearity range. The concentration of each drug was calculated by substitution in the corresponding regression equation, from which mean % recovery can be calculated. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.113-117.1832 Taha et al. / European Journal of Chemistry 10 (2) (2019) 113-117 115 Table 1. System suitability parameters by the proposed HPLC method. Parameter Ramipril Felodipine Retention time (min) 2.522 3.87 Capacity factor (K') 3.2 4.3 Resolution 6.43 6.43 Theoretical plates 1269 13096 Table 2. Validation parameters of the proposed HPLC method for the determination of Ramipril and Felodipine. Parameter Ramipril Felodipine Linearity range (µg/mL) 10-80 5-80 LOQ (µg/mL) 2.15 1.21 Regression parameters Slope 30.67 55.30 Intercept 16.66 30.60 Correlation coefficient (r2) 0.9998 0.9992 Accuracy (R% of added standard±SD) 100.88±0.84 100.76±1.19 Precision (RSD %)* Intraday 0.43-0.83 0.21-0.96 Interday 0.95-1.44 1.03-1.34 *Average of 9 determinations. Figure 2. HPLC chromatograms of RAM and FLD using a mobile phase of phosphate buffer: methanol: acetonitrile (70:15:15, v:v:v). (a) RAM, (b) FLD and (c) RAM and FLD binary mixture. 2.5.6. Robustness To prove the reliability of the analytical method during normal usage, some small but deliberate changes were made in the analytical method (e.g., flow rate, mobile phase composition and column temperature). Changes in the chromatographic parameters were evaluated for the studies. 3. Results and discussion In the present work, HPLC method was applied to the method development and validation study of binary mixture of ramipril with felodipine. 3.1. Method development Different chromatographic conditions were experimented to achieve better efficiency of the chromatographic system. Parameters such as mobile phase composition and pH, wavelength of detection, column and diluents were optimized. Choice of retention time, tailing, theoretical plates, and run time were the major tasks while developing the method. Hyperchem C18 column (250 mm, 4.6 mm i.d., 5 μm) was used for the elution. Several solvents (water, methanol and acetonitrile) were tried and different proportions of solvents were evaluated in order to obtain suitable composition of the mobile phase. Buffers like potassium di-hydrogen phosphate, di-potassium hydrogen phosphate, and di-sodium hydrogen phosphate were tried; potassium di-hydrogen phosphate (adjusted to pH = 3.4 using ortho-phosphoric acid): methanol: acetonitrile in the ratio 70:15:15 (v:v:v) gave perfectly eluted peaks. Trials were also done using different flow rates and detection was done at different wavelengths, in which 1.5 mL/min was optimum for separating mixture at a detection wavelength of 210 nm, simultaneously. Typical chromate- grams obtained for the cited drug mixture under final optimized HPLC conditions showed retention time of 2.522, and 3.87 min for RMP and FLD, respectively (Figure 2). 3.2. Method validation The proposed method was subjected to validation process to satisfy the requirements of ICH guidelines [33]. Freshly prepared stock solutions were used to establish system suitability tests. The variation in selectivity, retention time, resolution, and theoretical plates were well within the acceptable ranges for all analytes (Table 1). The drugs concentrations and peak areas were plotted to construct the calibration curves. Good linearity was established with excellent correlations (>0.999) within the concentration range of 10-80 and 5-80 μg/mL for RMP and FLD, respectively. Regression parameters were computed and presented in Table 2. The LOQ was determined for all the analytes (Table 2). The low quantification concentrations reflected the good sensitivity of the reported procedure. The mean percentage recovery was calculated to assess the accuracy of the newly developed method. 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.113-117.1832 116 Taha et al. / European Journal of Chemistry 10 (2) (2019) 113-117 Table 3. Determination of ramipril/felodipine in their laboratory prepared mixtures by the proposed HPLC method. Lab-prepared mixture (Ratio of Ramipril:Felodipine) Recovery% Ramipril Felodipine 10:01 98.67 101.06 03:01 99.20 101.80 02:01 101.77 99.32 01:01 * 101.49 101.87 01:02 99.75 100.67 01:03 98.61 98.70 01:08 98.77 101.52 Mean±S.D 99.75±1.24 100.7±1.15 * Ratio in pharmaceutical formulation. Table 4. Robustness study for the proposed HPLC method. Parameters Changed condition %RSD Ramipril Felodipine Flow rate, 1.5 mL/ min ±10% 1.18 0.86 Mobile phase ratio, 0.01 M KH2PO4 (pH = 3.4), methanol: acetonitrile (15:15:70, v:v:v) ±2% 1.56 1.00 Column temperature, 30 °C ±5 °C 1.29 1.85 Table 5. Statistical analysis of results obtained by the proposed and reported method [32] for the determination of ramipril/felodipine in their pharmaceutical formulation. Parameters HPLC method Reported method Ramipril Felodipine Ramipril Felodipine Mean% 100.57 100.70 99.68 100.13 N 5 5 5 5 SD 0.84 0.66 1.04 1.22 Variance 0.70 0.44 1.08 1.50 t-test 1.49 0.92 - - F-test 1.55 3.41 - - The theoretical t- and F-values at p = 0.05 (1.860) and (6.39), respectively [34]. The mean recoveries were from 100.76 to 100.88 %±0.84- 1.19 for the added drugs (Table 2), representing good accuracy of the method. Intraday and interday precision were undertaken to determine the reproducibility of the process. The % RSD values for the inter-day and intra-day measurements were ranging from 0.21-1.44%. The results listed in Table 2 showed that the proposed procedure is precise. Specificity was determined by applying the proposed method to laboratory prepared mixtures containing different ratios of each of the two drugs in the two mixtures. Good recoveries for the studied drugs in both mixtures were obtained and results were presented in Table 3. The robustness of the suggested method was confirmed by performing the analysis with modifications to the flow rate of the mobile phase (±10%), mobile phase composition (±2%) and column temperature (±5 °C). The results showed in Table 4 declares that slight modifications did not affect the resolution and tailing factor, indicating good robustness of the HPLC method. Table 5 showed statistical comparisons of the results obtained by the proposed method and reported method [32]. The calculated t- and F-values were less than the theoretical ones indicating that there was no significant difference between the proposed and the reported methods with respect to accuracy and precision. 4. Conclusion A newly developed and validated HPLC method for simultaneous analysis of RAM and FLD in pharmaceutical preparations was very simple, rapid, accurate, and precise. The method was successfully applied for determination of RAM and FLD in their pharmaceutical tablet formulation. Hence, this method can be conveniently used for routine quality control analysis of RAM and FLD in their pharmaceutical formulation. The validation studies as per ICH guideline in accordance to linearity, accuracy, precision, LOD, LOQ and robustness proved suitability of the method for the intended use. Also, the non-interference of additives and excipients makes it suitable for determination of the studied drugs in bulk and in their combined dosage forms. Acknowledgements The authors would like to express their gratitude to National Organization for Drug Control and Research (NODCAR, Egypt) for providing instruments and chemicals. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Elham Anwar Taha http://orcid.org/0000-0002-8588-8242 Manal Mohammed Fouad http://orcid.org/0000-0002-7991-0706 Ali Kamal Attia http://orcid.org/0000-0002-9833-3414 Zainab Mahmoud Yousef http://orcid.org/0000-0002-9717-7798 References [1]. Raja, A. P.; Venkateshwar, R. J. Asian J. Pharm. Clin. Res. 2013, 6(3), 178-181. [2]. British Pharmacopoeia. 9th edition, The Council of Europe: 2018; Vol. I 768-771& II 1003-1005. [3]. Brunton, L.L.; Goodman and Gilman’s., 2011. The Pharmacological Basis of Therapeutics, The McGraw-Hill Co., Inc., China. [4]. Whalen, K., Finkel, R., Panavelil, T.A., 2015. Lippincott’s Illustrated Reviews: Pharmacology, Lippincott Williams and Wilkins, Philadelphia, Baltimore, New York, London. [5]. Salama, F. M.; El-Sattar, O. I. A.; El-Aba, S. N. M.; Fuad, M. M. Al-Azhar J. Pharmaceu. 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Miller, J. N.; Miller, J. C. Statistics and Chemometrics for Analytical Chemistry. 6th edition, Pearson Education Limited: Harlow, England, 2010. Copyright © 2019 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). 2019 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.10.2.113-117.1832 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Instrumentation 2.2. Materials and reagents 2.2.1. Pure samples 2.2.2. Market samples 2.2.3. Solvents 2.3. Solution preparation 2.3.1. Preparation of mobile phase 2.3.2. Stock solution preparation 2.3.3. Tablet solution preparation 2.4. Chromatographic conditions 2.5. Method validation 2.5.1. Linearity 2.5.2. Accuracy 2.5.3. Precision 2.5.4. Limit of quantification 2.5.5. Specificity 2.5.6. Robustness 3. Results and discussion 3.1. Method development 3.2. Method validation 4. Conclusion Acknowledgements Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: