Analytical Method Development and Validation for Simultaneous Estimation of Amlodipine Besylate and Indapamide by using UV VIS Spectrophotometry and RPHPLC in Bulk and Dosage Form

The objective of this study was to develop a rapid and sensitive UV spectroscopic and RP-HPLC method for the analysis of Amlodipine Besylate and Indapamide in bulk drug and pharmaceutical dosage form by using the most commonly employed C-18 column with UV-detection. Acetonitrile: acetate buffer pH-5 (40: 60 % v/v) with 1.2 mL / min flow rate was selected as mobile phase.In the HPLC method, the conditions were optimized to obtain an adequate separation of components. Initially, various mobile phase compositions were tried to separate active ingredients. This method has been found to be better than previously reported methods, due to its wider range of linearity, use of readily available mobile phase, lack of extraction procedures. Hence thesemethodscould be used in quality control for routine analysis of finished products of amlodipine besylate and Indapamide simultaneously without any interference.


INTRODUCTION
Analytical method development and validation play important roles in the discovery, development, and manufacture of pharmaceuticals.
Pharmaceutical products formulated with more than one drug, typically referred to as combination products, are intended to meet previously unmet patients need by combining the therapeutic effects of two or more drugs in one product. These combination products can present daunting challenges to the analytical chemist responsible for the development and validation of analytical methods. The official test methods that result from these processes are being used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products.

BASIC CRITERIA FOR NEW METHOD DEVELOPMENT OF DRUG ANALYSIS
 The drug or drug combination may not be official in any pharmacopoeias.  A proper analytical procedure for the drug may not be available in the literature due to patent Regulations.  Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.  Analytical methods for the quantization of the drug in biological fluids may not be available.  Analytical methods for a drug in combination with other drugs may not be available.  The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.

MATERIALS
Amlodipine besylate (Gift sample procured from Matrix Laboratories Ltd., Hyderabad.),Indapamide (Gift sample procured from Yarrow Chem Laboratories Ltd.), Methanol AR grade (S. D. Fine-chem limited, Mumbai), HPLC grade Methanol (Merck specialties private limited, Mumbai), Double distilled water (Merck specialties private limited, Mumbai) employed for the method development.

Instruments
Axis Ag N 204-PO digital balance, Elico LI 120 pH meter, 1.5LH Ultrasonic bath sonicator, Elico SL 218 double beam UV-Vis spectrophotometer, With wide range photodiode detection and fixed 10 mm path holders for reference and sample. (Instrument-1), Elico SL 210 double beam UV-Vis spectrophotometer, with wide range photodiode detection and fixed 10 mm path holders for reference and sample. (Instrument-2), Agilent 1120 compact LC system.

Drug profile of Amlodipine besylate
Amlodipine besylate is a dihydropyridine calciumchannel blocker.

Drug profile of Indapamide Structure
Chemically it is 3-(aminosulfonyl)-4-chloro-N-(2,3-dihydro-2-methyl-1H-indol-1-yl). It is used for treatment of Hypertension. Indapamide blocks the slow component of delayed rectifier potassium current (IKs) without altering the rapid component (IKr) or the inward rectifier current. Specifically it blocks or antagonizes the action the proteins KCNQ1 and KCNE1. Indapamide is also thought to stimulate the synthesis of the vasodilatory hypotensive prostaglandin PGE2 [1-3].

Solvent selection
In order to select suitable solvent for determination of Indapamide, various solvents were selected for the solubility studies [4,5] and it was found that Indapamide was freely soluble in the methanol, ethanol, and slightly soluble in water. In the present investigation, methanol was used as primary solvent and distilled water was used as secondary solvent [4,6].

Preparation of standard stock solution
Standard stock solution was prepared by dissolving accurately weighed 25mg of Indapamide in methanol and the volume was made up to 25 mL with distilled water in a 25 mL volumetric flask (Stock solution-I, 1000 µg/ mL). 1.0 mL of stock solution-I was diluted to 10 mL with solvent (Stock solution-II, 10 µg/ mL).

Determination of λ max
The solution (10µg/mL) was scanned in the UV region of 200-400 nm, which showed maximum absorbance at 280 nm and the absorbance curve.

Study of Beer-Lambert's law
Aliquots of standard solution of ranging from 10-50µg/ mL were transferred into a series of 10 mL volumetric flasks. The volume in each flask was made up to 10 mL with solvent and the absorbance's were measured at 280 nm against solvent blank and the absorbance values were shown in Table 1. The obtained absorbance values are plotted against the concentration of Indapamide to get the calibration curve and were represented in Fig. 1. The concentration of the unknown sample was determined from the calibration graph [5].

Optical parameters of the calibration curve:
The regression equation and correlation coefficient were determined and were given in Table 2.

Application of proposed method for analysis of tablet formulation
For the analysis of commercial formulation, 20 tablets of Indapamide were weighed, powdered in glass mortar and the powder equivalent to 25mg of Indapamide was weighed accurately and transferred into a 25mL standard volumetric flask. The contents were dissolved in solvent and sonicated for few minutes. This solution was filtered through (0.45µ) Whatmann filter paper no. 41. 1.0 mL of the filtrate was diluted to 10 mL with solvent to get the solution of 100 µg/ mLl. An appropriate aliquot of 3.0mL of test solution was diluted to 10mLlto produce the concentration 30µg/mL. The absorbance of the solution was recorded at 280 nm and the concentration of the Indapamide was determined by linear regression equation. Results were shown in Table 3.

Validation of Spectrophotometric Method
The following parameters were determined to validate the developed analytical method as per ICH guidelines (ICH Q2B, 1996) [7,8].

Accuracy
Accuracy is the closeness of the test results obtained by the method to the true value. To assess the accuracy of the proposed method, recovery experiment was performed at three different levels i.e. 50%, 100% and 150%. To the pre analyzed sample solution a known amount of standard drug solution was added at three different levels and absorbance were recorded. The recovery values were summarized in Table 4.

Precision
The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of random error results and was expressed as coefficient of variation (CV).

Intra and Inter-day precision:
The precision of the proposed method was ascertained by actual determination of six replicates of fixed concentration (50 µg/mL) of the drug within the Beer's range and variation of results within the same day (intra-day), variation of results between days (inter-day) was analyzed and was shown in Table 5.

Linearity
This is the method ability to obtain results which are either directly, or after mathematical transformation proportional to the concentration of the analyte within a given range.
The linearity of the method was demonstrated over the concentration range of 10-50 µg/mL of the target concentration. Aliquots of 10, 20, 30, 40 and 50 µg/ mL are prepared from Stock solution (1000µg/mL); Calibration curve was plotted and presented in Fig. 2.

Ruggedness
Ruggedness is the degree of reproducibility of results obtained by the analysis of the same sample under a variety of normal test conditions i.e. different analysts, laboratories, instruments, reagents, assay temperatures etc.
The solution of 50µg/mL was prepared and analyzed with change in the analytical conditions like different Instruments (Elico SL 218 and Elico SL 210) and different analysts (Analyst-1 and Analyst-2). The results were given in Table 6.

LOD and LOQ:
The LOD and LOQ values were determined by the formulae LOD = 3.3 σ/S and LOQ = 10 σ/S (Where, σ is the standard deviation of y intercepts obtained from the replicate measurements (n=6) and S is mean of the slopes of the calibration curves) and were given in Table 2.

Molar extinction coefficient = Equation 20
Where, A= Absorbance of drug, C= Concentration of drug in gm/100mL,

L= Path length
The results were given in Table 2 The results were given in Table 2.

Solvent Selection
In order to select suitable solvent for determination of Amlodipine Besylate, various solvents were selected for the solubility studies and it was found that Amlodipine Besylate was freely soluble in the following solvents: Methanol, Ethanol, acetone, acetonitrile…etc., and slightly is soluble in water. In the present investigation Methanol was used as primary solvent and distilled water was used as secondary solvent.

Preparation of Standard Stock Solution
Standard stock solution was prepared by dissolving accurately weighed 25mg of Amlodipine Besylate in Methanol and the volume was made up to 25 ml with distilled water in a 25ml volumetric flask (Stock solution-I, 1000 µg/ mL). 1.0ml of stock solution-I was diluted to 10 ml with distilled water (Stock solution-II, 100 µg/ mL).

Determination of λ max
The solution (10µg/mL) was scanned in the UV region of 200-400 nm, which shows maximum absorbance at 360 nm and the absorbance curve was given in Fig.2

Study of Beer-Lambert's law
Aliquots of standard solution of Amlodipine Besylate ranging from 10-50µg/mL were transferred into a series of 10 ml volumetric flasks. The volume in each flask was made up to 10 ml with solvent and the absorbance were measured at 360 nm against solvent blank and the absorbance values were shown in Table 7.
The obtained absorbance values are plotted against the concentration of Amlodipine Besylate to get the calibration curve and were shown in Fig. 10. The concentration of the unknown sample was determined from the calibration graph [7].

Optical Parameters of the Calibration curve
The regression equation and correlation coefficient were determined and were given in Table 8.   Table 9.

Validation of spectrophotometric method
The following parameters were determined to validate the developed analytical method as per ICH guidelines (ICH Q2B, 1996).

Accuracy:
Accuracy is the closeness of the test results obtained by the method to the true value. To assess the accuracy of the proposed method, recovery experiment was performed at three different levels i.e. 50%, 100% and 150%. To the pre analyzed sample solution a known amount of standard drug solution was added at three different levels and absorbance were recorded. The recovery values were summarized in Table  10.

Precision
The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of random error results and was expressed as coefficient of variation (CV).

Intra and Inter-day Precision:
The precision of the proposed method was as certained by actual determination of six replicates of fixed concentration (50µg/ml) of the drug within the Beer's range and variation of results within the same day (intra-day), variation of results between days (inter-day) was analyzed and was shown in Table 11.

Linearity:
This is the method ability to obtain results which are either directly, or after mathematical transformation proportional to the concentration of the analyte within a given range.
The linearity of the method was demonstrated over the concentration range of 10-50 µg/ mL of the target concentration. Aliquots of 10, 20, 30, 40 and 50 µg/ mL are prepared from Stock solution (1000µg/mL); Calibration curve was plotted and presented in Fig. 4.

Ruggedness
Ruggedness is the degree of reproducibility of results obtained by the analysis of the same sample under a variety of normal test conditions i.e. different analysts, laboratories, instruments, reagents, assay temperatures etc.   The solution of 50µg/mL was prepared and analyzed with change in the analytical conditions like different instruments (Elico SL 218 and Elico SL 210) and different analysts (Analyst-1 and Analyst-2). The results were given in Table 12.

LOD and LOQ
The LOD and LOQ values were determined by the formulae LOD = 3.3 σ/S and LOQ = 10 σ/S (Where, σ is the standard deviation of y intercepts obtained from the replicate measurements (n=6) and S is mean of the slopes of the calibration curves) and were given in Table 8.

Molar Extinction Coefficient (lit. mol -1 cm -1 ):
It can be calculated as, Where, A= Absorbance of drug, C= Concentration of drug in gm/100mL, L= Path length The results were given in Table 8.

Sandell's sensitivity = Equation 21
Where, C= Concentration of drug, A= Absorbance of drug.
The results were given in Table 8.

Simultaneous Estimation of Amlodipine
Besylate and Indapamideby UV-Spectrophotometric Method

Simultaneous Equation Method (Vierodt's method)
If sample contains two absorbing substances (x and y) and each of which absorbs at the λmax of the other, then it may be possible to determine both the drugs by the technique of simultaneous equation. The information required is: λ 1 : Wavelength maxima for drug x λ 2 : Wavelength maxima for drug y ax 1 and ax 2 : Absorptivity of X at λ 1 and λ 2 ay 1 and ay 2 : Absorptivity of Y at λ 1 and λ 2 A 1 : Absorbance of sample at λ 1 A 2 : Absorbance of sample at λ 2 Let Cx and Cy be the concentration of X and Y respectively in the diluted sample: Two equations are constructed based upon the fact that at λ1 and λ2, the absorbance of the mixture is the sum of the individual absorbances of X and Y.
At λ 1 A 1 = ax 1 bCx + ay 1 bCy (3) At λ 2 A 2 = ax 2 bCx + ay 2 bCy (4) For measurements in 1 cm cells, b = 1 Concentrations of X and Y can be determined by the equations 3.6.1 Selection of sampling wavelength for analysis and preparation of standard

Solvent Used
Distilled water

Preparation of Standard Stock Solution
25 mg each of standard Amlodipine Besylate and Indapamidewere weighed accurately and transferred in to two separate 25mL flasks,

Selection of Analytical Wavelengths
Appropriate dilution of the standard stock solutions 'A1' and 'A2' were scanned separately in the entire ultraviolet range. The λmax of each standard was selected in such a way that at each absorption maxima the difference in absorption of the two components should be as large as possible. The two wavelengths were 360nm and 280nm for Amlodipine Besylate and Indapamiderespectively. At 360nm Amlodipine Besylate has higher absorbance than Indapamideand at 280nm Indapamidehas higher absorbance than Amlodipine Besylate which was shown in Fig. 11.

Selection of Analytical Concentration Range and Construction of Calibration Graph
Amlodipine Besylate: Appropriate aliquots ranging from 0.1 mL to 0.5mL (1mL=100 µg/mL) was pipetted out in to a series of 10mL volumetric flasks. The volume was made up to the mark with distilled water to obtain a concentration range, ranging from 10-50µg/mL (10, 20, 30, 40, 50 µg/mL). Absorbance of the above solutions was measured at 360 nm and a calibration curve of absorbance against concentration was plotted.
Indapamide: Appropriate aliquots ranging from 0.1 mL to 0.5mL (1mL=100 µg/mL) was pipetted out in to a series of 10mL volumetric flasks. The volume was made up to the mark with distilled water to obtain a concentration range, ranging from 10-50µg/mL (10, 20, 30, 40, 50 µg/mL). Absorbance of the above solutions was measured at 280 nm and a calibration curve of absorbance against concentration was plotted.
Both drugs follow Beer Lambert's law in the concentration range of 10-50 µg/mL. Regression equation was established and the correlation coefficient was determined. The results were given in Table 13 and 14and calibration curves of both the drugs were shown in Fig 6.

Analysis of tablet formulation
Twenty tablets of Amlodipine Besylate and Indapamide combination dosage forms (Amlodac-D) (Dose 5 mg) were weighed and their average weight was determined. The tablets were crushed in to fine powder. From the tablet triturate a tablet mass equivalent to 12.5mg of Amlodipine Besylate or 25 mg of Indapamide was transferred in to a 25mL volumetric flask , dissolved in a small quantity of methanol by sonication for 10 min and finally the volume was made up to the mark with methanol. The resultant solution was filtered through a Whatmann filter paper no. 41 and used as sample stock solution 'A' (500µg/mL Amlodipine Besylate and 1000µg/mL Indapamide).
From the above stock solution 1mL aliquot was transferred in to a 10 mL volumetric flask, dissolved in a small quantity of distilled water and the volume was made up to the mark with distilled water to obtain a final concentration of 50µg/mL Amlodipine Besylate and 100µg/mL Indapamide. This solution was used as the sample stock solution 'B'.
1.0mL of the sample stock solution 'B' was transferred in to a 10 mL volumetric flask, dissolved in a small quantity of distilled water and the volume was made up to the mark with distilled water. The absorbance of the resultant solution was measured at the two absorption maxima 360nm and 280nm. This absorbance was noted as A 1 and A 2 respectively and amount of the drugs present was calculated using simultaneous equation method and the results were given in Table 15.

3.6.3Method validation
The following parameters were determined to validate the developed analytical method as per ICH guidelines (ICH Q2B, 1996).

Accuracy
Accuracy is the closeness of the test results obtained by the method to the true value. To study the accuracy, 20 tablets were weighed and powdered and analysis of the same was carried out. Recovery studies were carried out by addition of known amount of the known amount of Amlodipine Besylate and Indapamideto the sample at three different concentration levels i.e. 50%, 100% and 150% (Standard addition method). The results were given in Table 16.

Precision
The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. It provides an indication of random error results and was expressed as relative standard deviation (coefficient of variation)

Procedure for the Determination of Intra-day Precision
In intraday precision six replicate sample matrices separately containing 50µg/mL of Amlodipine Besylate and Indapamide were analysed at different time intervals on the same day at 360nm and 280 nm respectively. The variation of the results within the same day was analysed and statistically validated.

Procedure for the Determination of Inter-day Precision
In inter-day precision six replicate sample matrices separately containing 50µg/mL of Amlodipine Besylate and Indapamide were analysed on different days at 360nm and 280 nm respectively. The variation of the results was analysed and statistically validated, which were given in Table 17 and 18.

Linearity and Range
The linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in the sample with in a given range. Appropriate aliquots ranging from 0.1mL to 0.5mL were pipetted out separately from the 'standard stock solution B1 and B2' out in to a series of 10mL volumetric flasks. The volume was made up to the mark with distilled water to obtain a concentration range, ranging from 10-50µg/mL (10, 20, 30, 40, 50µg/mL). Absorbance of the above solutions was measured at 360nm and 280 nm respectively.
A calibration curve of concentration vs. absorbance was established and shown in Fig. 6. Both drugs follow Beer's lamberts law in the concentration range of 10-50µg/mL. Regression equation was established and the correlation coefficient was determined. The optical and regression parameters were given in Table 14.

Ruggedness
Ruggedness is the degree of reproducibility of results obtained by the analysis of the same sample under a variety of normal test conditions i.e. different analysts, laboratories, instruments, reagents, assay temperatures etc.
The solution of 50µg/mL of Amlodipine Besylate and Indapamide was prepared separately and analyzed with change in the analytical conditions like different instruments (Elico SL 218 and Elico SL 210) and different analysts (Analyst-1 and Analyst-2) and the results were given in Table 19 and 20.

LOD and LOQ
The LOD and LOQ values were determined by the formulae LOD = 3.3 σ/S and LOQ = 10 σ/S (Where, σ is the standard deviation of y intercepts obtained from the replicate measurements (n=3) and S is mean of the slopes of the calibration curves) and were given in Table 14.

Determination of Molar Extinction Coefficient
The absorbance of all the concentrations of Amlodipine Besylate (10-50µg/mL) and Indapamide (10-50µg/mL) were determined at the absorption maximum 360nm and 280nm respectively. The molar extinction coefficient was determined using the formula Molar extinction coefficient = A/C × L ( 7) Where, A= Absorbance of drug, C= Concentration of drug in gm/100mL, L= Path length The results were given in Table 14. The results were given in Table 14.

Simultaneous
Estimation of Amlodipine Besylate and Indapamide by RP-HPLC Method 3.7.1 Selection of sampling wavelength for analysis and preparation of standard calibration curves

Selection of Mobile Phase
The standard solutions containing Amlodipine Besylate and Indapamide were injected into the HPLC system and run in different solvent systems. By studying literature survey, different mobile phases in different proportions and different pH were tried in order to find the best conditions for the separation.
It was found that acetonitrile and acetate buffer of pH-5 gives satisfactory results as compared to other mobile phases. This mobile phase system was tried with different proportions and using different flow rates. Finally, the optimal composition of the mobile phase was determined to be Acetonitrile:Acetate Buffer pH-5(40:60v/v)

Preparation of Mobile Phase
Mobile phase was prepared by mixing Acetonitrile and Acetate Buffer pH-5 in the ratio of 40:60 and was initially filtered through 0.45μmmillipore membrane filter and sonicated for 15 min before use.

Preparation of Acetate Buffer pH-5
Dissolve 13.6g of sodium acetate in 200ml of water and add 6.0ml of glacial acetic acid to adjust the pH and the volume is made upto 1000ml.

Preparation of Standard Stock Solution
The separate stock solutions of Amlodipine Besylate and Indapamide were prepared by accurately weighing 25mg each into a separate 25 ml volumetric flasks A and B and made up to the volume with mobile phase to get 1000µg/ml respectively. From the above standard stock solutions 1.0ml from volumetric flask A and 1.0 ml from volumetric flask B was transferred to a 10 ml volumetric flask and made up to the volume with same mobile phase to get 100µg/ml and 100µg/ml of Amlodipine Besylate and Indapamide respectively (Working stock solution).

Selection of Analytical Wavelength
By appropriate dilution of each standard stock solution with mobile phase, various concentrations of Amlodipine Besylate and Indapamide were prepared separately. Each solution was scanned using double beam UV visible spectrophotometer between the range of 200 nm to 400 nm and their spectra was overlaid. From the overlain spectra shown in Fig.  6 of Amlodipine Besylate and Indapamide, 268 nm was selected as analytical wavelength for Multicomponent analysis using HPLC method.

Optimized Chromatographic Conditions
Mobile phase consisting of Acetonitrile : Acetate Buffer pH-5 (40:60 v/v) was used in isocratic mode. The mobile phase was initially filtered through 0.45μm millipore membrane filter and sonicated for 15 min before use. The flow rate was maintained at 1 ml/min and the injection volume was 20μL. UV detection was performed at 268 nm and the separation was achieved at ambient temperature.

Selection of Analytical Concentration Range and Construction of Calibration Curve for Amlodipine Besylate and s
Appropriate aliquots ranging from 0.5mL to 2.5mL were pipetted out from the working stock solution (100 μg/mL of Amlodipine Besylate) and 1.0mL to 5.0mL (from 100μg/mL of Indapamide) in to a series of 10 ml volumetric flasks. The volume was made up to the mark with the mobile phase to get a set of solutions having the concentration range, ranging from 5-25 μg/mL of Amlodipine Besylate 10-50µg/mL of Indapamide Chromatograms representing linearity was shown in Fig. 13 to 17. Triplicate dilutions of each of the above mentioned concentrations were prepared separately and from these triplicate solutions, 20 μL of each concentration of the drug were injected into the HPLC system three times separately and their chromatograms were recorded under the same chromatographic conditions as described above.
Peak areas were recorded for all the peaks and a standard calibration curve of area against concentration was plotted as concentration of the drug Vs peak area. The results were shown in Table 22. Both the drugs follow the Beer's Lambert's law in the concentration range of 5-25μg/mL of Amlodipine Besylate and 10-50µg/mL of. Indapamide The linearity of calibration curves and adherence of the system to Beer's Lambert's law was validated by high value of correlation coefficient and less than 2% percent relative standard deviation (%RSD) for the intercept value which were shown in Table 22.

Analysis of tablet formulation
The tablets were initially powdered and an amount equivalent to 12.5mg of Amlodipine Besylate and 25 mg of Indapamide was accurately weighed into a 25 mL volumetric flask, mixed with 20mL of mobile phase. The solution was made up to the volume with mobile phase and sonicated for 5 minutes. The solution was then filtered through 0.45µm millipore membrane filter. Final stock containing 10µg/mL and 5µg/mL of Indapamide and Amlodipine Besylate respectively was prepared by subsequent dilution with the same mobile phase. 20µL of sample solution was injected into chromatographic system and the peak responses were measured. The solution was injected three times into the column. The amount present in each tablet was calculated by comparing the areas of test with that of the standard. A typical chromatogram of test solution containing 10 μg/mL of Indapamide and 5μg/mL of Amlodipine Besylate was shown in Fig. 20. The results were shown in Table 23.

Method validation
The method was validated according to ICH Q2 B guidelines for validation of analytical procedures in order to determine system suitability, linearity, sensitivity, precision, accuracy and robustness for the analytes (ICH Q2B, 1996).

System Suitability
The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from six replicate injections for Amlodipine Besylate and Indapamide retention times and peak areas.
System suitability was carried out by injecting 100% concentration (sample having 50µg/ml of Indapamide and 25µg/mL of Amlodipine Besylate) into the HPLC system. This was repeated for six times under similar condition. The tailing factor (T) and no. of theoretical plates (N) obtained were shown in Fig. 21 and the results were given in Table 24 and 25.

Accuracy
To confirm the accuracy of the proposed method, recovery experiments were performed by standard addition technique. In this method a known quantity of pure drug was added at three   Fig. 22 to 24. The results of recovery studies by proposed method were validated by statistical evaluation and were given in Table 26.

Linearity and Range
The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. Linearity of the method was determined by means of calibration curve using different concentration of the drugs. Linearity was evaluated by visual inspection of a calibration curve. The linearity of the method was determined in concentration range of 5-25µg/mL for Amlodipine Besylate and 10-50µg/mL for Indapamide. Each solution was injected in triplicate. Chromatograms representing linearity were shown in Fig. 13 to 17. The slope, intercept was reported as required by ICH which were given in Table 21 and 22.

Precision
The precision of an analytical method was studied by performing intraday and inter day precision.

Intraday Precision
Variation of results within the same day was analyzed. Intraday precision was determined by analyzing a set of six combined standard solutions of Amlodipine Besylate (25µg/mL) and Indapamide (50µg/mL) in linearity range as 100% concentration at three different time intervals on same day. Chromatogram representing intraday precision was shown in Fig. 25 and the results were given in Table 27.

Specificity and Selectivity
The specificity of the RP-HPLC method was determined by complete separation of Amlodipine Besylate and Indapamide with parameters like retention time (Rt), resolution (Rs) and tailing factor (T f ). Here tailing factor for peaks of Amlodipine Besylate and Indapamide was less than 2% and resolution was also more than 1%. The average retention time and standard deviation for Amlodipine Besylate and Indapamide were found to be satisfactory for six determinations of sample solution containing 25µg/mL of Amlodipine Besylate and 50µg/ml of Indapamide respectively. The peaks obtained for Amlodipine Besylate and Indapamide were sharp and have clear baseline separation as none of the excipients interfered with the analytes of interest. The chromatogram to represent specificity was shown in Fig. 26 and the results were given in Table 28.

Robustness
The evaluation of robustness should be considered during the development phase and depends upon the type of procedure under study. It should show the reliability of analysis with respect to deliberate variations in method parameters like different column temperature, different analytical wavelength, different flow rate. The solution containing 25μg/mL of Amlodipine Besylate and 50μg/mL of Indapamide was injected into sample injector of HPLC three times under different parameters like deliberate variations in flow rate (±0.2mL/min) and detection wavelength (± 2 nm).
Chromatogram representing robustness was shown in Fig. 27 and 28for change in flow rate and the results were given in Table 29. Chromatogram representing robustness was shown in Fig. 29 and 30 for change in detection wavelength and the results were given in Table  30.   Fig. 31 and 32 for change in analysts and the results were given in Table 31.

LOD and LOQ
The LOD and LOQ values were determined by the formulae LOD = 3.3 σ/S and LOQ = 10 σ/S (Where, σ is the standard deviation of the responses and S is mean of the slopes of the calibration curves). The results were given in Table 22.

Estimation of Indapamide by UV-Spectrophotometric Method
The objectives of the proposed work was to develop some new and sensitive analytical methods for the determination of Indapamide and to validate the methods according to USP and Amlodipine ICH guidelines and applying the same for its estimation in pharmaceutical formulations.
The absorption spectra were recorded in the wavelength region of 200-400 nm which was shown in Fig. 7 and the λmax was found to be at 280nm. Beer's law range was confirmed by the linearity of the calibration curve of Indapamide, which was shown in Fig. 8. Indapamide showed linearity in the concentration range of 10-50 µg/mL [7,9].  The optical characteristics such as absorption maxima, Beer's law limits, molar absorptivity, slope (b), intercept (C), correlation coefficient (r 2 ) obtained from different concentrations, percent relative standard deviation, LOD and LOQ values were given in Table 2 with satisfactory results. The quantitative estimation was carried out on formulation. The percentage purity of the marketed formulation was found to be 98.4%w/w. The results were given in Table 3.
The accuracy of the methods was confirmed by the recovery studies, by adding known amount of the pure drug to the formulation and the analytical data were given in Table 4.
The percentage recovery was found to be between 99.2 -100.6%w/w, shows that the method was free from the interference of excipients used in the formulation.
The % RSD values were less than 2 indicating the precision of the methodology and low standard error values indicates the accuracy of the method. The statistical data's were given in Table 5.
Results obtained for the proposed methods confirm the suitability of these methods for pharmaceutical dosage forms. The other active ingredients and excipients usually present in the pharmaceutical dosage forms did not interfere in the estimation, when commercial dosage forms were analyzed by these methods.
The results obtained in Ruggedness test (%RSD ≤ 2) expresses the reproducibility of the method. The ruggedness results were shown in Table 6.
The developed UV Spectrophotometric method was found to be rapid, simple, precise, accurate and economic for routine estimation of Indapamide in commercial dosage forms.

Estimation of Amlodipine Besylate by UV-Spectrophotometric Method
The objectives of the proposed work was to develop some new and sensitive analytical methods for the determination of Amlodipine Besylate and to validate the methods according to USP and ICH guidelines and applying the same for its estimation in pharmaceutical formulations.
The absorption spectra were recorded in the wavelength region of 200-400 nm which was shown in Fig. 9 and the λmax was found to be at 360 nm. Beer's law range was confirmed by the linearity of the calibration curve of Amlodipine Besylate, which was represented in Fig. 10. Amlodipine Besylate showed linearity in the concentration range of 10-50 µg/mL.
The optical characteristics such as absorption maxima, Beer's law limits, molar absorptivity, slope (b), intercept (C), correlation coefficient (r2) obtained from different concentrations, percent relative standard deviation, LOD and LOQ values were shown in Table 8 with satisfactory results.
The quantitative estimation was carried out on formulation. The percentage purity of the marketed formulation was found to be 98.2%w/w. The results were given in Table 9.
The accuracy of the methods was confirmed by the recovery studies, by adding known amount of the pure drug to the formulation and the analytical data are presented in Table 10.
The percentage recovery was found to be between 99.6 -101.4%w/w, shows that the method was free from the interference of excipients used in the formulation.
The % RSD values are less than 2 indicating the precision of the methodology. The statistical data's were given in Table 11. Results obtained for the proposed methods confirm the suitability of these methods for pharmaceutical dosage forms. The other active ingredients and excipients usually present in the pharmaceutical dosage forms did not interfere in the estimation, when commercial dosage forms were analyzed by these methods.
The results obtained in Ruggedness test (%RSD ≤ 2) expresses the reproducibility of the method. The ruggedness results were shown in Table 12.
The developed UV Spectrophotometric method was found to be rapid, simple, precise, accurate and economic for routine estimation of Amlodipine Besylate in commercial dosage forms.

Simultaneous Estimation of Amlodipine
Besylate and Indapamide by

UV-Spectrophotometric Method
The objective of the proposed work was to develop UV simultaneous method for the determination of Amlodipine Besylate and Indapamide, and to validate the methods according to ICH guidelines and applying the same for its estimation in marketed formulations. This is a very simple method and requires knowledge of molar absorptivity of the components which should be determined very accurately. It only requires measurement of absorbance at 280 nm and 360 nm which were selected as the maximum absorbance, the absorption curve was shown in Fig. 11 and few simple calculations, which can be done manually. The percent label claim in given tablet formulation was found to be 99.2% w/w for Amlodipine Besylate and 99.80% w/w for Indapamide, which were found to be satisfactory and the results were given in Table 15.
The method was validated as per ICH norms. Accurate results were obtained by utilizing the proposed methods for the quantization of Amlodipine Besylate and Indapamide and a good agreement with the results obtained by the reported methods was found. For UV spectrophotometric method, linearity was obtained in the concentration range of 10-50µg/mL for both the drugs and the results were given in Table 13 with calibration curves shown in Fig. 12 High % recovery greater than 98% (99.07-100.4% w/w for Amlodipine Besylate and 99.5-101.5% w/w for Indapamide) showed that the method is free from the interference of excipients used in the formulation. The results were given in Table 16. The value of standard deviation and % RSD were found to be < 2%, indicated the high precision of the method. The results were given in Table 17and 18.
Detection limit and quantization limit were determined from the standard deviation of yintercepts of six calibration curves and average slope of the same. LOD and LOQ of Amlodipine Besylate were found to be 0.39µg/mL and 1.12µg/mL at 360nm. LOD and LOQ of Indapamide were found to be 1.43 µg/mL and 4.40µg/mL at 280nm. The reliability of the proposed method was evaluated and the results were shown in Table 14.
The results obtained in Ruggedness test (%RSD < 2) expresses the reproducibility of the method. The ruggedness results were shown in Table 19 and 20.
From the validation results the developed UV Spectrophotometric method was found to be rapid, simple, precise, accurate and economic for routine estimation of Amlodipine and Indapamide in commercial dosage forms.

Simultaneous Estimation of Amlodipine Besylate and Indapamide by RP-HPLC Method
In HPLC method, the conditions were optimized to obtain an adequate separation of eluted compounds. Initially, various mobile phase compositions were tried to separate active ingredients [10][11][12].
The objective of this study was to develop a rapid and sensitive RP-HPLC method for the analysis of Amlodipine Besylate and Indapamide in bulk drug and pharmaceutical dosage form by using the most commonly employed C-18 column with UV-detection.
The optimum wavelength selected was 268 nm from the overlain spectra obtained which was shown in Fig. 13 at which better detector response for the drug was obtained. The retention time for Amlodipine Besylate and Indapamide was found to be 5.2 min and 6.7 min respectively which were shown in Fig. 14 and15. The linearity was observed in concentration range of 5-25 µg/ mL and 10-50 µg/ mL for Amlodipine Besylate and Indapamide respectively. Calibration curves of the respective drugs were shown in Fig. 21 and 22.Summary of linearity results were given in Table 22.
System suitability was assessed by injecting 5 replicate injections of 100% test concentration. Number of theoretical plates was more than 2000 for both the drugs and tailing factor was less than 1.5 for both Amlodipine Besylate and Indapamide was reported. A Resolution of greater than 1 was observed. The relative retention times of six replicate injections and system suitability parameters were given in Table 24 and 25.
Specificity of the chromatographic method was tested by injecting sample concentration prepared from marketed formulation. The response was compared with that obtained from the standard drug. The chromatogram confirms the presence of Amlodipine Besylate and Indapamide at 5.2min and 6.7min, respectively without any interference. Thus the developed method was specific to Amlodipine Besylate and Indapamide and the parameters were given in Table 28.

Fig. 20. RP-HPLC Chromatogram of Test Formulation
The robustness of an analytical method was determined by analysis of aliquots from homogenous lots by differing physical parameters such as change in flow rate to 1.2 ± 0.2 mL and changing detection wavelength 268nm ± 2nm. The obtained values were given in Table 29 and 30. These values with low % RSD (<2) indicated that the method was quite robust.
Ruggedness of the proposed method was determined by analysis of aliquots from homogeneous slot by different analysts, using similar operational and environmental conditions; the % RSD reported was found to be less than 2 and these values were listed in Table 31.
The proposed method was validated in accordance with ICH parameters and was applied for analysis of the same in marketed formulations. The content of each component in the formulation was estimated by comparing the peak area of the test sample with that of the peak area of the standard and the results were given in Table 23 which were found to be 99.8% w/w for Amlodipine Besylate and 99.2% w/w for Indapamide, respectively. High % recovery and low % RSD suggested that the method can be applicable for the routine analysis of commercial formulations.
Hence, the developed HPLC method can be adopted for the routine analysis of Amlodipine Besylate and Indapamide in pharmaceutical formulations [31-37].
Drug combinations are commonly used clinically and analyst is required to develop suitable methods of their analysis. For routine analytical purposes it is always of interest to establish methods capable of analyzing a large number of samples in a short time period with good accuracy and precision. The commonly used tests of pharmaceutical analysis generally entail compendia testing method development, setting specifications, and method validation.
Analytical testing is one of the more interesting ways for scientists to take part in quality process by providing actual data on the identity, content and purity of the drug products. New methods are now being developed with a great deal of consideration to worldwide harmonization. As a result, new products can be assured to have comparable quality and can be brought to international markets faster.
A liquid chromatographic technique coupled with spectrophotometric analysis is a versatile tool that can generate extensive analytical data that is highly useful in the routine drug analysis. For routine analytical purposes it is always of interest to establish methods capable of analyzing a large number of samples in a short time period with good accuracy and precision. The results were summarized as follows:

Conclusions
Development of methods to achieve the final goal of ensuring the quantity of drug substances and drug products is not a trivial undertaking. The capabilities of the methods developed were complementary to each other. Hence they can be regarded as simple, specific and sensitive methods for the estimation of Amlodipine Besylate and Indapamide in single and combined pharmaceutical dosage forms. The developed UV Spectrophotometric methods and RP-HPLC methods were validated according to ICH guidelines and were found to be applicable for the routine analysis of Amlodipine Besylate and Indapamide in their single and combined dosage forms.
The proposed UV methods were simple, sensitive and reliable with good precision and accuracy. This method was specific while estimating the commercial formulations without interference of excipients and other additives. Hence, this method can be used for the estimation of Amlodipine Besylate and Indapamide in bulk samples and their pharmaceutical formulations individually and in combination by simultaneous equation method. This method has been found to be better than previously reported methods, due to its wider range of linearity, use of readily available mobile phase, lack of extraction procedures. Hence above method can be used in quality control for routine analysis of finished products of Amlodipine Besylate and Indapamide simultaneously without any interference.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.