INTRODUCTION:

Atenolol is (figure. 1) chemically (RS) -4-(2-hydroxy-3-isopropylaminopropoxy) phenylacetamide. Atenolol is a beta-adrenergic receptor antagonist, or a more commonly known as a beta blocker. Atenolol is used to treat angina, hypertension and acute myocardial infarction, supraventricular tachycardia, ventricular tachycardia and alcohol withdrawal symptoms¹. Atenolol was the main beta blocker identified as carrying a higher risk of provoking type-2 diabetes². The literature survey reported that atenolol individually and combined with other drugs by Spectrophotometry^3-5, HPLC^6-7, HPTLC^8-9 and LC-MS^10-11 methods for estimation of atenolol in its pharmaceutical formulation. Thus the present study was undertaken to develop and validate a simple, sensitive, accurate, precise, and reproducible visible methods for atenolol

Figure No. 1 Structure of Atenolol

MATERIALS AND METHODS:

Instrument and materials:

The present work was carried out on ElicoSL164 UV Visible spectrophotometer having double beam detector configuration. Amlodipine besylate pure drug obtained from Spectrum Labs, Hyderabad as gift sample with 99.99% w/w assay value and was used without further purification. The absorption spectra of reference and test solution were carried out in a 1 cm quartz cell over the range of 400-800 nm. All chemicals of analytical grade used as it is.

Preparation of standard stock solution:

The standard stock solution was prepared by dissolving accurately weighed 100 mg of atenolol in methanol and the volume was made up to 100 ml with methanol (Stock solution-I, 1000 mcg/ml). 10 ml of solution took from stock-I and then diluted to 100 ml with water (Stock solution-II, 100 mcg/ml). 1ml of stock solution-II, 1 ml of concentrated hydrochloride, 1ml of 1% NaNo₂solution, 1 ml of 0.1% vanilline in a10 ml volumetric flask were added and diluted to 10 ml with distilled water so that to produce the concentration 10 mcg/ml. This method was done on an ice bath and maintain temperature below at 8⁰ C was transferred to a 10ml volumetric flask and the final volume was diluted to 10 ml with water, so that to produce the concentration 10 mcg/ml. The absorbance of red chromogen obtained was measured against respective blank solution in the visible region of 400-800 NM, which shows maximum absorbance at 650 nm.

Figure No. 2 Determination of λ-max of Atenolol React with Vanilline

Preparation of calibration curve:

Aliquots of standard solutions of atenolol ranging from 0.2-1.0 ml (1 ml = 100 mg) was transferred into a series of 10 ml volumetric flasks. The volume in each flask was made up to 10 ml with distilled water and the absorbances were measured at 650 nm against solvent blank. The obtained absorbance values when plotted against the concentration of atenolol give the calibration graph. The concentration of the unknown sample was determined from the calibration graph.

Preparation of sample solution:

20 tablets of one brand of atenolol were taken, and all the tablets were crushed to fine powder by using a pestle and mortar. Powder equivalent to 25 mg of atenolol was weighed accurately and transferred into a 25 ml standard volumetric flask. The contents were dissolved in 40ml of ethanol and make up to 100 ml of ethanol. Heating the resulting solution to 60⁰c and shake for 15 minutes and cool and sonicated for five minutes. This solution was filtered through 0.45 μm watchman filter paper. 10 ml of the filtrate was diluted to 100 ml with distilled water to get the solution of 100 mcg/ml. An aliquot of 1 ml of test solution, 1 ml of concentrated hydrochloride, 1ml of 1% NaNo₂solution, 1 ml of 0.1% β-napthol in a10 ml volumetric flask were add and diluted to 10 ml with distilled water so that to produce the concentration 10 mcg/ml. This method was done on an ice bath and maintain temperature below at 8⁰ C. The absorbance of red chromogen obtained was measured against respective blank solution in the visible region of 590-650 nm, which shows maximum absorbance at 650 nm.

Figure No 3- Calibration curve of atenolol (2-10mcg/ml).

RESULT AND DISCUSSION:

Precision:

The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogeneous samples. It provides an indication of random error results and was expressed as coefficient of variation (CV).

Intra and inter-day precision:

A variation of results within the same day (intra-day), variation of results between days (inter-day) was analyzed. Intra-day precision was determined by analyzing atenolol for five times in the same day at 365nm. Inter-day precision was determined by analyzing the drug daily once for five days at 650 nm. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.16, 99.75 and 0.26, 100.00 respectively (Table no. 4).

Accuracy (Recovery Test):

Accuracy is the closeness of the test results obtained by the method to the true value. The recovery experiments were performed by adding known amounts to the tablet. The recovery was performed at three levels, 50, 100and 150% of atenolol standard concentration. The recovery technique was performed to judge the accuracy of the proposed method. For this, known quantities of the atenolol solution were mixed with definite amounts of pre-analyzed formulations and the mixtures were analyzed. The total amount of atenolol was determined by using the proposed method and the amount of added drug was calculated by the difference. The recovery values for atenolol ranged from 99.97 ± 0.3969 (Table no. 3) was studied by recovery experiments.

Table No.1 - Calibration curve parameter

S.No.	Concentration (mg/ml)	Absorbance ± SD	% Relative standard deviation
1	2	0.410± 0.046	1.4
2	4	0.826± 0.053	1.8
3	6	1.230± 0.042	1.6
4	8	1.642± 0.064	1.5
5	10	2.080± 0.073	1.1

Linearity:

The linearity of the response of the drug was verified at 2 to 40 _g/ml concentrations, but linearity was found to be between 5-25 g/ml concentration. The calibration graphs were obtained by plotting the absorbance versus the concentration data and were treated by linear regression analysis (Table no. 2). The equation of the calibration curve for atenolol obtained Y = 0.0163C--0.00120, the calibration curve was found to be linear in the precedent concentrations. The correlation coefficient (r2) of determination was 0.9999.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ of amlodipine besylate were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines¹².The LOD and LOQ Was found to be as in table no.2.

Table No. 2 - Validation parameters

Sr. No	Parameter	Result
1	Absorption maxima (nm)	650
2	Linearity Range (mg/ml)	2-10
3	Standard Regression Equation	Y=0.2071C - 0.0043
4	Correlation Coefficient (r2 )	0.9999
5	Molar absorptivity	0.468 X104
6	Accuracy (% Recovery ±SD)	99.5± 0.16
7	Precision	99.75% (Intra-day precision) and 100.0% (Inter-day precision)
8	Specificity	A 10 mg/ml solution of candidate drug in water at Visible detection of 650 nm will show an absorbance value of 2.080 ± 0.073
9	Sandell’s Sensitivity (mg/cm2/0.001absorbance unit)	0.0048
10	LOD (mg/ml)	0.341
11	LOQ (mg/ml)	1.035

Table No. 3 - Determination of Accuracy by percentage recovery method

Ingredient	Tablet amount (mg/ml)	Level of addition (%)	Amount added (mg)	Drug found (mg/ml)	% Recovery	Average % recovery
Atenolol	10	50	2	1.99	99.5
	10	100	4	3.98	99.5	99.5± 0.16
	10	150	6	5.97	99.5

Table No.4 - Determination of Precision

Sample number	Assay of Atenolol	As % labelled amount
Sample number	Analyst-I (Intra-day precision)	Analyst-II (Inter-day precision)
1	99.71	99.79
2	99.83	99.85
3	99.84	99.78
4	99.51	99.93
5	99.61	100.23
6	100.10	100.42
Mean	99.75	100.0
RSD	0.16	0.26

CONCLUSION:

From the results the method described in this paper for the determination of Amlodipine besylate from tablet formulation is simple, accurate, sensitive and reproducible. The proposed method could be applied for routine analysis in quality control laboratories.

ACKNOWLEDGEMENTS:

We are thankful to Spectrum Labs at Hyderabad for providing the gift sample of Atenolol. We would also like to thank Dr. G. Vijay Kumar, Principle, A. K. R. G. College of Pharmacy for providing all the facilities to complete our work successfully.

REFERENCES:

1. Agon P, Goethals P, Van HD and Kaufman JM. Permeability of the blood brain barrier for atenolol by positron emission tomography. J Pharm Pharmacol. 43(8); 1991: 597-600.

2. Carlberg B, Samuelsson O and Lindholm LH. Atenolol in hypertension is it a wise choice. Lancet. 364(9446); 2004: 1684-9.

3. Lalitha G, Salomi P and Ravindra RK. Development of an analytical method and its validation for the analysis of atenolol in tablets dosage form by UV-Spectrophotometry. International Journal of Pharmacy and Pharmaceutical Sciences. 5(3); 2013: 197-199.

4. Lalitha KV, Jyothi RK, Padma B. UV spectrophotometric method development and validation of atenolol and losartan potassium by q analysis. International Bulletin of Drug Research. 3(4); 2013: 54-62.

5. Dey S, Sarkar S, Malakar J and Ghosh A. Spectrophotometric method for simultaneous of atenolol and atorvastatin in tablet dosage forms, International Journal of Pharm Biomedicl Research. 3(1); 2012: 40-43.

6. Vidhya BK and Sunil DR. Validated hplc method for simultaneous quantitation of amlodipine besylate, atenolol and aspirin in bulk drug and formulation. J P B M S. 17(9); 2012: 1-6.

7. Kavita J, Muralidharan S. Development and validation of new method for atenolol, hydrochlorothiazide and losartan potassium by RP-HPLC. International Journal of Chemtech Research. 2(2); 2010: 880-884.

8. Vidhya KB and Sunil RD. Validated hptlc method for simultaneous estimation of atenolol and aspirin in bulk drug and formulation. I S R N Analytical Chemistry. 2012, 10.5402/ 609706.

9. Nikita DP, Anandkumari DC, Kreny EP. Development and validation of hptlc method for simultaneous determination of atenolol and losartan potassium in bulk and in pharmaceutical dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 5(2); 2012: 325-331.

10. Sridharan D, Thenmozhi A, Sundarananda SV. Bioanalytical method development and validation of atenolol in human plasma by LCMS. Asian Journal of Pharmaceutical and Clinical Research, 3(2); 2010: 92-94.

11. Kallem RR, Mullangi R, Hotha KK, Spoorthy YN. Simultaneous estimation of amlodipine and atenolol in human plasma a sensitive LC-MS/MS method validation and its application to a clinical pk study. Bioanalysis. 5(7); 2013: 827-37.

12. Text on Validation of Analytical Procedures Q2 (R1) in, I.C.H. Harmonised Tripartite Guidelines; Nov. 1996.

Received on 07.05.2015 Modified on 10.06.2015

Asian J. Research Chem. 8(7): July- 2015 ; Page 445-448

DOI: 10.5958/0974-4150.2015.00071.1