Simultaneous Estimation of Amlodipine besylate and Perindopril erbumine by UV Spectrophotometric Method


Shweta Purohit Nayak* and Sujit Pillai

GRY Institute of Pharmacy, Borawan, Khargone

*Corresponding Author E-mail:



Two simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous estimation of Amlodipine Besylate and Perindopril Erbumine in tablet dosage form have been developed. First method employs formation and solving of simultaneous equation using 237 nm and 206 nm as two analytical wavelengths for both drugs in methanol. The second method is Q value analysis based on measurement of absorptivity at 227 nm (as an iso-absorptive point) and 237 nm. Beer’s law obeyed in concentration range of 5-40 μg/ ml and 15-60 μg/ ml for Amlodipine and perindopril respectively. Recovery studies was found close to100 % for Amlodipine Besylate and Perindopril Erbumine for both the methods. The proposed method is recommended for routine analysis of both the drugs in combined dosage form.


KEYWORDS: Amlodipine Besylate (AB), Perindopril erbumine(PE), Simultaneous equation method, Q analysis.




Amlodipine, chemically, 2-[(2- aminoethoxy) methyl]- 4- (2-chlorophenyl) -1, 4-dihydro- 6-methyl-3, 5- pyridinedicarboxylic acid 3-ethyl, 5-methyl ester, is an antihypertensive and an antianginal agent in the form of the besylate salt1. Various analytical methods have been reported for the analysis of Amlodipine besylate2 in pure form as well as in pharmaceutical formulations. This includes high performance liquid chromatography,3-8 reversed phase high performance liquid chromatography,9-12 high performance thin layer chromatography,13-16 gas chromatography,17 gas chromatography–mass spectrometry,18 liquid chromatography with tandem mass spectrometry19 and fluorimetry,20 derivative spectroscopy,21,22 simultaneous multicomponent mode of analysis and difference spectrophotometry23-25.

Perindopril erbumine is the tert-butylamine salt of perindopril, the ethyl ester of a nonsulfhydryl angiotensin-converting enzyme (ACE) inhibitor. Perindopril (2S,3aS,7aS)-1-[(S)- 1-carboxybutylalanyl] hexahydro-2-indolinearboxylic acid 1- ethyl ester compound with tert-butylamine (1:1)26.


Few analytical methods for determination of perindopril in pharmaceutical formulations which include gas chromatography27, gas chromatography mass spectrometry28, radioimmunoassay29 and derivatization-gas chromatography and spectrophotometric methods have been reported, but none of the method for estimation of amlodipine and perindopril in combined dosage form has been reported.


In the present work an attempt has been made to develop and validate two simple, sensitive and reproducible methods for the simultaneous estimation of Amlodipine Besylate and Perindpril Erbumine from combined dosage form.




UV-Visible double beam spectrophotometer, Shimadzu model 1700 with spectral bandwidth of 1nm and a pair of 10 mm matched quartz cell was used. Pure sample of Amlodipine Besylate were obtained as gift samples from Cadila health care Ankleshwar, Gujarat and Perindopril Erbumine were obtained as gift samples from Cipla Ltd Mumbai. The commercially available tablets, COVERYL - AM (Label claim: amlodipine besylate – 5 mg, perindopril erbumine– 4 mg) manufactured by SERDIA Pharmaceuticals Mumbai was procured from the local market. Methanol analytical grade was used as a solvent was of analytical grade.


Preparation of Standard Drug Solution:

For the preparation of standard solution 50 mg of amlodipine besylate (AB) and perindopril erbumine (PE) was accurately weighted and dissolved in methanol in respective 50 ml volumetric flask and volume was made up to the mark. From the above stock solution, 5 ml of each solution was further diluted to 50 ml in a volumetric flask with methanol to get a working standard solution containing 100 µg/ml of each drug.


Determination of Absorption Maxima:

From the standard stock solution of both drugs 2.0 ml was further diluted to 10 ml in a volumetric flask to obtain the final dilution of 20 µg/ml of each drug. Both solutions were scanned in the UV range 400-200 nm to determine the λmax of both drugs and overlain spectra of the two drugs was obtained. The λmax of AB and PE was found to be 237 nm and 206 nm respectively. From the overlain spectra, the isobestic point was found to be 227 nm.(fig.1)


Fig. 1- Overlain spectra of Amlodipine Besylate and Perindopril Erbumine


Method I (Simultaneous equation method):

Two wavelengths selected for the method are 237 nm and 206 nm that are absorption maximas of AB and PE respectively in methanol. The stock solutions of both the drugs were further diluted separately with methanol to get a series of standard solutions of 5-40 µg/ml for AB and 15-60 µg/ml for PE concentrations. The absorbance of these dilutions were measured at the selected wavelengths and absorptivities (A 1%, 1 cm) for both the drugs at both wavelengths were determined as mean of three independent determinations. Concentrations in the sample were obtained by using following equations-

A1= aX1bcx + aY1bcY                               …………1

A2= aX2bcx + aY2bcY                               .….……..2

Where, A1 and A2 are absorbances of mixture at 206 nm and 237 nm respectively, ax1 and ax2 are absorptivities of AB at  206nm and 237nm respectively and ay1 and ay2 are absorptivities of PE at 206nm and 237nm respectively. Cx and Cy are concentrations of AB and PE respectively.


Method II (Absorbance ratio or Q-analysis method):

From the overlain spectrum of AB and PE, two wavelengths were selected one at 227 nm which is the isoabsorptive point for both the drugs and the other at 237 nm which is λmax of AB. The absorbances of the sample solutions prepared in a similar manner as in the previous method, were measured and the absorptivity values for both drugs at the selected wavelengths were also calculated. The method employs Q values and the concentrations of drugs in sample solution were determined by using the following formula,

For AB,

CX== (QM – QY)/ (QX – QY)*A1/aX1 …………3

For PE,

CY=   (QX – QY)/ (QY – QX)*A1/aY1…………4


               Absorbance of sample at 237 nm

Qm=  --------------------------------------------------------

                Absorbance of sample at 227 nm


               Absorptivity of AB at 237 nm

Qx =  --------------------------------------------------------

               Absorptivity of AB at 227nm


                  Absorptivity of PE at 237nm

Qy =----------------------------------------------------------

                   Absorptivity of PE at 227nm


A = Absorbance of sample at isoabsorptive point, a1 and a2 = Absorptivities of AB and PE respectively at isoabsorptive point.


Analysis of tablet formulation:

Twenty tablets containing AB 5mg and PE 4mg were accurately weighed and average weight per tablet was determined. Tablet were powdered and powder equivalent to 5 mg of AB was transferred to 10 ml volumetric flask and dissolved in 8 ml of solvent with frequent shaking for 15 minutes and then final volume was made up to the mark. The sample solution was then filtered through Whatmann filter paper No.41. The above solution was further diluted to get concentration 25 µg/ml of AB and 20 µg/ml of PE.


The absorbances of tablet sample solution were recorded at 237 nm and 227 nm respectively and concentration of two drugs in the sample were determined using the simultaneous equation (method I). The procedure was repeated five times with tablet formulation. Results of analysis are repeated in table.


For Method II, the concentration of both AB and PE were determined by measuring absorbance of the sample at 227 nm and 237 nm and values were substituted in the respective formula to obtain concentrations. Results of tablet analysis are reported in Table 3.


Table 1: Quantitative parameters for analysis of AB and PE.


Method I

Method II





Beer’s law limit(µg/ml )





Correlation coefficient (r)





Molar absorptivity (lit/mole/cm)















% Concentration*






Table 2: Results of analysis of tablet sample



Label Claim %

Label Claim  ±  R. S. D

Recovery* (Mean ± R.S.D.)





104.28 ± 0.05668

100.22 ±




99.45  ± 


99.97 ±






104.8 ±


100.51 ±




100.2 ±


99.24 ±


*Average of five determinations; R.S.D.; Relative Standard Deviation.


Table 3: Results of intermediate precisions.



Method I

Method II

% Label claim estimated

(Mean ± % R.S.D.)

% Label claim estimated

(Mean ± % R.S.D.)























*Average of  five determinations, R.S.D.; Relative Standard Deviation


Table 4: Results of Ruggedness



Amount Found (% ) ± RSD



Analyst I

Analyst II



100.20 ± 0.43

100.25 ±0.43



99.99± 1.50

98.99 ± 1.30



99.21 ± 1.21

99.81 ± 1.21



100.26± 0.52

100.25 ± 1.04



The method was validated according to ICH Q2B guidelines for validation of analytical procedures in order to determine the linearity, sensitivity, precision and accuracy for the analyte.


Accuracy: To check the accuracy of the proposed method, recovery studies were carried out at different concentration as per ICH guidelines. The recovery study was performed three times at each level.


Linearity: The linearity of measurement was evaluated by analyzing different concentration of the standard solution of AB and PE. For simultaneous equation method and Q analysis, the Beer- Lambert’s concentration range was found to be 5-40 µg/ml for AB and 15-60 for PE.



Precision was studied to find out intra and inter-day variations in the test method of AB and PE. Calibration curves prepared in medium were run in triplicate in same day and for three days. %RSD (relative standard deviation) were calculated which should be less than 2 %.



The overlain spectra of AB and PE exhibit λmax of 237 nm and 206 nm for AB and PE respectively which are quite separated from each other. Additionally one isoabsorptive point was observed at 227 nm. This wavelength was selected for simultaneous estimation of AB and PE for Q value analysis and it is assume to be sensitive wavelength. Standard calibration curves for AB and PE were linear with correlation coefficients (r) values in the range of 0.999 - 0.999 at all the selected wavelengths. The results show the % RSD values < 2.0% signifies the precision of the method. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by standard deviation of response and slope of calibration curve. LOD and LOQ of AB were found to be 0.1512, 0.458 for method I and 0.0696, 0.2111 for method II respectively. LOD and LOQ of PE were found to be 0.0603, 0.106 for method I and 0.182, 0.323 for method II respectively. System reproducibility was determined by five replicate applications and five times measurement of a laboratory mixture at the analytical concentration.  The accuracy of the method was confirmed by recovery studies from tablet at three different levels of standard additions; recovery in the range of 95 – 110% justifies the accuracy of method. The proposed method for simultaneous estimation of AB and PE in combined dosage form was found to be simple, accurate and rapid and can be employed for estimation of pharmaceutical formulations in quality control departments.



The most striking feature of this method is its simplicity and rapidity, non- requiring- consuming sample preparations such as extraction of solvents, heating, degassing which are needed for HPLC procedure. These are new and novel methods and can be employed for routine analysis in quality control analysis. The described methods give accurate and precise results for determination of Amlodipine besylate and Perindopril erbumine mixture in marketed formulation.



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Received on 24.09.2010       Modified on 12.10.2010

Accepted on 24.10.2010      © RJPT All right reserved

Research J. Pharm. and Tech. 4(5): May 2011; Page 735-738