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Development and validation of RP-HPLC and UV-spectrophotometric methods for rapid simultaneous estimation of amlodipine and benazepril in pure and fixed dose combination
⁎Corresponding author. Tel.: +91 79 27439375, 27416409, mobile: +91 9099069333. mtbitat@gmail.com (Manju Misra)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
High-performance liquid chromatographic (HPLC) and UV spectrophotometric methods were developed and validated for the quantitative determination of amlodipine besylate (AM) and benazepril hydrochloride (BZ). Different analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines. The RP-HPLC method was developed by the isocratic technique on a reversed-phase Shodex C-18 5e column. The retention time for AM and BZ was 4.43 min and 5.70 min respectively. The UV spectrophotometric determinations were performed at 237 nm and 366 nm for AM and at 237 nm for BZ. Correlation between absorbance of AM at 237 nm and 366 nm was established and based on developed correlation equation estimation of BZ at 237 nm was carried out. The linearity of the calibration curves for each analyte in the desired concentration range was good (r2 > 0.999) by both the HPLC and UV methods. The method showed good reproducibility and recovery with percent relative standard deviation less than 5%. Moreover, the accuracy and precision obtained with HPLC co-related well with the UV method which implied that UV spectroscopy can be a cheap, reliable and less time consuming alternative for chromatographic analysis. The proposed methods are highly sensitive, precise and accurate and hence successfully applied for determining the assay and in vitro dissolution of a marketed formulation.
Keywords
Amlodipine besylate (AM)
Benazepril hydrochloride (BZ)
Correlation equation
RP-HPLC
1 Introduction
Amlodipine besylate (Fig. 1a), chemically 2-[(2-amino ethoxy)-methyl]-4-(2-cholophenyl)-1,4-dihydro-6-methyl-3,5-pyridine dicarboxylic acid 3-ethyl-5-methyl ester, benzosulfonate, is a 1,4-dihydropyridine calcium channel blocker which blocks the calcium entry by preventing the opening of voltage gated L-type and T-type Ca-channels. It mainly affects heart and smooth muscles inhibiting calcium entry caused by depolarization in these tissues (Joel and Alfred, 2001; Williams AD, 2002). Benazepril HCl (Fig. 1b), chemically (3S)-3-[(1S)-1-ethoxycarbonyl-3 phenylpropylamino]-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepin-1-yl] acetic acid hydrochloride, is an antihypertensive drug, which belongs to the group of angiotensin convertase inhibitors. It acts on the renin–angiotensin–aldosterone system by inhibition of the conversion of the inactive angiotensin I to the highly potent vasoconstrictor angiotensin II. It also reduces the degradation of bradykinin. It is applied in pharmacotherapy as a first choice drug for treatment of arterial hypertension, ischemic heart disease, hypertrophy of the left heart ventricle and post infarction heart dysfunction (Healey et al., 2005; Joel and Alfred, 2001; Williams AD, 2002). Combination of both drugs into fixed dose (FDCs) has been an essential constituent in treatment of hypertension.
Literature survey reveals stability indicating UV spectrophotometric (Gupta et al., 2010; Ramesh and Ramakrishna, 2011; Shama et al., 2009), RP-HPLC (Çelebier M et al., 2010; Chitlange et al., 2008; Kamble et al., 2010; Kasawar and Farooqui, 2009; M Sarat and Rambabu, 2012; Shaalan et al., 2017), HPLC in human serum (Bahrami and Mirzaeei, 2004), HPTLC (Chabukswar et al., 2010; Kamble et al., 2010; Meyyanathan and Suresh, 2005) and LC–MS method (Pilli et al.) for the estimation of AM alone and in combinations. Several analytical methods have been reported for the quantitative determination of BZ such as high performance liquid chromatography (HPLC) (Naidu et al., 2005), HPTLC-densitometry (Meyyanathan and Suresh, 2005) and LC–MS (Pilli et al., 2011). A major problem associated with simultaneous estimation of AM and BZ is that AM has λmax at 237 nm and it also shows absorbance at 366 nm and BZ has λmax at 237 nm. As AM and BZ are having common λmax (237 nm), direct estimation of BZ is not possible. So for the estimation of AM and BZ simultaneously, a new analytical method needs to be established. The spectrophotometric method based on simultaneous equation and the absorption ratio method for simultaneous estimation of both drugs (Pawar et al., 2011) is reported. Several RP-HPLC methods reported for simultaneous estimation of AM and BZ involve estimation of drug content at pH 3 which can reduce life span of column (Kasawar and Farooqui, 2009; M Sarat and Rambabu, 2012; Naidu et al., 2005). In the present work we have developed a simple UV method for rapid simultaneous estimation of AM and BZ using a correlation equation; obtained from absorbance maxima of both AM and BZ. The values obtained from the developed equation were also verified using HPLC. The existing HPLC method was further modified to reduce the retention time. Both these methods are used to calculate drug content in marketed formulation and in dissolution media.
2 Materials and methods
2.1 Reagent and chemicals
AM (Torrent Pharma Ltd.) and BZ (Astron Research Ltd.) were received as gift sample. Marketed formulation (AMACE-BP tablet, Madras Pharmaceuticals Ltd., India), containing 5 mg of AM and 10 mg of BZ was procured from local market. HPLC grade acetonitrile and purified grade potassium di-hydrogen phosphate were purchased from Fischer chemicals Ltd., India and Merck, India respectively. All other reagents employed were of high purity analytical grade. All weighing was done on a calibrated analytical balance. Calibrated glass wares were used throughout the work. Double distilled water and Mili-Q water were used in the UV method and RP-HPLC method respectively.
2.2 RP-HPLC method
2.2.1 Instrumentation
The HPLC method was performed on a system equipped with a universal loop injector (Rheodyne 7725 i), Jasco UV-975 Intelligent UV/Vis detector and HPLC pump. The column used was Shodex-RP C18 5e column (250 × 4.6 mm, 5 μm). The mobile phase used was potassium dihydrogen phosphate buffer: Acetonitrile (55:45 v/v) and the final pH adjusted was 5.3 by using orthophosphoric acid. Injection volume was 50 μL. The flow rate was set to 1 ml/min and detection of both drugs was carried out at 237 nm by UV detector.
2.2.2 Chromatographic condition
The optimal composition of the mobile phase was determined to be potassium dihydrogen phosphate buffer pH 5.3: Acetonitrile (55:45 v/v). The mobile phase was filtered through nylon 0.22 μm membrane filter and was degassed before use (30 min). Stock solution was prepared by dissolving AM and BZ (10 mg each) that were weighed accurately and separately transferred into 100 ml volumetric flasks. Both drugs were dissolved in 25 ml of mobile phase to prepare standard stock solutions. After the immediate dissolution, the volume was made up to the mark with mobile phase. These standard stock solutions were observed to contain 100 μg/ml of AM and BZ. Appropriate volume from this solution was further diluted to get appropriate concentration levels according to the requirement. From the above stock solutions, dilutions were made in the concentration range of 1–6 μg/ml of both drugs. A volume of 50 μL of each sample was injected into column.
2.2.3 Preparation of buffer
0.02 M potassium dihydrogen phosphate buffer of pH 5.3 was used for method development. Buffer was prepared by dissolving 13.6 g of potassium dihydrogen phosphate by diluting with Mili-Q water to 1000 ml. Then stock solution was further diluted to get 0.02 M Phosphate buffer. The pH was adjusted by ortho-phosphoric acid using pH meter (Eutech Instruments, Singapore). The prepared buffer was passed through 0.22 μm membrane filter (Milipore, USA) and the same was used for mobile phase preparation.
2.2.4 Preparation of mobile phase
Mobile phase was prepared by mixing 0.02 M potassium di-hydrogen phosphate buffer (pH 5.3) and acetonitrile (HPLC grade) in 55:45 (v/v) proportions. Mixture was shaken vigorously and sonicated for 30 min prior to use.
2.2.5 Preparation of stock solutions and test solutions (AM, BZ and binary mixture)
Aqueous solution (100 μg/ml) of AM, BZ and its binary mixture was prepared by adding accurately weighed 10 mg of AM and BZ and binary mixture of both drugs in 50 ml of mobile phase, then sonicated for 10 min and diluted up to 100 ml. Series of test solutions were prepared in the concentration range of 1–6 μg/ml. by diluting appropriate volume of the stock solution (100 μg/ml) with mobile phase. The dilutions were first vortexed and then used for further analysis.
2.2.6 Preparation of calibration curve
The calibration curve was prepared by injecting concentration of 1–6 μg/ml for AM, BZ and binary mixture solutions manually in triplicate to the HPLC system at detection wavelength of 237.0 nm. Mean of n = 6 determinations was plotted as the standard curve. The calibration curve was tested by validating it with inter-day and intra-day measurements. Linearity, accuracy and precision were determined for both inter-day and intra-day measurements.
2.3 UV spectrophotometric method
2.3.1 Instrumentation
The UV method was performed on SHIMADZU double beam spectrophotometer (Model: UV-1800) with 2 nm spectral bandwidth using 10 mm matched quartz cuvettes. Data acquisition was done by using UV-probe software version 2.42. The absorption spectra of reference and test solution were carried out over the range of 200–400 nm.
2.3.2 Determination of wavelength of maximum absorbance (λmax) of AM and BZ
Wavelength of maximum absorption was determined by scanning 10 μg/ml solution of AM and BZ using UV–visible double beam spectrophotometer from 200 to 400 nm using 0.01 N HCl as blank.
2.3.3 Preparation of stock solutions and test solutions (AM, BZ and binary mixture)
Aqueous solutions (100 μg/ml) of AM, BZ and its binary mixture were prepared by adding accurately weighed 10 mg of AM and BZ and binary mixture of both drugs in 50 ml of 0.01 N HCl, then sonicated for 10 min and diluted up to 100 ml. Series of test solutions were prepared in the concentration range of 2–24 μg/ml by diluting appropriate volume of the stock solution (10 μg/ml) with 0.01 N HCl. The dilutions were first vortexed and then used for further analysis.
2.3.4 Preparation of calibration curve
The calibration curve was prepared by scanning test samples ranging from 2–24 μg/ml at 237 nm and 366 nm for AM and at 237 nm for BZ. The calibration curve was tested by validating it with inter-day and intra-day measurements. Linearity, intra-day and inter-day measurements, accuracy and precision were determined for both. Mean of n = 6 determinations was plotted as the standard curve.
2.3.5 Correlation equation method
Seven standard solutions of each drug having concentration in the range of 2–24 μg/ml were prepared in 0.01 N HCl and absorbance at 237 nm and 366 nm was measured. Mixed standards containing AM and BZ were also prepared (2–24 μg/ml) and absorbance at 237 nm and 366 nm was measured. Using these data a correlation was established between absorbance of AM at 237 nm and 366 nm. From the developed correlation equation, absorbance of AM at 237 nm was generated with the help of absorbance at 366 nm. The method validation was done on binary mixture of AM and BZ by observing absorbance of AM at 366 nm and subtracting this absorbance from total absorbance of binary mixture to obtain absorbance of BZ, which was measured at 237 nm.
2.4 Method validation
2.4.1 Linearity
The methods were validated according to International Conference on Harmonization Q2B guidelines (2005) for validation of analytical procedures in order to determine the linearity, sensitivity, precision and accuracy for each analyte. Calibration curves were generated with appropriate volumes of working standard solutions for both UV and HPLC with the range of 2–24 and 1–6 μg/ml respectively. The linearity was evaluated by the least square regression method using unweighted data.
2.4.2 Precision and accuracy
Both precision and accuracy were determined with standard quality control samples (in addition to calibration standards) prepared in triplicates at different concentration levels covering the entire linearity range. Precision is the degree of repeatability of an analytical method under normal operational conditions. The precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day) and reported as %R.S.D. for a statistically significant number of replicate measurements (Swartz and Krull, 1998). The intermediate precision was studied by comparing the assays on 3 different days and the results documented as standard deviation and %R.S.D.
Accuracy is the percent of analyte recovered by assay from a known added amount. For the measurement of accuracy data from nine determinations over three concentration levels covering the specified range were determined (Swartz and Krull, 1998).
2.4.3 Specificity
The method specificity was assessed by comparing the chromatograms (HPLC) and scans (UV) obtained from the drug and the most commonly used excipient mixture with those obtained from blank (excipient solution in water without drug). The excipients chosen are the ones used commonly in tablet formulation, which included di-calcium phosphate (DCP), lactose, starch, micro- crystalline cellulose (MCC), polyvinyl pyrrolidone (PVP), sodium starch glycolate (SSG) and magnesium stearate. The drug to excipient ratio used was similar to that in the commercial formulations.
2.4.4 LOD and LOQ
The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from background levels. The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with acceptable accuracy, precision and variability (ICH guideline Q2B, 2005). The LOD and LOQ were calculated as
2.5 Analysis of marketed tablet formulation (AMACE-BP)
Twenty tablets of marketed formulation (AMACE-BP) were weighed and grounded to obtain fine power. Accurately weighed powder sample equivalent to 5 mg of AM and 10 mg of BZ was dissolved in a 100 ml volumetric flask containing 0.01 N HCl. The solution was kept for sonication for 20 min, filtered through Whatmann filter paper No. 41. Aliquot of this solution was diluted to produce the concentration of 5 μg/ml for AM and 10 μg/ml for BZ (n = 6). The absorbance of sample solutions at 237 and 366 nm was measured and the amount of drug present in the sample solution was calculated in the same manner as that of pure mixed standard solution. The results of analysis and statistical validation for the marketed tablet formulation are reported in Table 3. The results of recovery studies conducted by the addition of different amounts of pure drugs at three different levels to a tablet solution were found to be satisfactory and are given in Table 4.
| RP-HPLC | UV-spectrophotometry | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Drug | Amt (μg/ml) | Intra-day variation (n = 6) | Inter-day variation (n = 6) | Amt (μg/ml) | Intra-day variation (n = 6) | Inter-day variation (n = 6) | ||||
| % Precision ± SD⁎ | % RSD⁎⁎ | % Precision ± SD | %RSD | % Precision ± SD | % RSD | % Precision ± SD | %RSD | |||
| A M | 2 | 98.43 ± 0.62 | 0.62 | 99.58 ± 0.44 | 0.45 | 8 | 98.93 ± 0.67 | 0.68 | 98.58 ± 0.58 | 0.59 |
| 3 | 99.33 ± 1.61 | 1.63 | 99.32 ± 1.55 | 1.56 | 12 | 98.73 ± 1.04 | 1.06 | 98.42 ± 1.24 | 1.26 | |
| 4 | 98.58 ± 0.49 | 0.49 | 98.4 ± 0.74 | 0.75 | 16 | 99.58 ± 0.43 | 0.43 | 99.4 ± 0.34 | 0.35 | |
| B Z | 2 | 98.41 ± 0.68 | 0.68 | 99.83 ± 1.67 | 1.69 | 8 | 99.41 ± 0.58 | 0.58 | 98.83 ± 0.47 | 0.48 |
| 3 | 97.87 ± 0.57 | 0.58 | 98.57 ± 1.61 | 1.63 | 12 | 98.87 ± 0.57 | 0.58 | 98.87 ± 0.61 | 0.62 | |
| 4 | 98.51 ± 0.78 | 0.69 | 98.12 ± 0.86 | 0.87 | 16 | 99.51 ± 0.68 | 0.68 | 99.32 ± 0.36 | 0.37 | |
| Method | Drug | Amt. present (μg/ml) | Amt. added (μg/ml) | Amt. found⁎ (μg/ml) | Amt. recovered⁎ (μg/ml) | % Recovery⁎ | SD | % RSD |
|---|---|---|---|---|---|---|---|---|
| HPLC method | AM | 2 | 1 | 2.993 | 0.993 | 99.3 | 1.587 | 1.591 |
| 2 | 2 | 3.967 | 1.967 | 98.35 | 1.516 | 1.525 | ||
| 2 | 3 | 4.973 | 2.973 | 99.1 | 1.512 | 1.516 | ||
| BZ | 2 | 1 | 2.987 | 0.987 | 98.7 | 1.676 | 1.685 | |
| 2 | 2 | 3.997 | 1.997 | 99.85 | 0.896 | 1.897 | ||
| 2 | 3 | 4.967 | 2.967 | 98.9 | 1.796 | 1.805 | ||
| UV method | AM | 10 | 8 | 17.967 | 7.967 | 99.58 | 1.043 | 1.047 |
| 10 | 12 | 21.933 | 11.933 | 99.44 | 1.967 | 1.972 | ||
| 10 | 16 | 25.966 | 15.966 | 99.78 | 1.588 | 1.589 | ||
| BZ | 10 | 8 | 17.967 | 7.967 | 99.58 | 1.236 | 1.241 | |
| 10 | 12 | 21.933 | 11.933 | 99.44 | 1.176 | 1.183 | ||
| 10 | 16 | 25.966 | 15.966 | 99.78 | 1.433 | 1.436 |
2.6 In-vitro dissolution study
In-vitro dissolution study was performed on marketed formulation AMACE-BP (6 tablets) by using USP type II apparatus. Dissolution media used were 0.01 N HCl at 37 ± 2 °C. Samples were taken at specific time intervals as per the FDA guideline and were analyzed by using the UV method and HPLC method. Results were plotted as cumulative percentage release (CPR) vs. time for both methods and were compared.
3 Result and discussion
3.1 RP-HPLC and UV-method validation
RP-HPLC and UV-Spectrophotometric methods were developed for AM and BZ which can be conveniently employed for routine analysis in pharmaceutical dosage forms and will eliminate unnecessary tedious sample preparations. The chromatographic conditions were optimized in order to provide a good performance of the assay. The retention times (Rt) of AM and BZ were 4.43 min and 5.70 min respectively. The chromatograms have been shown in Fig. 2. A seven point calibration curve was constructed with working standards and was found linear (r2 ⩾ 0.999) for each of the analytes over their calibration ranges. The slopes were calculated using the plot of drug concentration versus area of the chromatogram. The developed HPLC method was accurate, precise, reproducible and very sensitive.
Fig. 3 shows overlay spectra of both drugs of the UV-Spectrophotometric method. The regression coefficient of the correlation equation curve was greater than 0.999 (Fig. 4) and the method was validated by using binary mixture of both drugs with less than 2% RSD (Table 1). All the method validation parameters are well within the limits as specified in the ICH Q2B guidelines as shown in Table 2. The intra- and inter-day precision (%R.S.D.) at different concentration levels was found to be less than 2% (Table 3). Table 4 lists the percent recovery (content uniformity) of both drugs in the commercial formulations by HPLC and UV methods. Moreover the %R.S.D. (less variation) shows good precision of both developed methods. The calculated LOQ and LOD concentrations confirmed that the methods were sufficiently sensitive. The methods were specific as none of the excipients interfered with the analytes of interest (Table 5). Hence, the methods were suitably employed for assaying both the drugs in commercial marketed formulation (Table 6).
| Concentration (μg/ml) | Absorbance of amlodipine at 366 nm (n = 6) | Absorbance of amlodipine at 237 nm based on equation(y = 2.6751x − 0.0024) | Absorbance of binary mixture at 237 nm (n = 6) | Absorbance of benazepril in binary mixture | Absorbance of benazepril as per calibration curve (n = 6) | % RSD (Relative standard deviation) |
|---|---|---|---|---|---|---|
| 2 | 0.033 | 0.087 | 0.132 | 0.044 | 0.045 | 1.220 |
| 4 | 0.064 | 0.170 | 0.258 | 0.087 | 0.086 | 0.550 |
| 8 | 0.129 | 0.342 | 0.514 | 0.171 | 0.174 | 1.019 |
| 12 | 0.195 | 0.521 | 0.787 | 0.266 | 0.264 | 0.396 |
| 16 | 0.261 | 0.697 | 1.046 | 0.348 | 0.354 | 0.782 |
| 20 | 0.321 | 0.858 | 1.291 | 0.433 | 0.446 | 1.481 |
| 24 | 0.385 | 1.028 | 1.545 | 0.516 | 0.529 | 1.216 |
| Parameters | HPLC method | UV-method | ||
|---|---|---|---|---|
| AM | BZ | AM | BZ | |
| Working λmax | 237 nm | 237 nm | 366 nm | 237 nm |
| Beer’s law limit | 1–6 μg/ml | 1–6 μg/ml | 2–24 μg/ml | 2–24 μg/ml |
| Regression equation⁎ | y = 271730x + 80905 | y = 175439x − 6287.2 | y = 0.0161x + 0.0015 | y = 0.0222x − 0.001 |
| Regression coefficient (r2)⁎ | 0.999 | 0.9991 | 0.999 | 0.999 |
| SD of slope⁎ | 545 | 1614 | 0.0002 | 0.0001 |
| SD of intercept⁎ | 1092 | 4383 | 0.0017 | 0.0011 |
| Data point | 6 | 6 | 7 | 7 |
| Absorptivity⁎ | NA | NA | 0.017 | 0.0215 |
| Retention time (min)⁎ | 4.43 | 5.70 | NA | NA |
| LOD⁎ | 0.074 | 0.053 | 0.291 | 0.070 |
| LOQ⁎ | 0.223 | 0.161 | 0.883 | 0.212 |
| Excipient | % AM ⁎ ± SD | % RSD | % BZ⁎ ± SD | % RSD |
|---|---|---|---|---|
| PVP | 98.54 ± 0.72 | 0.73 | 98.48 ± 0.87 | 0.88 |
| Mg stearate | 99.49 ± 0.58 | 0.59 | 98.10 ± 0.93 | 0.94 |
| SSG | 98.43 ± 1.09 | 1.10 | 99.96 ± 1.03 | 1.03 |
| Starch | 97.92 ± 1.52 | 1.55 | 98.77 ± 1.18 | 1.19 |
| MCC PH102 | 99.25 ± 1.57 | 1.58 | 99.49 ± 1.82 | 1.83 |
| DCP | 99.27 ± 1.76 | 1.77 | 95.70 ± 1.35 | 1.41 |
| Lactose | 97.16 ± 1.93 | 1.98 | 97.88 ± 1.56 | 1.59 |
3.2 In-vitro dissolution study
The average percentage drugs released within 75 min as detected by the proposed UV method as well as the HPLC method after in vitro dissolution of tablets containing combination drug product are depicted in Fig. 5. AM and BZ were completely released within 75 min of dissolution study and the same could be measured using both the methods with almost similar release pattern.
3.3 Statistical comparison of HPLC and UV methods
Statistical comparison was done on assay results obtained from UV and HPLC methods for marketed formulation (AMACE-BP) by using student’s t-test. Calculated values for t-test were 1.47 and 2.01 for AM and BZ respectively which is less than ttable value (2.306) indicating that there was no significant difference between the HPLC method and UV method.
4 Conclusion
Simple, rapid, accurate and precise RP-HPLC as well as spectrophotometric methods have been developed and validated for the routine analysis of AM and BZ in API and tablet dosage forms. Both methods are suitable for the simultaneous determination of AM and BZ in multi-component formulations without interference of each other. The developed methods are recommended for routine and quality control analysis of the investigated drugs in two component pharmaceutical preparations. The amount found from the proposed methods was in good agreement with the label claim of the formulation. Also the value of standard deviation and coefficient of variation calculated were satisfactorily low, indicating the suitability of the proposed methods for the routine estimation of tablet dosage forms. The developed method can also be conveniently adopted for dissolution testing of AM and BZ in commercial formulation.
Conflict of interest
The authors declare no conflict of interests.
Acknowledgement
Abhi Kavathia would like to acknowledge the Department of Pharmaceuticals, Ministry of Chemical and Fertilizer, Govt. of India for providing financial assistance during this project.
References
- Simple and rapid HPLC method for determination of amlodipine in human serum with fluorescence detection and its use in pharmacokinetic studies. J. Pharm. Biomed. Anal.. 2004;36(1):163-168.
- [Google Scholar]
- HPLC method development for the simultaneous analysis of amlodipine and valsartan in combined dosage forms and in vitro dissolution studies. Braz. J. Pharm. Sci.. 2010;46(4):761-768.
- [Google Scholar]
- Simultaneous HPTLC estimation of telmisartan and amlodipine besylate in tablet dosage form. Appl. Sci. Res.. 2010;2:94-100.
- [Google Scholar]
- Stability indicating RP-HPLC method for simultaneous estimation of valsartan and amlodipine in capsule formulation. Asian J. Res. Chem.. 2008;1(1):15-18.
- [Google Scholar]
- Simultaneous UV spectrophotometric determination of valsartan and amlodipine in tablet. Int. J. Chem. Tech. Res.. 2010;2(1):551-556.
- [Google Scholar]
- Role of the renin–angiotensin–aldosterone system in atrial fibrillation and cardiac remodeling. Curr. Opin. Cardiol.. 2005;20(1):31-37.
- [Google Scholar]
- ICH Guidelines: Validation of Analytical Procedures: Text and Methodology Q2 (B), 2005.
- Goodman and Gilman’s the pharmacological basis of therapeutics. New York, USA: McGraw Hill; 2001.
- Validated HPLC and HPTLC method for simultaneous quantitation of amlodipine besylate and olmesartan medoxomil in bulk drug and formulation. Anal. Lett.. 2010;43(2):251-258.
- [Google Scholar]
- Simultaneous determination of amlodipine besylate and benazepril hydrochloride in pharmaceutical dosage form by LC. Anal. Sci.. 2009;25(12):1495-1498.
- [Google Scholar]
- Development and validation of RPHPLC method for simultaneous estimation of amlodipine besylate and benazepril HCl in tablet dosage form. Int. J. Curr. Pharm. Res.. 2012;4(3):80-84.
- [Google Scholar]
- HPTLC method for the simultaneous determination of amlodipine and benazepril in their formulations. J. Chromotogr. Sci.. 2005;43(2):73-75.
- [Google Scholar]
- Stability indicating RP-HPLC method for simultaneous determination of amlodipine and benazepril hydrochloride from their combination drug product. J. Pharm. Biomed. Anal.. 2005;39(1):147-155.
- [Google Scholar]
- Simultaneous spectrophotometric estimation of amlodipine besylate and benazepril HCl in pure and pharmaceutical dosage form. Der Pharmacia Lettre. 2011;3(3):397-403.
- [Google Scholar]
- Simultaneous determination of atorvastatin, amlodipine, ramipril and benazepril in human plasma by LC MS/MS and its application to a human pharmacokinetic study. Biomed. Chromatogr.. 2011;25(4):439-449.
- [Google Scholar]
- New spectrophotometric methods for simultaneous determination of amlodipine besylate and atorvastatin calcium in tablet dosage forms. Int. J. Pharm. Pharm. Sci.. 2011;2:215-219.
- [Google Scholar]
- Shaalan, R.A., Belal, T.S., El Yazbi, F.A., Elonsy, S.M., 2017. Validated stability-indicating HPLC-DAD method of analysis for the antihypertensive triple mixture of amlodipine besylate, valsartan and hydrochlorothiazide in their tablets. Arabian J. Chem. 10, S1381–S1394.
- Utility of oxidation–reduction reaction for the spectrophotometric determination of amlodipine besylate. Arabian J. Chem.. 2009;2(1):59-63.
- [Google Scholar]
- Foye’s Principles of Medicinal Chemistry. Philadelphia: Lippincott Williams & Wilkins; 2002.