RP-HPLC Method Development and Validation for Determination of Atenolol in Bulk Drug


Seema Jadhav*, Pournima Morey, Manisha Karpe, Vilasrao Kadam

Bharati Vidyapeeth’s College of Pharmacy, University of Mumbai, Sector-8, C.B.D., Belapur,

Navi Mumbai- 400614, Maharashtra, India

*Corresponding Author E-mail:- smjadhav226@gmail.com




Atenolol is a hydrophillic β-1 cardioselective adrenergic receptor blocker. It is prescribed widely in treatment of various cardiovascular diseases such as hypertension, angina pectoris, arrhythmias and myocardial infarction. A sensitive, simple, precise and accurate Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method was developed and validated for the analysis of Atenolol in bulk drug. The HPLC separation achieved on HiQSil C18HS (4.6 mm ϕ X 250 mm) at temperature of 25°C employing mobile phase consisting of 20 mM Ammonium Acetate Buffer (pH 5): Acetonitrile (80:20) with flow rate of 0.75 ml/min was used. The UV detection was done at 273 nm. The retention time was found to be 4.606 min. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to Linearity and Range, System suitability, Selectivity, Precision, Accuracy, Detection limit and Quantitation limit. The method was found to be simple, precise and accurate. The calibration curve was found to be linear in the concentration range of 5 – 40 µg/ml with r2 value of 1. The method was accurate (98.02 to 99.80 %), precise (the % Relative Standard Deviations (% RSD) of repeatability and intermediate precision were 0.3508 and 1.2135 respectively). The detection limit and quantitation limit by standard deviation method for Atenolol drug was found to be 0.075 μg/ml and 0.23 μg/ml.


KEYWORDS Atenolol, RP-HPLC, development and validation



Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice. Atenolol is chemically known as (RS)-4-[2-(hydroxyl-3-isoproplyaminopropoxy) phenyl]acetamide.1 Atenolol is a selective β1 receptor antagonist, a drug belonging to the group of beta blockers, a class of drugs used primarily in cardiovascular diseases.2 It is reported to lack intrinsic sympathomimetic activity and membrane-stabilising properties. Atenolol is prescribed widely in treatment of various cardiovascular diseases such as hypertension, angina pectoris, arrhythmias and myocardial infarction. Structure of atenolol depicted in figure-1.


Figure 1: Structure of Atenolol


Various analytical HPLC methods have been developed and validated for simultaneous determination of Atenolol with other drugs in formulation such as simultaneous determination of Atenolol and Hydrochlorothiazide3 in pharmaceutical dosage form, simultaneous determination of Atenolol and Nitrendipine4 in tablet dosage form, simultaneous determination of Atenolol and Lercandipine HCl in pharmaceutical dosage form and simultaneous determination of Atenolol and Amlopidine5   in tablet dosage form etc. Aim of this work to develop and validate a RP-HPLC method for determination of Atenolol alone in bulk drug.



Pharmaceutical grade ATE was supplied as a gift sample by IPCA laboratories, Mumbai. Methanol HPLC grade, Acetonitrile HPLC grade, Ammonium acetate AR grade and Acetic acid AR grade (S.D. Fine Chemicals Limited, Mumbai) were used. Acetonitrile, ammonium acetate buffer and double distilled water were filtered through 0.45 µm membrane filter and used for analysis.


An Agilent HPLC 1200 series equipped with Quaternary pump, Autosampler and UV visible detector was utilized. EZ Chrom Elite software was used for data acquisition and mathematical calculations. Column used was HiQSil C18HS (4.6 mm ϕ X 250 mm) at 25°C. Membrane filter was used to filter the mobile phase (PALL Life Sciences, Ultipor N66, Nylon 6, 6 membrane, 0.45 µm).


Preparations of different solutions for analysis:

·         Standard stock solution of Atenolol: Stock solution of concentration 1000 µg/ml was prepared by dissolving 50 mg of Atenolol in 50 ml of methanol HPLC grade and different concentrations were prepared by appropriately diluting stock solution with mobile phase.

·         5 M Acetic acid: 30 g of acetic acid AR grade was diluted to 100 ml with double distilled water to make final volume to 100 ml.

·         Ammonium acetate buffer (pH 5): Accurately weighed quantity of ammonium acetate for producing 20 mM ammonium acetate buffer was dissolved in double distilled water and pH was adjusted to 5 with 5 M acetic acid and final volume was made. Double distilled water, Ammonium acetate buffer (pH 5) and ACN HPLC grade were filtered through 0.45 µm membrane filter and degassed using a sonicator for 30 min.


Method validation 6, 7

Linearity and range:

The calibration curves were constructed in the range of 5 – 40 µg/ml. The linearity was evaluated by linear regression analysis which was calculated by least square regression method. The peak areas obtained were then plotted against concentration to get calibration curve.


System suitability:

The system suitability was assessed by six replicate analysis of the drug at concentration of 10 µg/ml. Different parameters such as number of theoretical plates, peak asymmetry and tailing factor were calculated from obtained data.



Placebo microspheres were prepared, containing the ingredients other than the drug and subjected to HPLC to check if any interferences from the excipients existed to interpret the selectivity of the method. 10 µg/ml solution of placebo microspheres was injected into the column.



Repeatability: Repeatability was assessed by using nine determinations of three different concentrations. Three concentrations of 10 µg/ml, 25 µg/ml and 40 µg/ml and three replicates of each were carried out to see variation in their peak areas within the same day.

Intermediate Precision: The intermediate precision was assessed by using nine determinations of three different concentrations. Three concentrations of 10 µg/ml, 25 µg/ml and 40 µg/ml and three replicates of each were carried out to see variation in their peak areas for consecutive three days.



Recovery studies were carried out by applying the method to a placebo sample (blank microspheres) to which the known amount of ATE had been spiked. Recovery study was performed by spiking 80%, 100% and 120% amount of standard drug (considering 10 µg/ml as 100%) externally to the placebo samples. Placebo microspheres stock solution was prepared by dissolving 25 mg dose equivalent microspheres in sufficient amount of ethanol diluting to make final volume of 25 ml. The experiment was conducted in triplicate. This was conducted to check the recovery of drug at different levels.


Detection limit and Quantitation limit:

Detection limit and Quantitation limit were calculated according to ICH guidelines using following equations,


Where, S = the slope of the calibration curve

             σ = the standard deviation (SD) of the response



Table 1: Optimized chromatographic conditions


Optimized condition

Mobile Phase

20 mM Ammonium acetate buffer (pH 5): Acetonitrile (80:20)

Flow rate

0.75 ml/min

Injection volume

20 ml

Column temperature



UV detector at 273 nm

Column used

HiQSil C18HS (4.6 mm ϕ X 250 mm)



The data obtained in the linearity experiment was subjected to linear regression analysis. The linearity of the peak area responses versus concentration was studied from 5 – 40 μg/ml. The regression equation obtained was y = 7715x – 2652 with r˛ = 1. The linearity range was 5 – 40 μg/ml. The results showed good correlation between peak area and concentration of ATE. The data meet the acceptance criteria for coefficient of regression i.e. r2 ≥ 0.999. Results of linearity are depicted in the Table 2. Standard chromatogram of drug showed retention time of 4.606 min (Figure 2). Calibration curve obtained from plot of different concentration vs area is shown in Figure 3.


Figure 2: Standard chromatogram of atenolol


Figure 3: Calibration curve of atenolol


Table 2: Linear regression data for calibration curves



Linearity range (µg/ml)

5 – 40

Coefficient of regression (r2) ± SD

1 ± 0.00057

Slope ± SD

7715 ± 10.06

y-Intercept ± SD

2652 ± 176.89


System suitability:             

The results obtained for system suitability parameters were within the range. The % relative standard deviation (% RSD) of the peak area and retention time for the drug was within 2%, indicating suitability of system. It can be concluded that the method and system are adequate for the analysis to be performed. Table 3 explains the system suitability parameters. ‘n’ is equal to number of replicates.


Table 3: System suitability parameters [Atenolol (10 µg/ml)]



USP Plate counts


USP Tailing factor


Peak asymmetry


Retention time (min) (n=6)


SD of retention time


% RSD of retention time


Average peak area (mAU) (n=6)


SD of peak area


% RSD of peak area




Placebo chromatogram did not show any peak for excipients at the retention time of Atenolol confirming the selectivity of method. These excipients did not interfere with the proposed method as seen from chromatogram. Placebo chromatogram is shown in Figure 4.


Figure 4: Placebo chromatogram



The results of repeatability and intermediate precision are shown in Table 4 and Table 5 respectively. The measured concentrations showed % RSD values within 2%, indicating that the proposed analytical technique has a good repeatability and intermediate precision.


Table 4: Repeatability (n=3)

Concentration in µg/ml

Average area ± SD


Average % RSD


73110 ± 557.95




187495.333± 281.19



302779.333± 421.82







Table 5: Intermediate precision (n=3)

Concentration in µg/ml

Average area ± SD


Average % RSD


73385.56 ± 304.002









Table 6: Recovery studies (n=3)

% Level recovery

Amount of standard spiked µg/ml


peak area ± SD

Amount of standard recovered (µg/ml)  ± SD

% Recovery

± SD




80 %


58950.33 ± 562.71

7.984 ± 0.07

99.80 ± 0.91



100 %


74345.66 ± 378.96

9.980  ± 0.05

99.80 ± 0.49


120 %


88097 ± 269.90

11.762 ± 0.03

98.02 ± 0.29





The results of accuracy studies are shown in Table 6. After spiking the placebo microspheres with the standard drug (80%, 100% and 120%), the proposed method afforded recovery in the range of 98.02 to 99.80 % for ATE from the microspheres. % RSD was found to be within limits, thus indicating that the method is accurate within the desired range.


Detection limit:

DL by SD of the response and slope was found to be 0.075 µg/ml.


Quantitation limit:

QL by SD of the response and slope was found to be 0.23 µg/ml.



A precise and sensitive HPLC method using 20 mM ammonium acetate buffer (pH 5) and ACN (80:20) with flow rate of 0.75 ml/min was developed and validated. System suitability showed % RSD less than 2%. Linearity over a concentration range of 5 – 40 µg/ml with coefficient of regression 1 indicates that the developed HPLC method was linear. The values of % RSD were less than 2 % indicating the accuracy and precision of the developed method. The % recovery was in the range of 98.02 – 99.80% for Atenolol. The results obtained on the validation parameter met the requirements. It inferred that the method was found to be simple, specific, precise and linear. The method was found to have a suitable application in routine laboratory analysis with a high degree of accuracy and precision.



Authors extend their appreciation and wish to thank IPCA Laboratories, Mumbai for providing gift sample of Atenolol for this work. Authors are also thankful to Bharati Vidyapeeth’s College of Pharmacy to provide necessary facilities to carry out this work.



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Received on 02.04.2013          Modified on 18.04.2013

Accepted on 15.05.2013         © RJPT All right reserved

Research J. Pharm. and Tech 6(7): July 2013; Page 736-739