Development and Validation of Ratio Spectra Derivative Spectrophotometric Method for Determination of Ternary Mixture of Aspirin, Atenolol and Amlodipine Besylate in Formulation


Bhadoriya A.S., Padekar S.S., Patil V.D., Shinde S.N., Choudhari V.P.* and Kuchekar B.S.

Deparment of Pharmaceutical Analysis and Quality Assurance, MAEER’s Maharashtra Institute of Pharmacy, Kothrud, Pune-411038, India.

*Corresponding Author E-mail:



A simple, economical, precise and accurate ratio spectra derivative spectrophotometric method for Aspirin(ASP), Atenolol(ATN), and Amlodipine besylate (AML) ternary combination on laboratory prepared tablet formulation has been developed.  The method is based on obtaining first derivative of ratio spectra and measuring amplitude at selected wavelength. The derivative amplitudes at 312.5 nm, 315.5 nm and 347.0 nm were selected for measuring the response of Aspirin (ASP), Atenolol (ATN), and Amlodipine besylate (AML), respectively. Beer’s law is obeyed in the concentration range of 20-100 μg ml-1 for ASP and 10-50 μg ml-1 for ATN and 1-5 μg ml-1 AML. The % assay was found to be in the range 98.0 – 101.0 % for ASP and 98.91 – 101.72 % for ATN and 98.71-101.2 % for AML by the proposed method. The method was validated with respect to linearity, precision and accuracy. Recovery was found in the range of 98.72 - 100.64 % for ASP and 98.45 – 101.21 % for ATN and 98.32-101.12 % for AML.


KEYWORDS: Aspirin, Amlodipine, Atenolol, Ratio Spectra, First Derivative.




The resolution of complex multicomponent systems without separation of the constituting analytes is rather a difficult task. The resolution of binary mixtures of compounds with overlapped spectra by derivative technique is frequently made on the basis of zero-crossing measurements1–3.


However, in certain cases, the derivative technique cannot cope with the level of the interference especially when the spectra are strongly overlapped or in case of ternary mixtures.

A spectrophotometric method for resolving binary mixtures has been reported4. The method is based on the use of the first derivative of the ratio of the spectrum5-6.


Chemically, Aspirin (ASP) is 2-acetoxybenzoic acid, Atenolol (ATN) is (RS)-2-{4-[2-hydroxy-3-(propan-2-ylamino)propoxy]phenyl}acetamide and Amlodipine besylate (AML) is (RS)-3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3, 5-dicarboxylate, are formulated in laboratory as tablets in the ratio of 20:10:1.


Drugs in combination used in Cardiovascular diseases, acts by different mechanism, ASP acts as antithrombotic agent, ATN is a selective β1−receptor antagonist, slowdowns the heart, reduce workload, AML is a long-acting calcium channel blocker. The combination is being prescribed for heart diseases and literature survey revealed that there is no analytical method reported for this combination. Therefore the combination was selected for the study.



2.1. Apparatus:

The spectrophotometric measurements were carried out on an UV-Visible double beam sspectrophotometer (Varian Cary -100) with 10 mm matched quartz cells were used. All weighing Shimadzu AUW-220D balance was used.


2.2. Materials and Reagents:

All materials and reagents were of analytical reagent grade. Pure drug sample of drug were kindly supplied as a gift sample by Vapi Care Pharma Pvt Ltd, Vapi Gurajat, (ASP), Zest Pharma, Indore, India (ATN), and Sunmour Pharmaceutical Pvt Ltd, Thane, India (AML). These samples were used without further purification.


2.3. Preparation of standard solutions and Construction of calibration graphs:

Stock solutions containing 0.1 mg/ml AML, 1 mg/ml ATN, and 2 mg/ml ASP were prepared in methanol. Further dilutions were done using methanol. Into three sets of 10 ml volumetric flasks, different aliquots of the standard solutions of ASP, ATN, and AML within the concentration range in Table 1, was transferred. The solutions were then completed to the volume with methanol. The absorption spectrum of each solution was recorded and stored. For construction of calibration curve, solution 1- 5 of ternary standard mixture and solution 6-8 of binary mixture have prepared in methanol as given in table-1.


2.6. Spectrophotometric measurements:

The combination of drug ASP+ATN+AML is available in ratio of 100+50+5 mg respectively, in the international market and was selected for analysis. Due to non availability of this drug combination locally, tablets were formulated in laboratory. The dilution of standard drug and formulation mixture were made in the ratio 20:10:1 μg ml-1 for ASP+ATN+AML respectively, and such five replicates were prepared in increasing concentration (solution 1- 5). The absorbance of the standard solutions were recorded within the wavelength range 200–400 nm and stored.


2.5. Preparation of Sample Solution and Formulation Analysis

Accurately weighed powder of 20 tablets equivalent to 25 mg of ASP (12.5 mg of ATN and 1.25 mg of AML) was weighed and dissolved in the 25 ml of methanol, sonicated for 10 min. and solution was filtered through Whatman paper No. 41 into a 100 ml volumetric flask. Filter paper was washed with methanol, adding washings to the volumetric flask and volume was made up to the mark with methanol. Resulting solution was further diluted with methanol to obtain solution having concentration 3 μg/ml, 30 μg/ml and 60 μg/ml of AML, ATN and ASP respectively. The sample solution was scanned in the wavelength range of 200-400 nm.




3.1. Assay conditions:

This mixture contains ASP, ATN and AML. The absorption spectra of the three components are strongly overlapped that no zero-crossing point is present to determine any component in presence of the other two. On the other hand, this spectral overlapping was sufficiently enough to demonstrate the resolving power of the proposed method.


Figure1. Chemical structure of drugs


3.1. Ratio spectra first derivative spectrophotometry:

The method involves the obtaining five UV-spectrum of the formulation in increasing concentration in the same ratio selected for study and dividing the spectras of mixture by spectrum of two analytes other than the target analyte having middle concentration (e.g. 60+30 mg of ASP+ATN for determination of AML, latter obtaining the first derivative of ratio spectra and measurement of derivative amplitude at 347nm). Likewise ratio spectra were obtained for ASP and ATN and derivative amplitude was measured at selected wave length (Table 1). Scheme of obtaining of ratio spectra first derivative of drugs in mixer shown in figure.


Figure 2. Zero order spectra of Aspirin, Atenolol, Amlodipine besylate (solution 5-8).


5 = Mixture of AML+ ATN + ASP (5+50+100),

6= ASP+AML(100+5),

7= ASP+ATN(100+50), 8= ATN+AML(50+5)



Figure 3. Ratio spectra of aspirin (20 – 100 μg/ml, solutions as per Table 1)



Sr. No. of solution

Drug conc.  in formulation/ standard  mixture (μg/ml)

Ratio Spectra obtained by dividing spectra obtained of solution 1 –  5 with…

1st order derivative Amplitude measured  at




Target Analyte

Divisor Analyte   mixture (conc. μg mL-1)






ATN+AML(30+3) (solution -6)

312.5 nm







ASP+AML(60+3) (solution -7)

315.5 nm










ASP+ATN(60+30) (solution -8)

347.0 nm














Table 1- Solutions used for acquiring Spectra of three drug mixture and two drug mixture for the method.


Table -2 Regression equation and results of assay, precision and recovery






Formulation Assay

( % RSD)

Tablet from Lot I

101.67  (0.698)

99.27 (0.618)

100.74 (1.46)

Tablet from  Lot II

99.45 (0.825)

100.24 (1.23)

99.67 (0.92)






















( %RSD)





Intra Day




Inter Day








1.87 (0.26304)

0.04 (0.13152)

0.092 (0.00396)





Correlation coefficient  (r)





















a=slope and b= intercept   SE= standard error


Figure 4. First derivative ratio spectra of Aspirin (20 – 100 μg/ml)


3.3. Method validation:

Method was initially applied to mixtures of standard drug solutions, after getting satisfactory results it was then applied for laboratory formulated tablet formulation.


Figure 5. First derivative ratio spectra of Atenolol(10 -50 μg/ml)


In order to test the accuracy of the proposed method, synthetic mixtures of each combination were prepared in different proportions. The resulting mixtures were assayed according to the above stated procedure and the results were calculated as the percentage of analyte recovered. The good recovery values and the low values of the standard deviation assure the high accuracy of the proposed method


The linearity of the proposed method was evaluated for each drug by analyzing different concentrations of each of ASP, ATN and AML, within the concentration range in Table 2 in the absence and presence of certain concentrations of other components in the mixture. The assay was performed according to the previously stated conditions. The corresponding first derivative amplitude for each drug was measured at the specific wavelength, and plotted against its concentration (Table 2). A straight line was obtained in each case. Statistical analysis of these graphs showed excellent linearity of the calibration graph and agreement to Beer’s law. Moreover, the slope was independent of the concentration of each component in the mixture.


Statistical data in the result shows precision, recovery and %RSD was always less than 2% indicate precision and accuracy of the method.


Method selectivity was detected by preparing different mixtures within the linearity range such that the mixture contains variable amounts of one component and constant amounts of the other two components. The mixtures were then analyzed according to the above stated procedures and the first derivative ratios were obtained. Statistical analysis of these data showed that the slope of the calibration graph for each drug is independent on the concentration of the other components (Table 2). It only shows that the first derivative amplitudes were only a function of the concentration of the assigned drug at the specified wavelength. Consequently, the results obtained verified the high selectivity of the proposed method and its potential for the simultaneous determination of this mixture.


Figure 6. First derivative ratio spectra of Amlodipine besylate (1 - 5 μg/ml)



The proposed method is simple, accurate, and reproducible for quantitative analysis of ASP, ATN and AML as a ternary mixture. The proposed method is simple as there is no need for solvent extraction and it estimates each component independent of the other. In addition, the method is rapid, of low cost, and harmless to the environment. So it could be applied in quality control laboratories where economy and time are essential.



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Received on 10.01.2010        Modified on 09.02.2010

Accepted on 12.03.2010        © AJRC All right reserved

Asian J. Research Chem. 3(3): July- Sept.  2010; Page 562-565