Zero Order and Area Under Curve Spectrophotometric methods for determination of Atenolol in Pharmaceutical Formulation.

Jadhav Santosh¹*, Patil Sanjay², Yeligar Virendra³, Tamboli Ashpak⁴

¹Department of Pharmaceutics, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India.

²Department of Pharmaceutics, Ashokrao Mane College of Pharmacy, Peth-Vadgaon-416112, Tal. Hatkananagale, Dist. Kolhapur, Maharashtra, India.

³Department of Quality Assurance Technology, Ashokrao Mane College of Pharmacy, Peth-Vadgaon-416112, Tal. Hatkananagale, Dist. Kolhapur, Maharashtra, India.

⁴Department of Pharmaceutical chemistry, Sahyadri College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India.

*Corresponding Author E-mail: jadhavsan88@gmail.com

ABSTRACT:

Simple, fast and reliable spectrophotometric methods were developed for determination of Atenolol in bulk and pharmaceutical dosage forms. The solutions of standard and the sample were prepared in Methanol. The quantitative determination of the drug was carried out using the zero order derivative values measured at 224 nm and the area under the curve method values measured at 222-228 nm (n=2). Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of Atenolol using 2-10μg/.ml (r²=0.9994 and r²=0.9995) for zero order and area under the curve spectrophotometric method. All the proposed methods have been extensively validated as per ICH guidelines. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations. Developed spectrophotometric methods in this study are simple, accurate, precise and sensitive to assay of Atenolol in tablets.

KEYWORDS: Atenolol, UV visible spectrophotometry, AUC, Method Validation.

INTRODUCTION:

Atenolol, chemically known as 4-(2-hydroxy-3-isopropylamino propoxy)phenyl acetamide is a cardio selective adrenoreceptor antagonistic drug used for anti angina treatment to relieve symptoms, improve tolerance and as an anti arrythemic to help regulate heart beat and infections. It is also used in management alcohol withdrawal in anxiety states, migraine, and prophylaxis.

Literature survey reveals that, several spectrophotometric method^[1-3]TLC- densitometry ^[4], UV spectrophotometric and HPLC-DAD methods^[5], HPLC method^[6-8]High Performance Thin Layer Chromatography–Densitometry ^[9], have been reported for the estimation of atenolol in pharmaceutical formulations. A few analytical methods were reported in literature for the determination of atenolol and other combination drugs which include spectrophotometric method ^[10-26], and spectrofluorimetric method.

To our notice, no UV- spectrophotometric method using Zero Order and Area under Curve (AUC) has been reported for the determination of Atenolol in bulk and tablets. Hence an attempt has been made to develop new Zero Order and Area under Curve Spectrophotometric methods method for estimation of Atenolol in bulk and pharmaceutical formulations with good accuracy simplicity, precision and economy.

MATERIALS AND METHODS:

Apparatus and Instrumentation

A Shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (CONTECH, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India).Calibrated volumetric glassware (Borosil®) was used for the validation study.

Materials

Reference standard of Atenolol API was supplied as gift sample by Lupin Laboratory Park Aurangabd. Methanol was getting from Research - Lab Fine Chem Industries, Islampur, Mumbai, Maharashtra. Tablet sample with label claim 25 mg per tablet were purchased from local market Pune.

Method development

Preparation of Standard and Sample Solutions:-

Stock solution of 10μg/ml of Atenolol was prepared in Methanol, for zero order and area under the curve spectrophotometric analysis. The standard solutions were prepared by dilution of the stock solution with Methanol in a concentration range of 02, 04, 06, 08, and 10μg/ml with Methanol for zero order and area under the curve spectrophotometric methods. Methanol was used as a blank solution.

Area under curve (Area calculation)

Area under curve method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelengths such as λ1 and λ2 representing start and end point of curve region. The area under curve between λ1 and λ2 was calculated using UV probe software. In this study area was integrated between wavelength ranges from 222 to 228 nm.

Area calculation: (α+β) =

Where, α is area of portion bounded by curve data and a straight line connecting the start and end point, β is the area of portion bounded by a straight line connecting the start and end point on curve data and horizontal axis, λ1 and λ2 are wavelength range start and end point of curve region^[27].

Assay Procedure:

Twenty tablets each containing 25mg of Atenolol were weighed crushed to powder and average weight was calculated. Powder equivalent to 10mg of Atenolol was transferred in 100 ml of volumetric flask. A 50 ml of Methanol was added and sonicated for 15minutes. Then solution was further diluted up to the mark with Methanol. The solution was filtered using Whatman filter paper no. 41; first 5 ml of filtrate was discarded. This solution was further diluted to obtain 15µg/mL solution with water subjected for UV analysis using Methanol as blank. Appropriate dilutions were made with methanol from stock solution for both zero order and area under the curve spectrophotometric methods.

RESULTS AND DISCUSSION:-

The zero order and area under the curve spectra for Atenolol were recorded at the wavelength of 224nm and 222-228nm respectively [Fig. 1 and 2].

Linearity and Range:

Under the experimental conditions described, the graph obtained for zero order and area under the curve spectra showed linear relationship. Regression analysis was made for the slope, intercept and correlation coefficient values. The regression equations of calibration curves were y=0.037x+0.0517 (r²=0.9994) at 224nm for zero order derivative spectrophotometry and y=0.008x+0.0086 (r²=0.9995) at 222-228nm for area under the curve spectrophotometry. The range was found to be 2-10μg/ml for both zero order and area under the curve spectrophotometric methods.

Table 1: Assay of tablet dosage form

Sr. No.	Sample Solution Concentration (µg/ml)	*Amount found (%) Zero derivative**	*Amount found (%) AUC**	Mean % Found zero derivative	Mean % Found AUC	%RSD zero derivative	%RSD AUC
1	15	98.91	98.21
2	15	99.89	97.23	99.50	97.94	0.5242	0.6373
3	15	99.71	98.39

*n=3, % RSD = % Relative Standard Deviation.

Fig. 3 Zero order derivative spectrum of Atenolol in Methanol dosage form (15µg/ml).

Fig.4 Linearity of Atenololby Absorbance

Fig.5 Linearity of Atenolol by AUC.

Table 2: Stastical data for the calibration graphs for determination of Atenolol by Proposed methods.

Parameters	Zero order derivative	Area Under the Curve
Linearity range (µg/ml)*	2-10	2-10
r²± S.D*	0.9994	0.9995

Accuracy

To study the accuracy of the proposed methods, and to check the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. The accuracy for the analytical method was evaluated at 80%, 100% and 120% levels of 15µg/ml standard solution. For Area under curve (AUC) was measured in wavelength range 222-228 nm and For Zero order derivative at 224nm and results were obtained in terms of percent recovery. Three determinations at each level were performed and % RSD was calculated for each level.

Precision:

To determine the precision of the method, Atenolol solutions at a concentration of 10μg/ml were analysed each three times for both zero order and area under the curve spectrophotometric methods. Solutions for the standard curves were prepared fresh every day.

Fig. 6 Zero order derivative overlay of Atenololat diff. Concentration.

Table 3: Accuracy results for Atenolol.

Accuracy level	Sample conc (µg/)	Std. conc	Total amnt. Added (µg/m)	%Recovery zero derivatie	% Recovery AUC*	Mean of Zero derivative*	Mean of Auc	% RSD Zero derivative	% RSD AUC
80	15	12	27	97.18	98.41
100	15	15	30	97.14	98.36	97.01	98.43	0.269	0.094
120	15	18	33	96.71	98.54

*n=3, % RSD = % Relative Standard Deviation.

Table 4: Results of Intra and Inter Day Precision

Parameters	Intra Day Precision		Inter Day Precision
Parameters	S.D*	% RSD*	S.D*	% RSD*
Zero derivative	0.002	0.4773	0.0011	0.2523
Area under the curve	0.0011	1.2505	0.0020	1.8206

Table 5: Summary of validation parameters

Parameter	Zero derivative	AUC
λ range	200-400 nm	222-228nm
Regression Equation (y=mx+c)	Y= 0.037x+0.0517	Y= 0.008x+0.0086
Measured wavelength	224 nm	224nm
Linearity range	2-10µg/ml	2-10µg/ml
Slope	0.0370	0.008
Intercept	0.0517	0.0086
Correlation coefficient (R²)	0.9994	0.9995
Limit of Detection (LOD) µg/ml	0.2962	0.2608
Limit of Quantitation (LOQ) µg/ml	0.8977	0.7905
Accuracy (Mean % Recovery)	97.01	98.43
Precission (%RSD)	0.4773	1.2505

Sensitivity:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the equations LOD = 3xσ/ S and LOQ = 10xσ/S, where σ is the standard deviation of intercept, S is the slope. The LOD and LOQ were found to be 0.2962μg/ml and 0.8977μg/ml respectively for zero order derivative and The LOD and LOQ were found to be 0.2608µg/ml &0.7905µg/ml for area under the curve methods respectively.

Analysis of the Marketed Formulation:

There was no interference from the excipients commonly present in the tablets. The drug content was found to be 97.01% and 98.43% zero order and area under the curve spectrophotometric methods respectively. It may therefore be inferred that degradation of Atenolol had not occurred in the marketed formulations that were analysed by this method. The low % R.S.D. value indicated the suitability of this method for routine analysis of Atenolol in pharmaceutical dosage form.

CONCLUSION:

No UV or Area under Curve spectrophotometric methods have been described for the determination of Atenolol. Therefore simple, fast and reliable derivative spectrophotometric methods were developed for the routine determination of Atenolol. The developed methods can be concluded as accurate, sensitive and precise and can be easily applied to the pharmaceutical formulation.

ACKNOWLEDGEMENT:-

The authors are highly thankful to the Sahyadri College of Pharmacy, Methwade, Sangola, Solapur, Maharashtra, India for proving all the facilities to carry out the research work.

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Received on 20.04.2015 Modified on 01.05.2015

Res. J. Pharm. Dosage Form. & Tech. 7(3): July-Sept., 2015; Page 185-189

DOI: 10.5958/0975-4377.2015.00027.0