*Corresponding Author:
N. Mallikarjuna Rao
Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, Kakinada-533 003, India
E-mail: mallimpharmmba@gmail.com
Date of Submission 06 Dec 2015
Date of Revision 29 Feb 2016
Date of Acceptance 04 Apr 2016
Indian J Pharm Sci, 2016;78(2):217-223  


A Simple, accurate, specific and rugged reverse phase liquid chromatographic method was developed for the simultaneous estimation of atenolol, lisinopril, aspirin and simvastatin in bulk and tablet dosage form. A reverse phase gradient program has been developed to separate all the four active ingredients. A gradient programming has been done using 0.05M Phosphate buffer pH 2.5 adjusted with dilute phosphoric acid, acetonitrile in the ratio 70:30 from 0 min to 10 min, further increase the acetonitrile ratio from 30 to 70 from 10 min to 20 min, on a reverse phase C8 column (250×4.6 mm, 5 µ) with a flow rate 1 ml/min, monitored at 232 nm. The mean retention times of atenolol, lisinopril, aspirin and simvastatin were found to be 3.9, 5.8, 9.5 and 18.3min, respectively. The linearity was established for atenolol 12.5 to 75 µg/ml, lisinopril 2.5 to 15 µg/ml, aspirin 18.75 to 112.5 µg/ml, simvastatin 5 to 30 µg/ml. The proposed method was validated in terms of linearity, range, accuracy, precision, specificity, robustness and ruggedness and the method was successfully applied to the estimation of atenolol, lisinopril, aspirin and simvastatin in combined tablet dosage form.


Atenolol, lisinopril, aspirin, simvastatin, HPLC-DAD, tablet

Atenolol (Tenormin) belongs to a group of drugs called β-blockers. β-blockers have an effect on the heart and circulation of blood flow through arteries and veins. It is used to treat angina (chest pain) and hypertension (high blood pressure). It is also used to treat or prevent heart attack. Lisinopril belongs to a group of drugs called angiotensin-converting- enzyme (ACE) inhibitors. ACE stands for angiotensin converting enzyme. Lisinopril is used to treat high blood pressure (hypertension), congestive heart failure, and to improve survival after a heart attack. Aspirin is a salicylate. It works by reducing substances in the body that cause pain, fever, and inflammation. Aspirin is used to treat pain, and reduce fever or inflammation. It is sometimes used to treat or prevent heart attacks, strokes, and chest pain (angina).

Simvastatin belongs to a group of drugs called HMG CoA reductase inhibitors, or "statins." It reduces levels of "bad" cholesterol (low-density lipoprotein, or LDL) and triglycerides in the blood, while increasing levels of "good" cholesterol (high-density lipoprotein, or HDL). Simvastatin is used to lower cholesterol and triglycerides (types of fat) in the blood. Simvastatin is also used to lower the risk of stroke, heart attack, and other heart complications in people with diabetes, coronary heart disease, or other risk factors.

Atenolol, lisinopril, aspirin and simvastatin are now among the most frequently prescribed agents for reducing morbidity and mortality related to cardiovascular diseases and analysis of these drugs is a current problem. The major therapeutic action of statin drugs is reduction of circulating atherogenic lipoproteins as a result of inhibition of 3-hydroxy- 3-methylglutaryl coenzyme A (HMG-CoA) reductase [1]. The key enzyme catalyzes the conversion of HMG-CoA to mevalonate, a critical intermediary in the cholesterol biosynthesis. This mechanism was discovered in 1976, when Endo and co-workers isolated a compound mevastatin from Penicillium citrinum that exhibited cholesterol-lowering effects [2]. Clinical studies have shown that these drugs significantly reduce the risk of heart attack and death in patients with proven coronary artery disease, and can also reduce cardiac events in patients with high cholesterol levels [3]. Beside lipid-lowering activity, statins improve endothelial function, maintain plaque stability and prevent thrombus formation. There is also an increased interest in statins nonlipid activities such as an anti-inflammatory action [3-8].

A variety of chromatographic methods were developed to resolve drugs and their related impurities in the bulk drug forms and pharmaceutical formulations. About all methods used for the severance of drugs are based on high performance liquid chromatography. In drug industry UV detection was most commonly used. This approach to the analysis was chosen most probably because drugs are not used in combination with other molecules during therapy (rationale for the development of new method in comparison to existing methods is not included). The chemical structures of the atenolol, lisinopril, aspirin and simvastatin are shown in fig 1.

IJPS-Chemical structures

Figure 1: Chemical structures of analytes.
Chemical structures of (a) atenolol, (b) lisinopril, (c) simvastatin and (d) Aspirin

Materials and Methods

Pharmacopoeia grade working standards of atenolol, lisinopril, aspirin and simvastatin were obtained as a gift from Mylan laboratories, Hyderabad, India. Fixed dosage combination tablet containing 50 mg atenolol, 10 mg lisinopril, 75 mg aspirin and 20 mg simvastatin was purchased from local market Hyderabad, India. All the chemicals were HPLC grade purchased from S. D. Fine-Chem Ltd., Mumbai. MilliQ water was used.

Waters e 2695 series HPLC consisting pump, Auto sampler, Auto injector, VWD and photodiode array detector, thermostatic column compartment connected with Empower 2 software connected with a Hypersil BDS C8 250×4.6 mm, 5 μ, 100 A.

Mobile phase

Potassium dihydrogen orthophosphate (6.8 g) was weighed and dissolved in 1000 ml of water. The pH was adjusted to 2.5 using dilute phosphoric acid. The solution is filtered through 0.22 μ nylon filter and sonicated to degas it. The buffer is considered as mobile phase A and acetonitrile was considered as mobile phase B. A gradient program represented in Table 1 was used to elute the analytes maintained at 30º and detection was carried out at 232 nm with an injection volume of 20 μl.

Time (min) Mobile Phase A Mobile Phase B  
0 70 30 Isocratic
10 30 70 Linear gradient
20 30 70 Linear gradient
30 30 70 Isocratic
35 70 30 Isocratic

Table 1: Gradient Table

Standard solution preparation

An equivalent of 50 mg of atenolol, 10 mg lisinopril, 75 mg aspirin and 20 mg simvastatin were weighed and dissolved in 100 ml volumetric flask, 60 ml of mobile phase was added and dissolved, further made the volume with mobile phase. Further dilutions were made from this stock solution.

Sample preparation

Twenty tablets were weighed and crushed and transferred the 100 mg tablet powder in to 100 ml volumetric flask. 60 ml of mobile phase, sonicated for 10 min to dissolve. Further volume was made with mobile phase. The resulting solution was filtered through 0.22 μ filter. Working standard of the analytes was prepared from the above solution.

Results and Discussion

In order to achieve good separation between all the four components, different buffer pH conditions and different proportions of solvents like methanol, acetonitrile and water containing binary and tertiary eluents. However, in 0.05 M phosphate buffer pH 2.5, pH adjusted with dilute phosphoric acid and acetonitrile achieved good satisfactory results at a flow rate of 1.0 ml/min measured at 232 nm as detection wavelength. The chromatogram of optimized standard mixture are shown in Figure 2.


Figure 2: Representative chromatogram of standards.
Representative chromatogram of standards, where atenolol at 3.97 min, lisinopril at 5.82 min, aspirin at 9.49 min and simvastatin at 18.36 min

System suitability is an integral part of the method validation to evaluate the parameters like tailing factor, theoretical plates, resolution and % RSD for replicate injections. The results were within the limits and were presented in Table 2 and Figure 2 shows the system suitability chromatogram. In the placebo chromatogram (Figure 3) there were no peaks observed at the retention times of atenolol, aspirin, lisinopril and simvastatin and also the degradation studies showed that there was no interference with degradants, peak purities were found to be >0.99 for the sample solution indicating that the method is specific.


Figure 3: Placebo chromatogram

Parameter Results Required limits
Atenolol Lisinopril Aspirin Simvastatin
RSD of peak area 0.17 0.21 0.93 0.49 <1.0 for n≥6
RSD of retention time 0.06 0.03 0.02 0.01 <1.0 for n≥6
USP Tailing factor (T) 0.99 1.28 1.20 1.15 T < 2
USP Plate Count (N) 2196 4138 5168 8221 >2000
USP Resolution (R)   5.04 8.08 12.95 R > 2

Table 2: System Suitability Results

To determine the accuracy of the proposed method, recovery studies were conducted. Known amount of pure drug concentrations was spiked in placebo at three different levels, i.e. 50, 100 and 150% and was calculated. Accuracy was calculated as the percentage of recovery. The results were tabulated in Table 3.

Parameter Amount Added (µg) Amount Recovered (µg) % of recovery
50% level 25 25.16 100.64
100%level 50 49.76 99.51
150%level 75 73.95 98.6
50% level 5 4.99 99.88
100%level 10 9.962 99.62
150%level 15 15.12 100.8
50% level 37.5 37.55 100.13
100% level 75 74.93 99.9
150% level 112.5 112.41 99.92
50% level 10 9.975 99.75
100% level 20 19.97 99.83
150% level 30 30.01 100.03

Table 3: Accuracy Data

Precision was evaluated at three levels, repeatability, reproducibility and intermediate precision. Each level of precision was investigated by six replicate injections of concentrations 50, 10, 75 and 20 μg/ ml atenolol, lisinopril, aspirin and simvastatin, respectively. The result of precision was expressed as % of RSD and was tabulated in Table 4.

Parameter RESULTS
Atenolol Lisinopril Aspirin Simvastatin
RSD of Retention time 0.06 0.03 0.02 0.01
RSD of Peak Area 0.45 0.43 0.46 0.45
RSD of Retention time 0.05 0.03 0.02 0.01
RSD of Peak Area 0.45 0.43 0.46 0.45
Intermediate Precision  
RSD of Retention time 0.07 0.02 0.03 0.02
RSD of Peak Area 0.48 0.43 0.47 0.46

Table 4: Precision Studies

Linearity was evaluated by measuring different concentrations (25 to 150%) of the standard solutions to atenolol, lisinopril, aspirin and simvastatin. The calibration curve was constructed by plotting concentration of standard solutions against mean peak areas and the regression equation was computed. The summary of the parameters is shown in Table 5.

Parameter Atenolol Lisinopril Aspirin Simvastatin
Linearity range (µg/ml) 12.5 to 75 2.5 to 15 18.75 to 112.5 5 to 30
Correlation co-efficient 0.999 0.999 0.999 0.999
Slope 33757 22110 12637 24803
Y-intercept -21686 -35913 -10006 -65938

Table 5: Regression Equation Parameters

Estimation of detection limit (DL) and quantitation limit (QL) considered the acceptable signal-to-noise ratios 3:1 and 10:1, respectively. The limit of detection and quantitation were determined as 0.7546 and 2.2869 μg/ml for atenolol, 1.4089 and 4.2695 μg/ml for lisinopril, 11.2096 and 33.9685 μg/ml for aspirin and 2.9398 and 8.9086 μg/ml for simvastatin, respectively.

The robustness of the method was unaffected when small, deliberate changes like, flow change, mobile phase composition, column temperature were performed at 100% test concentration. The results were shown in Tables 6 and 7.

Parameter Variation Chromatographic Conditions
Retention time Area
Flow Change 0.9 ml/min 3.969 5.827 9.518 18.393 1731399 2184824 9461021 4943624
1 ml/min 3.341 5.402 8.511 17.344 1631535 2082343 8876544 3834252
1.1 ml/min 3.005 5.058 7.908 17.533 1531342 2084323 8434534 3832344
Temp. Change 25° 3.357 5.512 7.344 18.545 1423255 2084212 8765434 3846767
30° 3.341 5.402 8.632 18.345 1535443 2112344 8423456 3834356
35° 3.339 5.499 9.435 18.453 1556877 2141445 8654332 3835345
Wavelength Change 230 nm 3.337 5.496 8.345 17.435 1233453 2034533 8423454 3235353
232 nm 3.341 5.402 8.545 17.546 1238676 2084424 8476544 3563633
234 nm 3.323 5.495 8.425 17.324 1223457 2083521 8465433 3345333

Table 6: Robustness Study

Parameter Variation Chromatographic Conditions
Theoretical Plates Tailing Factor
Flow Change 0.9 ml/min 2257 4303 5333 8501 0.98 1.30 1.21 1.15
1 ml/min 2123 4243 5213 7467 0.64 1.28 1.12 1.12
1.1 ml/min 2132 4212 5212 7643 0.65 1.22 1.18 1.12
Temp. Change 25° 2187 4223 5243 7533 0.75 1.23 1.17 1.13
30° 2185 4223 5233 7655 0.67 1.23 1.18 1.12
35° 2153 4243 5254 7543 0.75 1.24 1.19 1.11
Wavelength Change 230 nm 2132 4221 5224 7543 0.76 1.27 1.16 1.12
232 nm 2143 4243 5235 7554 0.78 1.28 1.20 1.14
234 nm 2113 4233 5243 7548 0.75 1.26 1.18 1.13

Table 7: Robustness Study

The stability of the standard solution was to test for an interval 24 and 48 h at room temperature. There were no significant changes observed in the system suitable parameters like theoretical plates, tailing factors, retention time and resolution. Hence the standard solution was stable up to 48 h at room temperature.

The stability of the mobile phase was to test for intervals 24 and 48 h at room temperature. There were no significant changes observed in peak areas, theoretical plates, tailing factors, retention time and resolution. Hence the mobile phase was stable up to 48 h at room temperature. The proposed method was applied for the analysis of atenolol, lisinopril, aspirin and simvastatin in tablet dosage forms, the results were found to be between 98 and 100%, and the results were summarized in Table 8.

Drug Labeled Amount
Amount found
% of Assay
Atenolol 50 49.41 98.83
Lisinopril 10 9.96 99.67
Aspirin 75 74.79 99.73
Simvastatin 20 19.92 99.61

Table 8: Assay Results

Stress studies were performed at concentrations 500 μg/ml atenolol, 100 μg/ml lisinopril, 750 μg/ml aspirin and 200 μg/ml simvastatin drug substances to provide an indication of the stability indicating property and specificity of the proposed method. Degradation was attempted under stress condition of heat (60º), acid (5.0 N HCl at 27º), base (5.0 N NaOH at 27º) and oxidation (2% H2O2 at 27º) to evaluate the ability of the proposed method to separate analytes from their degradation products. For heat studies, study period was 10 days whereas for acid hydrolysis approximately 40 h; base hydrolysis 30 h and oxidation 2 h. Peak purities of the stressed samples were checked by using PDA detector and the purity angle was found to be within the purity threshold limit in all stressed samples which demonstrates the homogeneity of analyte peak. Assay was calculated for spiked samples of analytes with respect to test concentration. Representative chromatograms of stress studies of various conditions were shown in Figure 4 and Table 9 shows the results of the experiments.


Figure 4: Chromatograms of Stress studies.
Chromatograms of acid degradation (a); base degradation (b); oxidative (H2O2) degradation (c) and thermal degradation (d), where atenolols at 3.96 min, lisinopril at 5.83 min, aspirin at 9.62 min and simvastatin at 18.36 min

  Atenolol %Rec Lisinopril %Rec Aspirin %Rec Simvastatin %Rec
Acid 1656065 96.42 2114192 96.98 9192434 97.7 4688306 95.31
Base 1662314 96.91 2085714 96.12 8949969 95.4 4717757 96.21
Peroxide 1662919 97.01 2106821 97.09 9008315 96.02 4747175 97.03
Heat 1596322 93.12 2072012 95.48 8866665 94.48 4660845 95.13

Table 9: Forced Degradation And Stability

A simple, specific and reliable isocratic HPLC-DAD method was developed for the estimation of atenolol, lisinopril, aspirin and simvastatin in their pharmaceutical formulation. The four compounds were subjected to forced degradation applying several stress conditions. The proposed method was successfully separated all the three compounds with degradants, estimate the active contents. The proposed method is specific and stability-indicating power. Hence the developed method can be adapted to regular quality control analysis.


The authors are thankful to Mylan laboratories, Hyderabad for providing standards and lab facilities. The authors are also thankful to Department of Pharmaceutical Analysis, J.N.T. University, Kakinada, Department of Pharmaceutical Analysis and Quality Assurance, Andhra University, Vishakhapatnam, India for encouragement.

Financial Assistance


Conflict of Interests

None declared.