Application of Hydrotropic Solubilisation in Simultaneous Estimation of Atenolol and Amlodipine Besylate


Gurumurthy V., Deveswaran R.*, Bharath S., Basavaraj B.V. and Madhavan V.

M.S. Ramaiah College of Pharmacy, M.S.R. Nagar, M.S.R.I.T Post, Bangalore-560054, India

*Corresponding Author E-mail:



The present investigation was aimed to estimate atenolol and amlodipine besylate simultaneously from bulk drug and tablets using hydrotropic solubilization technique employing 4M urea as hydrotropic solvent. Atenolol showed maximum absorbance at 224.5nm, whereas amlodipine besylate showed maximum absorbance at 237nm. The solubility of both atenolol and amlodipine besylate was increased to greater extent in 4M urea. Both the drugs showed linearity in the concentration range of 4-20g/ml. The analysis of tablets indicated good correlation between the estimated and label claim. The values of LOD and LOQ were low, which confirmed the good sensitivity of the proposed method. The results of recovery studies showed that any small changes in drug concentration can be determined accurately by the proposed method. The method adopted is new, simple, accurate and cost-effective in the simultaneous estimation of atenolol and amlodipine besylate using hydrotropic solubilization phenomenon. This method can be successfully employed for routine analysis of atenolol and amlodipine besylate tablets.


KEYWORDS: Simultaneous estimation, Hydrotropy, Urea, Atenolol, Amlodipine besylate




Increasing the aqueous solubility of poorly soluble and slightly soluble drugs is of major importance1. This is a prime concern especially for Biopharmaceutical classification system- Class II drugs. Because of their low aqueous solubility and high permeability, dissolution is the rate limiting step in their absorption and systemic bioavailability. Hydrotropes are a class of chemical compounds which affect an increased aqueous solubility by several folds of drugs which are sparingly soluble in water under normal conditions2. Hydrotropy can be considered to be a potentially and industrially attractive technique since the observed increase in solubility is much higher than that affected by other solubilization methods3. A number of poorly water-soluble drugs have been solubilized by use of various aqueous hydrotropic solutions such as sodium benzoate, sodium salicylate, niacinamide, sodium hydroxide, sodium citrate, and urea4,5,6. Various organic solvents like methanol, chloroform, ethanol, dimethyl formamide, benzene, hexane, acetone, toluene, carbon tetrachloride, acetonitrile were widely used in spectrophotometric estimations of poorly water soluble drugs. But the organic solvents suffer from drawbacks such as higher cost, toxicity and sources of pollution7.


Simultaneous equation method is used when a sample contains two absorbing drugs(x and y) each of which absorbs at the λ max of the other (λ1 and λ2), it may be possible to determine both the drugs by this technique8. Atenolol is an anti-hypertensive, anti-anginal and anti-arrhythmic drug. Chemically it is 4-(2-hydroxy propyl-3-isopropyl aminopropoxy)-phenyl acetamide9. Amlodipine is calcium antagonist and chemically it is 5-methyl-(4RS)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxlate benzene sulphonate10. Combination of atenolol and amlodipine besylate was approved by the drug regulating bodies and the same are available in the market as tablets in different brand names. So it is necessary that estimation of these two drugs simultaneously by hydrotropic solubilisation would markedly reduce the cost of solvents used in analysis. With this objective the present study was aimed at developing a new, simple, accurate, cost-effective, environmental friendly simultaneous spectrophotometric estimation of atenolol and amlodipine besylate in bulk and tablets using 4M urea as hydrotropic solvent.




Shimadzu UV/Visible recording spectrophotometer (model-UV-1601) with 1cm matched silica cells was employed. Atenolol and Amlodipine besylate bulk drug samples were purchased from Yarrow Chem. Products Ltd, Mumbai. Tablets of atenolol with amlodipine besylate were procured from the local market. All other chemicals and solvents used were of analytical grade.


Experimental method:

Saturation solubility studies of the drug:

Solubility of atenolol and amlodipine besylate was determined separately by saturation aqueous solubility method in 4M urea and distilled water. An excess amount of drug was added separately to 50ml beakers containing 4M urea and distilled water. The beakers were shaken for 12 hours at 2810C. The solutions were filtered through Whatman filter paper # 41, and the resulting filtrates were suitably diluted and analyzed spectrophotometrically against solvent blank at their respective wavelengths.


Preparation of standard stock and calibration curve:

The standard stock solution of atenolol was prepared by dissolving 50mg of drug in 50 ml of 4M urea. From this solution 5ml of solution was taken and diluted to 100ml with distilled water to get a solution containing 50μg/ml and scanned in the entire UV range of 400-200 nm to determine the absorption maxima of the drug. The absorption maxima of atenolol were found to be 224.5 nm. Five working standard solutions having concentrations 4, 8, 12, 16 and 20μg/ml was prepared from the stock solution using distilled water. The absorbances of resulting solutions were measured at wavelength of 224.5 nm and a calibration curve was plotted to get the linearity and regression equation. Similarly stock solution of amlodipine was prepared using 4M urea and the solution was scanned in the entire UV range of 400-200 nm to determine the absorption maxima of the drug. The absorption maxima of amlodipine were found to be 237 nm. From the stock solution, five working standard solutions in the concentration range of 4-20g/ml were prepared and absorbances were measured, calibration curve was plotted.


Simultaneous equation method:

Simultaneous equation method was based on the absorption of drugs at the wavelength maximum of each other. Two wavelengths selected for the development of the simultaneous equations were 224.5nm for atenolol and 237nm for amlodipine besylate respectively. The absorptivity values E (1%, 1cm) determined at 224.5nm (λ1) for atenolol was found to be 0.0428 (ax1), 0.0207 (ax2) and at 237nm (λ2) for amlodipine besylate was 0.0123 (ay1), 0.0498 (ay2) and the isobestic point was determined. At λ1 and λ2 the absorbance of the mixture is the sum of individual absorbances of atenolol and amlodipine besylate. Based on this the following equations were framed.

At λ1, A1= ax1 bCX + ay1 bCy (1)

At λ2, A2=ax2 bCx + ay2 bCy (2)


Where, A1 and A2 are absorbances of mixture at 224.5nm and 237nm respectively. On rearrangement of the above equations,

Cx = (A2 ay1A1 ay2)/ (ax2 ay1ax1 ay2) (3)

Cy = (A1 ax2A2 ax1)/ (ax2 ay1ax1 ay2) (4)


Analysis of atenolol and amlodipine besylate in tablets using 4M urea:

Twenty tablets were weighed and powdered. Powder equivalent to 55mg, containing 50mg of atenolol and 5mg of amlodipine besylate was transferred to 50ml volumetric flask. To this 40ml of 4M urea was added. The flask was shaken for about 10min to solubilize the drug. Then volume was made up to the mark with distilled water. The resulting solution was diluted to 100 times with distilled water and the absorbances were measured at 224.5 nm and 237 nm respectively against solvent blank.


Validation of the proposed method:

The proposed method was validated for parameters like linearity, recovery studies, precision studies, LOD, LOQ and robustness11.



From the stock solution, serial dilutions were made and the absorbances of solutions were measured at the respective wavelength as per the developed method to confirm the linearity.


Recovery studies:

Tablet powder equivalent to 50 mg of atenolol was transferred to a 50ml volumetric flask containing 4M urea solution. Pure drug samples of atenolol weighing 20 mg were added to the same volumetric flask. The flask was shaken for 10min to solubilize the drug and filtered through Whatman filter paper # 41. The filtrate was diluted with distilled water appropriately and absorbance was measured at 224.5 nm against corresponding solvent blank. Drug content and percent recovery was calculated. Similarly tablet powder equivalent to 5mg of amlodipine besylate was transferred to a 50ml volumetric flask containing 4M urea solution. Pure drug samples of amlodipine besylate weighing 20 mg were added to the same volumetric flask. The flask was shaken for 10min to solubilize the drug and filtered through Whatman filter paper # 41. The filtrate was diluted with distilled water appropriately and absorbance was measured at 237 nm against corresponding solvent blank and percent drug content was calculated. Similar procedure was repeated using 40 mg and 60 mg of pure drug samples as spiked concentration.



Precision was determined by studying the repeatability and intermediate precision. The standard deviation, coefficient of variance and standard error were calculated for both the drugs.


Inter- day and Intra- day precision:

The intra-day concentration of the drugs was calculated on the same day at an interval of one hour, whereas the inter day concentration of drug was calculated on three different days within the laboratory conditions.


Limit of detection (LOD) and Limit of quantitation (LOQ):

The LOD and LOQ of atenolol and amlodipine besylate by the proposed method were determined using calibration standards. LOD was calculated by the formula 3.3σ/S, whereas LOQ was calculated by 10σ/S, where S is the slope of the calibration curve and σ is the standard deviation of response.



Both the drugs, atenolol and amlodipine besylate obeyed Beer-Lamberts law in the concentration range of 4-20g/ml at 224.5nm and 237nm respectively. The overlay spectra showed isobestic point at 235.5nm, whose absorbance is 0.428 (Fig.1). The solubility of atenolol and amlodipine besylate was found to have marked enhancement in hydrotropic solution of 4M urea as compared to distilled water. The solubility of pure atenolol in distilled water was found to be 1.85mg/ml, whereas in 4M urea, the solubility was found to be 19.48 mg/ml. Similarly solubility of amlodipine in distilled water was found to be 2.65mg/ml, whereas in 4M urea, the solubility was found to be 24.5mg/ml. The solubility of atenolol has been increased by more than 12 folds and the solubility of amlodipine has been increased by 10 folds. To check the drug stability and precipitation of drug in solvent, a part of solution were kept in room temperature for 48 hours and estimated.


Fig.1. Overlay spectra of atenolol and amlodipine in 4M urea.


The results revealed that estimation of atenolol and amlodipine can be done without substantial effect on drug stability as no precipitation was observed. From this study it is obvious that there was no interference of urea in estimation of drugs at their respective wavelengths. The estimated percent label claim in 4M urea was found to be 102.6% for atenolol and 102.4% for amlodipine besylate as shown in table 1, indicating good correlation between estimated claim and those claimed by the manufacturers. The recovery studies showed that the proposed method was accurate and reproducible.



Table 1: Analysis of tablet formulations of atenolol and amlodipine besylate

Tablet Formulation


Label claim


Amount founda


Percent label claim estimateda

(mean S.D)



Commercial tablet






Amlodipine besylate





a Average of six determinations


Table 2: Recovery studies of the proposed method

Tablet formulation

Amount of standard drug

added (mg)

Percent recovery estimateda (mean S.D.)

Standard Error


Amlodipine besylate

Commercial tablet













a Average of 3 determinations


Table 3: Optical characteristics data and validation parameters


Values for


Values for

amlodipine besylate

Working λmax (nm) in 4M urea



Beers law limit (μg/ml)



Molar Absorptivity



Correlation coefficienta









LODa (μg/ml)



LOQa (μg/ml)



Intra-daya (precision) (Co-eff. of variation)



Inter-daya ( precision) (Co-eff. of variation)






a Average of 6 determinations


The results of recovery study revealed that any small change in the drug concentration in the solution could be accurately determined by the proposed method (Table 2). Repeatability results indicated the precision under the same operating conditions over a short interval of time. Intra-day precision has a co-efficient of variation value of 0.3287 for atenolol and 0.131 for amlodipine besylate, whereas inter-day precision has a co-efficient of variation value of 0.331 for atenolol and 0.128 for amlodipine besylate. These low values of co-efficient of variation confirmed that there was not profound variation between analysis thereby proving the accuracy of the method. The value of LOD and LOQ was 0.298μg/ml and 0.904μg/ml for atenolol and 0.110μg/ml and 0.336μg/ml respectively indicated good sensitivity of the proposed method (Table 3).



Thus a new has been developed that is precise, simple, cost-effective, accurate and safe which has been successfully developed and validated. This proposed method that was developed on the principle of hydrotropic solubilisation concept can well be employed in routine analysis of simultaneous estimation of atenolol and amlodipine in tablets.



The authors are thankful to Gokula Education Foundation for providing necessary facilities to carry out the research work.



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Received on 21.11.2011 Modified on 18.12.2011

Accepted on 30.12.2011 AJRC All right reserved

Asian J. Research Chem. 5(1): January 2012; Page 57-60