Development and Validation of Spctrophotometric Method for Simultaneous Estimation of Amlodipine Besylate and Indapamide in Tablet Dosage Form

 

Raj Manish C. * and Chaudhari Bharat G.

Department of Quality Assurance, Shree S. K. Patel Collage of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidyanagar – 384 012, Mehsana, Gujarat, India.

*Corresponding Author E-mail: manish21288@gmail.com

ABSTRACT:

The present manuscript describes simple, sensitive, rapid, accurate, precise and economic dual wavelength spectrophotometric method for simultaneous determination of amlodipine besylate and indapamide in combined tablet dosage form. The utility of dual wavelength data processing program is its ability to calculate unknown concentration of components of interest in a mixture containing an interfering component. The principle for dual wavelength method is “the absorbance difference between two points on the mixture spectra is directly proportional to the concentration of the component of interest”. The wavelengths selected for determination of amlodipine besylate were 255 nm and 360 nm, whereas the wavelengths selected for determination of indapamide were 241.6 nm and 217.6 nm. The two drugs follow Beer-Lambert’s law over the concentration range of 10-60 µg/ml for AMLO and 3-18 μg/ml for INDA. The method was successfully applied to pharmaceutical dosage form because no interference from the tablet excipients was found. The results of analysis have been validated statistically and by recovery studies.

 

KEYWORDS: Amlodipine besylate, Indapamide, dual wavelength spectrophotometric method, validation, tablet.

 


INTRODUCTION:

Amlodipine besylate (AMLO) is chemically 3-ethyl 5-methyl (4RS)-2-[(2 aminoethoxy)methyl]-4-(2-chlorophenyl) 6-methyl-1,4 dihydropyridine-3,5-dicarboxylate benzenesulphonate8, is a Calcium channel blocker, used in the treatment of hypertension9. It is official in IP, BP and USP. IP1, BP3  and USP2 describe HPLC method for its estimation. Literature survey reveals UV spectrophotometry4, RP-HPLC5, spectrophotometric6 method for simultaneous determination of AMLO with other drug and RP-HPLC7 method for simultaneous determination of AMLO with other drug methods for determination of AMLO in pharmaceutical dosage forms as well as in biological fluids. Indapamide (INDA) is chemically 4-Chloro-N-[(2RS)-2-methyl-2,3-dihydro-1H-indol-1-yl]-3-sulphamoylbenzamide18, is a Thiazide diuretics for the treatment of hypertension19. Indapamide is  official in IP10, BP12 and USP11. IP, BP, and USP describe HPLC method for its estimation. Literature survey reveals LC-MS13, spectrophotometric14 and HPLC15 method for simultaneous estimation of INDA in whole human blood, RP-HPLC16 method for simultaneous estimation of INDA, LC-ESI-MS17 methods for the determination of INDA in human plasma.

 

This combination is not official in any pharmacopoeia hence official and reported methods of analysis are not available for this combination. The present manuscript describes simple, sensitive, accurate, precise, rapid and economic dual wavelength spectrophotometric method for simultaneous estimation of AMLO and INDA in tablet dosage form.

 

MATERIALS AND METHODS:

Apparatus:

A Shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software (UV Probe version 2.10). A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India) was used in the study.

 

Reagents and Materials:

AMLO and INDA bulk powder was kindly gifted by Torrent Research Centre, Ahmadabad, India. The commercial fixed dose combination product was procured from Serdia Pharmaceuticals (INDIA) Pvt ltd. Methanol AR grade was procured from S. D. Fine Chemicals Ltd., Mumbai, India and Whatman filter paper no. 41 (Millipore, USA) were used in the study.

Preparation of standard stock solutions:

An accurately weighed quantity of AMLO (10 mg) and INDA (10 mg) were transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with methanol to obtain standard solution having concentration of AMLO (100 μg/ml) and INDA (100 μg/ml).

 

Preparation of sample solution:

Twenty tablets were weighed and powdered. The powder equivalent to 5 mg of AMLO and 1.5 mg of INDA transferred to 50 ml volumetric flask. Methanol (25 ml) was added to it and sonicated for 20 min, and volume was made up to the mark with methanol. The solution was filtered through Whatman filter paper no. 41 and filtrate was suitably diluted with methanol to achieve a final concentration of 20 μg/ml of AMLO and 6 µg/ml of INDA. The absorbance of final solution was recorded at selected wavelengths for determination of AMLO and INDA. The analysis procedure was repeated three times with tablet formulation.

 

Development of the method:

Absorbance spectrum of pure AMLO was scanned in the spectrum basic mode. Using cursor function, the absorbance corresponding to 360 nm (wavelength λ1) was noted from the spectrum and the cursor function was moved along with the peak curve until the absorbance equal to that of absorbance at 360 nm was found. The wavelength obtained was corresponding to this absorbance value was 255 nm (λ2). Absorbance spectrum of pure INDA was scanned in the spectrum basic mode. INDA shows the absorbance corresponding to 241.6 nm (wavelength λ3) was noted from the spectrum and the cursor function was moved along with the peak curve until the absorbance equal to that of absorbance at 241.6 nm was found. The wavelength obtained was corresponding to this absorbance value was 217.6 nm (λ4). The absorbance of various dilutions of AMLO and INDA in methanol was measured at λ3, λ4 and λ1, λ2 respectively as depicted in Fig. 1. At λ1 and λ2 wavelengths, the absorbance difference for AMLO at any concentration was found to be zero, while for INDA at λ3 and λ4 wavelengths, the absorbance difference for INDA at any concentration was found to be zero.

 

Validation of the proposed method:

The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines20.

 

Linearity (Calibration curve):

Aliquots from the standard stock solutions (100 μg/ml) of AMLO and INDA were used to prepare different sets of dilutions. Mixed series consisted of different concentration of AMLO and INDA in the range of 10-60 μg/ml and 3-18 μg/ml, respectively. The solutions were prepared by pipetting out 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 ml  for AMLO and 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 ml for INDA from the stock solutions of both drugs in to a series of 10 ml volumetric flasks and the volume was made up to the mark with methanol.

Range:

Range is the interval between upper and lower concentration (amount) of analyte in sample for which it has been demonstrated that the analytical method has suitable level of precision accuracy and linearity. (The linear response was observed over a range of 10 -60 μg/ml for AMLO and 3-18 μg/ml for INDA respectively).

 

Method precision (repeatability)

The precision of the instrument was checked by repeated scanning and measurement of the absorbances of solutions (n=6) of AMLO (20 µg/ml) and INDA (6 μg/ml for both drugs) without changing the parameters.

 

Intermediate precision (reproducibility):

The intraday and interday precisions of the proposed method was determined by estimating the corresponding responses 3 times on the same day and on 3 different days over a period of one week for 3 different concentrations of standard solutions of AMLO (20, 30 and 40 µg/ml) and INDA (6, 9 and 12 μg/ml). The results were reported in terms of relative standard deviation (% RSD).

 

Accuracy (recovery study):

The accuracy of the method was determined by calculating the recoveries of AMLO and INDA by the standard addition method. Known amounts of standard solutions of AMLO and INDA were at added at 50, 100 and 150 % level to prequantified sample solutions of AMLO (20 µg/ml) and INDA (6 μg/ml). The amounts of AMLO and INDA were estimated by applying obtained values to the respective regression line equations.

 

Limit of detection and Limit of quantification:

The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines.

                 LOD = 3.3 × σ/S

                 LOQ = 10 × σ/S

Where, σ = the standard deviation of the response and S = slope of the calibration curve.

 

Analysis of AMLO and INDA in combined tablet dosage form:

The absorbance of sample solution of Amlodipine besylate and Indapamide was measured against methanol as blank at 217.6 nm, 241.6 nm and 255 nm, 360 nm for quantitation of AMLO and INDA, respectively.  The amount Amlodipine besylate and Indapamide present in the sample solutions were determined by using regression equation of AMLO and INDA.

 

RESULTS AND DISCCUSION:

Method Development

The utility of dual wavelength data processing program is its ability to calculate unknown concentration of components of interest in a mixture containing an interfering component. Four specific wavelengths were chosen: (1) First wavelength, λ1 (360 nm)  and (2) Second wavelength, λ2 (255 nm) at which the absorbance for AMLO was same as λ1, (3) Third wavelength, λ3 (241.6) and (4) Fourth wavelength, λ4 (217.6) at which the absorbance for INDA was same as λ3. In the proposed procedure the absorbance of AMLO and INDA in the mixture of AMLO and INDA was determined using dual wavelength data processing program. To remove the interference of AMLO to the absorbance at 255 nm λ2, the wavelength of the reasonable absorbance for INDA, another wavelength 360 nm λ1 was found out at which the absorbance of AMLO was same. While to remove the interference of INDA to the absorbance at 241.6 nm λ3, another wavelength 217.6 nm λ4 was found out at which the absorbance of INDA was same. This was confirmed by various dilution of AMLO and INDA in methanol at λ1, λ2 and λ3, λ4 respectively. The absorbance at these four wavelengths was found to be equal for AMLO and INDA. These four selected wavelengths were employed to determine the concentration of AMLO and INDA from the mixture of AMLO and INDA. The difference in absorbance at λ2 and λ1 (A255- A360) cancels out the contribution of absorbance of AMLO in measurement of INDA and the difference in the absorbance was proportional to the concentration of INDA in the mixture. While the difference in absorbance at λ4 and λ3 (A217.6- A241.6) cancels out the contribution of absorbance of INDA in measurement of AMLO and the difference in the absorbance was proportional to the concentration of AMLO in the mixture.


 

Fig. 1: Overlain absorption spectra of AMLO and INDA (20 µg/ml both) in methanol

 

 


.Fig. 2: Calibration curve of AMLO

 

Fig. 3: Calibration Curve of INDA


 

Table 1: Recovery data of proposed method (n=3)

Drug

Level

Amount of sample taken (µg/ml)

Amount of standard   spiked (µg/ml)

Total Amount recovered (µg/ml)

% Mean recovery±%RSD

(n=3)

AMLO

I

20

10

30.44

101.47±0.83

II

20

20

39.55

98.89±0.64

III

20

30

49.60

99.21±1.40

INDA

I

6

3

9.14

101.58±1.36

II

6

6

12.09

100.79±1.30

III

6

9

14.90

99.36±1.26

 

 


Table 2: Analysis of Formulation of AMLO and  INDA by Proposed   Method (n=6)

Sample No.

Label Claim

Amount Found

% Assay

AMLO(mg)

INDA(mg)

AMLO (mg)

INDA(mg)

AMLO(mg)

INDA(mg)

1

5

1.5

5.03

1.51

100.60

100.5

2

5

1.5

4.95

1.48

99.00

98.66

3

5

1.5

4.97

1.51

99.40

100.66

4

5

1.5

5.07

1.49

101.50

99.33

5

5

1.5

4.90

1.48

98.00

98.66

6

5

1.5

4.97

1.48

99.40

98.66

Mean

4.98

1.49

99.65

99.41

SD

0.061

0.014

1.23

0.94

 

Table 3:  Regression Analysis Data and Summaryof Validation Parameters for the Proposed Method

Parameters

AMLO

INDA

Wavelength  (nm)

Diff. in abs.(217.6-241.6)

Diff. in abs.(255-360)

Beer’s Law limit( µg /ml)

10-60

3-18

Sandell’s sensitivity

(µg/cm2/0.001 absorbance unit)

0.2857

0.1034

Regression equation (Y= mx+c)

Slop (m)

Intercept (c)

Y=0.006x-0.031
0.006

0.031

Y= 0.014x-0.014

0.014

0.014

Correlation coefficient (r2)

0.999

0.998

LOD (µg/ml)

0.89

0.22

LOQ (µg/ml)

2.71

0.67

Repeatability (RSD, n=6)

0.88

0.79

Precision (RSD)%

Intraday (n=3)

Interday (n=3)

0.34 – 0.63

0.61 – 0.82

0.38 - 0.64

0.63 – 0.86

Recovery (n=3)

99.85±0.95

100.57±1.30

Assay (n=6)

99.65±1.23

99.41±0.94

 


CONCLUSION:

The results of the analysis of pharmaceutical tablet formulation by the proposed method are highly reproducible and reliable and are in good agreement with the label claim of the drug. The additives usually present in the pharmaceutical formulations of the assayed samples did not interfere with determination of AMLO and INDA. The observations and results obtained from this study including

linearity, accuracy and precision (method precision as repeatability and intermediate precision as intra and inter day precision) are lie well within acceptable results. From the experimental studies it can be concluded that proposed method is sensitive, accurate and precise and can be adopted for the routine analysis of both drugs in tablet without interference of excipients.

 

ACKNOWLEDGEMENT:

The authors are thankful to Torrent Research Centre, Ahmadabad, India for providing gift sample of AMLO and INDA for research. The authors are highly thankful to Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidyanagar, Mehsana, Gujarat, India for providing all the facilities to carry out the work.

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Received on 27.04.2012       Modified on 19.05.2012

Accepted on 24.05.2012      © RJPT All right reserved

Research J. Pharm. and Tech. 5(6): June 2012; Page 795-799