Development and validation of stability indicating RP-HPLC method for simultaneous estimation of Atenolol and Nifedipine in bulk and pharmaceutical dosage form

 

Govindaraj Saravanan*, Sannihith Katari, Immadisetty Sri Krishnanjaneyulu, Dhanapal Visagaperumal

Department of Pharmaceutical Chemistry, Bapatla College of Pharmacy, Bapatla-522101, Andhra Pradesh, India. E-Mail: sarachem1981@gmail.com

*Corresponding Author E-mail: sarachem1981@gmail.com

A simple, rapid, selective, sensitive, linear, precise and accurate stability indicating RP-HPLC method was developed and validated for the simultaneous estimation of Atenolol and Nifedipine in pharmaceutical dosage form. Separation was attained on a STD Kromasil C18 (150 x 4.6 mm, 5 μ particle size) column at 30 °C using a mobile phase consisting of phosphate buffer (pH 5.0) and acetonitrile in the ratio of 45: 55 % v/v, at a flow rate of 1.0 ml/min. The UV detection wavelength was 232 nm and 10 μl of sample was injected. The linearity was found to be 15-90 μg/ml for Atenolol and 6-36 μg/ml for Nifedipine with a correlation coefficient of 0.9993 and 0.9994, respectively. Retention times were found to be 2.474 min and 4.553 min for Atenolol and Nifedipine, respectively. The overall mean % recoveries were found to be 99.69 % for Atenolol and 99.68 % for Nifedipine. The method was validated as per the ICH guidelines for sensitivity, linearity, accuracy and precision. The % RSD for precision, robustness and ruggedness of the proposed method was found to be less than 2 %. Further forced degradation studies were conducted for indicating the stability of the method developed. Hence the developed method can be successfully employed for routine quality control analysis of Atenolol and Nifedipine in pharmaceutical dosage forms.   

 

 

 

INTRODUCTION:

Chemically Atenolol is 2-(4-{2-hydroxy-3-[(propan-2-yl)amino]propoxy}phenyl)acetamide (Fig.1) with a molecular formula of C₁₄H₂₂N₂O₃ and molecular weight of 266.336. It is a white crystalline powder, soluble in water and freely soluble in ethanol and DMSO. It is a cardiovascular agent belonging to the group of β-blockers (particularly selective β₁ receptor antagonist) used in the treatment of hyper tension and heart failure. It works by slowing down the heart and reducing its workload.

 

Fig. 1: Structure of Atenolol

 

Nifedipine is chemically 3,5-dimethyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (Fig.2) with a molecular formula of C₁₇H₁₈N₂O₆ and molecular weight of 346.334. It is yellow crystalline powder, soluble in DMSO (50 mg/ml), ethanol (20 mg/ml) and insoluble in water. Nifedipine is a dihydropyridine calcium channel blocker that primarily blocks L-type calcium channels. Its main uses are as an anti-anginal (especially in Prinzmetal's angina) and antihypertensive. A large number of other indications have recently been found for this agent, such as Raynaud's phenomenon, premature labor and painful spasms of the esophagus such as in cancer and tetanus patients. It is also commonly used for the small subset of pulmonary hypertension patients whose symptoms respond to calcium channel blockers. It is on the WHO list of essential medicines, a list of the most important medication needed in a basic health system.

 

Fig. 2: Structure of Nifedipine

 

According to literature survey, various analytical methods were reported for the estimation of Atenolol^1-2 and Nifedipine^3-4 either alone or combination with other drugs^5-8. More over various analytical methods like UV,⁹ HPLC,¹⁰ HPTLC,¹¹ etc.¹² were reported for the simultaneous analysis of Atenolol and Nifedipine. Some RP-HPLC methods were not economical in terms of mobile phase composition, column dimensions and run times. Hence there is need for the development of newer method for estimation of Atenolol and Nifedipine present in tablet to overcome above discussed hurdles. So it is felt worthwhile to develop a simple, rapid, accurate, precise and more economical stability indicating high performance liquid chromatographic method for Atenolol and Nifedipine combination in bulk and its combined dosage form. Hence the present work is aimed to develop and validate the stability indicating assay method for Atenolol and Nifedipine using RP-HPLC technique. The present study was planned to develop a faster isocratic elution for estimation of Atenolol and Nifedipine. The developed method was planned to validate as per ICH guidelines in terms of accuracy, precision, specificity, limit of detection, limit of quantification, linearity, range and robustness. 

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Atenolol and Nifedipine were obtained from Spectrum Pharma Research Solutions, Hyderabad, India, as gift samples. Acetonitrile (HPLC grade), water (HPLC grade) and methanol (HPLC grade) were purchased from E. Merck (India) Ltd., Mumbai, India; while sodium dihydrogen orthophosphate (AR grade) and ortho phosphoric acid (AR grade) were purchased from Rankem, India.  

 

Instrumentation and chromatographic conditions:

The liquid chromatographic system consisted of Waters HPLC model (E-2695) with Empower 2 software containing variable wave length programmable UV detector. Shimadzu analytical balance, model (Unibloc) was used for weighing purpose. Chromatographic analysis was performed on STD Kromasil C₁₈ (150 x 4.6 mm, 5 μ particle size) column. The separation was carried at 30 °C using a mobile phase consisting of phosphate buffer (pH 5.0) and acetonitrile in the ratio of 45: 55 % v/v, at a flow rate of 1.0 ml/min. The UV detection wavelength was 232 nm, injection volume was 10 μl and runtime was 10 min. Mixture of acetonitrile and water in the ratio of 45: 55 % v/v was used as diluent.

 

Preparation of standard solutions:

Accurately weighed and transferred 6 mg of Atenolol and 2.4 mg of Nifedipine into a 10 ml clean dry volumetric flask, 7 ml of diluent was added and sonicated for 10 min to dissolve completely. Volume was then made up to the mark with diluent and filtered through 0.45 µ Millipore nylon filter to obtain a solution containing 600 μg/ml Atenolol and 240 μg/ml Nifedipine. Further 1 ml of  above standard stock solution was transferred into a 10 ml volumetric flask and made up to the volume with the diluent to obtain a solution containg 60 μg/ml of Atenolol and 24 μg/ml Nifedipine.

 

Preparation of sample solution:

20 tablets were accurately weighed and average weight of tablets were determined. The tablets were then crushed into a fine powder. Accurately weighed and transferred tablet powder equivalent to 50 mg of Atenolol and 20 mg of Nifedipine  into a 100 ml clean dry volumetric flask, 70 ml of diluent was added and sonicated for 30 min with intermittant shaking.Volume was then made up to the mark with the diluent and filtered through 0.45 µ Millipore nylon filter to obtain a solution containing 500 μg/ml Atenolol and 200 μg/ml Nifedipine. Further 1.2 ml of  both above sample stock solution was transferred into a 10 ml volumetric flask and made up to the volume with the diluent to obtain a solution containg 60 μg/ml of Atenolol and 24 μg/ml Nifedipine.

 

Fig. 3: A representative chromatogram of Atenolol and Nifedipine standard

 

Fig. 4: A representative chromatogram of Atenolol and Nifedipine sample

 

 

Method Development:

After several trails with various solvents, mobile phase system composed of phosphate buffer (pH 5.0): acetonitrile in the ratio of 45: 55 % v/v was chosen for the simultaneous estimation of Atenolol and Nifedipine in combined dosage form by RP-HPLC. This mobile phase composition offered good resolution for the drug at the detection wavelength of 232 nm and a flow rate of 1.0 ml/min gave less retention time and good peak shape. A reverse phase C₁₈ column was used as stationary phase. The retention time of Atenolol and Nifedipine were found to be 2.474 min and 4.553 min, respectively as shown in Fig. 3 and Fig 4. The run time was 10 min and total time of analysis was less than 5 min.

 

RESULTS AND DISCUSSION:

Method validation:

After method development, the validation of the proposed method has been performed in accordance with ICH guidelines which include accuracy, precision, specificity, linearity, limit of detection, limit of quantification, robustness and ruggedness.

 

System suitability:

System suitability was studied by injecting six replicates of the standard solution of 10 µl into the HPLC system after equilibrating the column with the mobile phase for 30 min prior to injection. The system suitability parameters were evaluated from standard chromatograms by observing theoretical plates, retention time, tailing factor and calculating % RSD of peak area and results obtained were within acceptable limits (Tailing factor below 2.0 and Theoretical plates > 2000) as shown in Table 1.

 

Table 1: System suitability parameters for Atenolol and Nifedipine

 

Specificity:

By comparing the chromatograms of blank, placebo, standard and sample, it was found that there is no interference due to excipients in the tablet formulation and also found good correlation between the retention times of standard and sample.

 

Precision:

The precision of the method was demonstrated by inter-day and intra-day variation studies by injecting six replicates of standard and sample solutions solution in to the chromatographic system and the response of drug peak and % RSD were calculated as shown in Table 2 and Table 3.

 

 

Table 2: Intraday precision study of Atenolol abnd Nifedipine

 

Table 3: Interday precision study of Atenolol and Nifedipine

Linearity:

Six point calibration graphs was constructed in the concentration range of 15-90 μg/ml for Atenolol and 6-36 μg/ml for Nifedipine by diluting aliquots of 0.25-1.50 ml of standard stock solutions into 10 ml volumetric flasks separately with the diluent. 10 µl of the standard solutions were injected into the HPLC system and analyzed. Linear relationship between the peak area response and the corresponding concentration was observed as shown in Fig.5 and Fig.6. The correlation coefficient, standard deviation of the slope and intercept were calculated as shown in Table 4 and Table 5.

 

 

Fig. 5: Linearity curve of Atenolol

 

Fig. 6: Linearity curve of Nifedipine

 

 

Table 4: Linearity data for Atenolol

 

Table 5: Linearity data for Nifedipine

 

 

Table 6: Accuracy data for Atenolol and Nifedipine

Level of accuracy	Atenolol			Nifedipine
	Peak area	% Recovery	Mean % recovery	Peak area	% Recovery	Mean % recovery
50 %	1169323	100.72	Mean: 100.39 S.D: 0.459 % RSD: 0.46	539576	100.53	Mean: 99.57 S.D: 0.925 % RSD: 0.93
	1167869	100.59		529676	98.69
	1159447	99.87		533996	99.49
100 %	2290288	98.64	Mean: 99.34 S.D: 0.851 % RSD: 0.86	1082424	100.84	Mean: 99.70 S.D: 1.023 % RSD: 1.03
	2328529	100.28		1066899	99.39
	2300835	99.09		1061216	98.86
150 %	3426629	98.38	Mean: 99.34 S.D: 1.053 % RSD: 1.06	1592457	98.90	Mean: 99.78 S.D: 0.858 % RSD: 0.86
	3499237	100.47		1607463	99.83
	3453868	99.17		1620064	100.62
Over all mean recovery	Mean: 99.69; S.D: 0.7876; % RSD: 0.793			Mean: 99.68; S.D: 2.806; % RSD: 0.94

 

 

Table 7: Robustness study of Atenolol and Nifedipine

 

 

Accuracy:

Accuracy of the method was determined by recovery studies. The solutions were analyzed in triplicate each level by spiking the sample with 50 %, 100 % and 150 % as per the proposed method. The % recovery and % RSD were calculated and results are shown in Table 6. Satisfactory recoveries were obtained by the proposed method which indicates that the proposed method is said to be accurate.

 

Robustness:

The robustness study was performed by changing variations in flow rate and temperature. The drug was analyzed under these experimental conditions and observed that there is no significant change in tailing factor and theoretical plates as shown in Table 7. 

 

Limit of detection and Limit of quantitation:

Limit of detection and limit of quantitation was determined by signal to noise ratio method using dilute solution of standard and results were shown in Table 8.

 

Table 8: LOD and LOQ of Atenolol and Nifedipine

 

Degradation studies:

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 24 h at room temperature. Further forced degradation studies were conducted for indicating the stability of the method developed. The results of the degradation studies are presented in Table 9 & the chromatograms are shown in Fig.7a-7f.

 

Fig. 7a: Chromatogram of acid degradation study

 

Fig. 7b: Chromatogram of base degradation study

 

Fig. 7c: Chromatogram of neutral degradation study

 

Fig. 7d: Chromatogram of peroxide degradation study

 

Fig. 7e: Chromatogram of heat degradation study

 

Table 9: Net % degradation of Atenolol and Nifedipine

 

Fig. 7f: Chromatogram of UV degradation study

 

 

CONCLUSION:

The new stability indicating RP-HPLC method was developed and validated as per guidelines for the simultaneous determination of Atenolol and Nifedipine in combined pharmaceutical dosage form. The proposed method was found to be accurate, precise, simple, economic, rapid and having good specificity. The developed method can be applied for the assay of commercial tablets containing Atenolol and Nifedipine in routine quality control analysis.

 

ACKNOWLEDGEMENTS:

Authors are thankful to the management of Bapatla College of Pharmacy for providing necessary facilities and Spectrum Pharma Research Solution, Hyderabad for providing gift samples of Atenolol and Nifedipine.

 

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Received on 23.11.2014         Modified on 06.12.2014

Accepted on 11.12.2014         © AJRC All right reserved