Scopus (CiteScore 2022 =3.0, Q3) , ISC

Document Type : Original Research Article


1 Department of Chemistry, College of Science, University of Mosul, Mosul, Iraq

2 Department of Chemistry, College of Education for Pure Science , University of Mosul, Mosul, Iraq


Atenolol, amlodipine, and furosemide are the most important medications that have been used to treat cardiovascular diseases. Due to this fact, we suggested a new method to estimate these three drugs in their pure form and their pharmaceutical dosages. The suggested spectrophotometric method was based on redox reaction of the mentioned drugs at the selected wavelength 516 nm of the resulting complex from the reaction of these three drugs in step I with cerium (IV) ions as an oxidizing agent in acidic medium, step II which is occurred between unreacted cerium (IV) with the reagent safranin. The linearity of this method was obeyed Beer’s law from (0.8-34), (1.6-40), and (2-40) μ of atenolol, amlodipine, and furosemide respectively, with molar absorptivity (2.66×104, 2.15×104, and 1.57×104) of atenolol, amlodipine, and furosemide, respectively. The limit of detection (LOD) is 0.915, 1.098, and 1.372 μ−1 for atenolol, amlodipine, and furosemide, respectively. The limit of quantification (LOQ) is 3.05, 3.66, and 4.57 μ−1 for atenolol, amlodipine, and furosemide, respectively. The method reveals a good accuracy and precision and it was applied successfully to the determination of these three drugs in pharmaceutical preparations. No interferences were observed from common additives found in pharmaceutical preparations. The Reddish-wine color of the product was highly stable and did not show a significant change in absorbance up to 60 min.

Graphical Abstract

A new spectrophotometric method to estimate atenolol, amlodipine, and furosemide in pharmaceutical dosages


Main Subjects


Patients with cardiovascular disease are commonly managed based on their medical conditions by clinicians by prescribing a cocktail of medications. Atenolol, amlodipine, and furosemide on their chemical structures depicted in Figure 1 are mostly involved as components of these cocktails [1]. These drugs have been used to treat cardiovascular diseases such as angina and hypertension. The first medication is atenolol, chemically named as (4-(2-hydroxy-3-isopropylaminopropoxy) phenyl-cetamide); it is commercially known as Tenormin. The second medication is amlodipine, or 2-[(2-aminoethoxy) methyl] -4-(2-chlorophenyl), which is trade named as Norvasc. Finally, furosemide or (4 chloro-N- [2-furyl methyl] -5-sulfamyl-anthranilic acid), the commercially name of furosemide is known as Lasix [1].

Many methods depending on various techniques were fixed in literature for estimating these three drugs, such as kinetic spectrophotometric method for estimating atenolol in dosage forms [2-7], a complex formation between atenolol and chloranilic acid depending on charge transfer reaction [8,9], or using sodium nitroprusside [10]. The other spectrophotometric method was based on dual wavelengths [11]. Flow-injection chemiluminescence was also used for estimated atenolol [12]. Furthermore, new techniques were utilized based on electrical method to assay atenolol [13,14]. Finally, chromatographic technique was used to determine atenolol in a dosage form [15].

Estimation of amlodipine in human plasma by HPLC, Liquid chromatography-mass, RP-HPLC, LC, and HPTLC method has been reported [7,11,16-24]. In addition, spectrophotometry technique have been used for estimating amlodipine via binary complex formation with eosin [25], or oxidation with cerium (IV) [26], charge-transfer complex formation with p-chloranilic acid [27], and 2, 3-dichloro 5,6-dicyano 1,4-benzoquinone and ascorbic acid [28]. The other methods were used for the determination of amlodipine in pure forms and tablet [29,30]. Furthermore, chromatographic methods have been reported to estimate amlodipine such as UHPLC method [31], HPLC [32,33], LC method [34], HPLC/MS/MS [35], HPTLC method [36], and RP-HPLC method [37].

Spectrophotometric method was studied for determination of furosemide depending on the use of charge transfer complex reaction [38], first order derivative [39], or the use of principal component regression [40].

Safranin is the reagent used to determine atenolol, amlodipine, and furosemide medications, as displayed in Figure 2. Safranin is a basic biological dye commonly used as a counter-stain in some staining protocols like gram staining. It is a crystalline solid with a characteristic green metallic luster and easily soluble in water [41].

In this paper, an indirect visible method was suggested to determine the studied drugs in pure and dosages form. These drugs were oxidized with cerium (IV) which was proportional inversely with safranin reagent, so that, the addition of an excess amount of cerium (IV) to the atenolol, amlodipine, and furosemide solutions in the presence of acidic medium, followed by the reaction of residual amount of cerium (IV) safranin reagent reduces the absorption of safranin dye which was measured at 516 nm.


Apparatuses: All spectrophotometric measurements were recorded in JASCOV-360 digital spectrophotometer equipped with 1-cm glass cells. Gilson micropipette with disposable tips was used to add samples, and A HANNA pH211 pH meter was employed to monitor the pH, and a BEL-sensitive balance was also employed to perform the appropriate weighing procedures.

Preparation of chemicals

Chemical solutions of a high degree of purity were used, atenolol, amlodipine, and furosemide drugs solutions have been prepared at 1000 µg/mL (SDI). When 0.1 g of these three drugs were dissolved in distilled water using a 100 mL volumetric flask, a simple dilution process of this solution was used to prepare working solution at 100 µg/ml, and then 0.002% of safranin reagent solution (BDH) was prepared when 0.002 g of safranin was dissolved in 100 ml distilled water. Oxidizing agent solution was prepared when 0.1353 g of ammonium cerric sulphate (Fluka) in 3 mL of concentrated sulphuric acid, and then the volume to the mark was completed with distilled water by using a 100 ml volumetric flask. Appropriate dilution of concentrated sulphuric acid with distilled water was used in a 100 mL volumetric flask to prepare 2N sulphuric acid solution. Finally, the preparation of dosage form [(Vascoten tablet) Edochemi LTD-Cyprus, Amloneer 5mg Tablet/Pioneer Co., Iraq, and furosemide 40mg/tab] was carried out by weighing ten tablets of each drug, and then it was grinded into a fine powder. After dissolving and filtering through filter paper, the solution was diluted to obtain 100 μg/mL as a concentration suitable for analysis [43].

Discussion of the results

To study the optimal conditions for the oxidation-reduction reaction between atenolol, amlodipine, and furosemide with an oxidizing agent and reagent, a 100 μg /25 mlof the mentioned three drugs have been used. The optimum condition of this reaction was studied and selected as follows:

Sulphuric acid

Sulphuric acid was selected after studying several types of acids, (acetic acid, hydrochloric acid, and sulphuric acid), which were prepared at a concentration of 2N, various amount (0.1-5.0) mL of sulphuric acid was studied, as depicted in Figure 3.

The practical results indicated in Figure 3, revealed that 2 mL of 2N sulphuric acid was adopted, depending on the highest absorbance value.

Oxidizing agent and time of oxidation process

Via the optimum amount of Ce(IV) ions prepared at 300 µg/ml, the optimum amount of Ce(IV) was estimated by taking a different quantity (1-4) mL of Ce(IV) solution in volumetric flasks of 25 mL containing different quantities of the studied drugs (50-700) µg, and then 2 mL of sulphuric acid was added at a concentration of 2N. After that, this mixture was left for 20 minutes (as an optimum time for completing oxidation process, Figure 4), 0.002% of safranin reagent was added, the experimental results proved that 1.5 mL of the oxidizing agent Ce (IV) gave the best value for absorption and correlation coefficient (0.9997), therefore 1.5 ml of Ce (IV) was selected for the subsequent experiments [6].

Optimum quantity of safranin

To determine the optimum amount of safranin used, Volumetric flasks of 25 mL were used containing different quantities ranging from (50-700) µg of each drug, and then sulfuric acid and the Ce(IV) ions were added in their optimal quantities, followed by the addition of various volumes (1-3) mL of 0.002 % safranin reagent, the measurements of the absorption intensity were carried out at the selected wavelength 516 nm after waiting 20 min to complete the oxidation step. The experimental results revealed that 2 ml of safranin reagent gave the best value for absorbance and correlation coefficient (0.9993). Therefore, 2 mL of 0.002 % safranin reagent was selected for the subsequent experiments [41].

Surfactants and addition sequence

The effect of adding different types of surfactants (positive, negative, and neutral) was studied with changing the sequence of addition, where the experimental results showed that the addition of all kinds of surfactants did not clear any effect on the intensity of absorption of the resulted visible complex, as represented in Table 1. Therefore, this study was neglected for the subsequent experiments [42].

Stabilization of the resulting complex

The effect of time on the intensity and stability of the resulted complex was studied for atenolol, amlodipine, and furosemide at 100 μ and 516 nm under the optimal conditions and for different periods of time at room temperature, as displayed in Figure 5, which indicates that the resulting colored complex is stable after 5 minutes and remained stable up to 2 hours.

Calibration curve and final spectrum

The present method was studied to determine the mentioned drugs under optimal conditions. Different quantities (25-1000) µg of these drugs were added to a series of 25 mL volumetric flasks, and then sulphuric acid, oxidizing agent, and reagent were added in their optimal quantities. After the dilution of all the flasks to the mark with distilled water, the measurements were carried out as shown in Figure 7 [10,30].

The proposed method follows Beer's law within at (20-850), (40-1000), and (50-1000) µg/25 mL of atenolol, amlodipine, and furosemide, respectively, as reveled in Figure 6, while Table 2 shows the values ​​of the analytical variables recorded at the selected wavelength for the present work.

Accuracy and precision measurements

The different concentrations of atenolol, amlodipine, and furosemide were studied for the purpose of verifying the accuracy and compatibility of the current method, as listed in Table 3 which indicates that the present method has a good accuracy and compatibility [42].

Continuous variations method

The reaction ratio of the studied drugs to the oxidizing agent and the oxidizing agent to the reagent ratio were studied by using the Job's method (continuous variables method), as shown in Figure 8 [12,38].

The results in Figure 5 indicate the reaction ratio between the cerium ions quaternary and the oxidizing agent is 1:1 depending on this fact the suggested equations, as shown below:

Medicines additives

Several chemical additives have been added to drugs in specific proportions to improve the smell, taste, or appearance of the pharmaceuticals, such as (gum acacia, starch, lactose, and gelatin). The effect of these substances on the proposed method was studied, as represented in Table 4, which indicated that the studied foreign substances do not interfere with the current method [9,40].

Application of the current method

The present work was applied for estimating the studied drugs (atenolol, amlodipine, and furosemide) in their pharmaceuticals, as indicated in Table 5.

The results shown in Table 5 reveal that the proposed method has good recoveries for the mentioned drugs. The t-test calculations of the present method when it compared with the literature methods indicate that t-test did not exceed the theoretical values at the 95% confidence level for five degrees of freedom, as represented in Table 6 [42].

Comparison of the method:

The current method has been compared with literature methods to determine atenolol, amlodipine, and furosemide, as depicted in Table 7 [38,42,43].

Table 7 indicates that the present method was sensitive and can be successfully applied for determining atenolol, amlodipine, and furosemide in their pharmaceuticals.


In this work, the most important drugs which were used to treat cardiovascular diseases have been studied by using a visible method, This method was depend on oxidation reduction reaction between atenolol, amlodipine, and furosemide and ceric (IV) ions in the presence of sulfuric acid and safranin dye. The reduced color of safranin was proportional to the concentration of the oxidizing agent and to the concentration of these three drugs, and thus has been measured at 516 nm. This method was applied in a very successful way to estimate atenolol, amlodipine, and furosemide in pure and pharmaceutical dosages forms.


We extend our sincere thanks to the University of Mosul, College of Science, Department of Chemistry.


Basima A. A. Saleem:


How to cite this article: Sahbaa Y. Majeed, Omar A. Salih, Basima A. A. Saleem*. A new spectrophotometric method to estimate atenolol, amlodipine, and furosemide in pharmaceutical dosages. Eurasian Chemical Communications, 2022, 4(12), 1285-1294. Link:


Copyright © 2022 by SPC (Sami Publishing Company) + is an open access article distributed under the Creative Commons Attribution License(CC BY)  license  (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



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