INTRODUCTION:

Losartan potassium, a potassium salt of 2-Butyl-4-chloro-1-[[2¢-(1H-tetrazol-5-yl) [1,1¢-biphenyl]-4-yl]methyl]-1H-imidazole-5-methanol [Figure - 1], represents the first of a new class of orally active non-peptide angiotensin II (Type AT₁ ) receptor antagonists employed in the management of essential hypertension^1,2. The individual determination of losartan has been carried out in tablets by HPLC, capillary electrophoresis and super-critical fluid chromatography³, in bulk and solid dosage forms by colorimetric method⁴, simultaneously with its degradates in stressed tablets by LC-MS/MS⁵ and HPTLC⁶and with its active metabolite in biological fluid by HPLC.^7-9

Atenolol, 4-[2-Hydroxy-3-[(1-methylethyl)amino]propoxy] benzeneacetamide is a selective β₁ -adrenoceptor antagonist¹⁰ applied in the treatment of numerous cardiovascular disorders such as hypertension and angina pectoris. Several analytical methods reported for the quantitative determination of atenolol individually in pharmaceutical formulations or in biological fluids, are HPLC^11-13, gas chromatography¹⁴, capillary zone electrophoresis¹⁵, titrimetry¹⁶ and spectrophotometry¹⁷.

No simultaneous spectrophotometric estimated of atenolol and losartan potassium in combine dosage forms in methanol solvent. Hence, an attempt has been made to develop new simultaneous UV methods for its estimation in pharmaceutical dosage formulations with good accuracy, simplicity and sensitivity.

MATERIALS AND METHODS:

Instrument:

A Shimadzu UV-1700 UV/VIS Spectrophotometer was used with 1 cm matches quartz cell.

Materials:

Gift samples of AT and LP were procured from Lupin Ltd Pune and Cipla Ltd Mumbai, respectively. Tablets containing both drugs i.e. Atenolol and Losartan Potassium were purchased from local pharmacy of commercial brand Losar*-Beta (Unichem Laboratory Ltd. Mumbai)

Solvent used: Methanol.

Preparation of stock Solution:

AT (2.5 mg) and LP (2.5 mg) were accurately weighed and transferred to two separate 50 ml volumetric flask, dissolved in Methanol solvent to obtained stock solution of 50 μg/ml each. The stock solutions of both the drugs were further diluted separately with solvent to obtain 10 μg/ml solutions each and scanned in spectrum mode from 400-200 nm. AT has λmax 226 nm while LP has λmax 208nm.

Fig.no 1 Wavelength range selected for Losartan potassium for Zero Order Method

Method I (Simultaneous Equation Method):¹⁸

From the stock solution, working standard solution of drugs were prepared by appropriate dilution and were scanned in the entire U.V. range .Two wavelengths selected for the method are 226 nm and 208 nm that are absorption maximas of AT and LP respectively in Methanol. A series dilution was prepared of standard solutions AT and LP 6-36 μg/ml and 2-16 μg/ml respectively. The absorptivity coefficients of AT within concentration range of 6-36 μg/ml and LP within concentration range of 2-16 μg/ml were determined at 226 and 208 nm by calibration curve.

Fig.no 2 Wavelength range selected of Losartan potassium for AUC method

For the estimation of drugs in the commercial formulations, ten tablets containing 50 mg of AT and 50 mg of LP were weighed and average weight was calculated. The tablets were crushed and powdered in glass mortar. Quantity of powder equivalent to 10mg of AT and 10 mg of LP was transferred to 100 ml volumetric flask, dissolved in sufficient quantity of methanol solvent, sonicated and volume was adjust up to mark with solvent to obtain a stock solution of 100 µg/ml of AT and 100 µg/ml of LP. This solution was then filtered through Whatmann filter paper # 41. Further dilutions were made from this stock solution to get required concentration. Absorbances of these solutions were measured at appropriate wavelengths, and values were substituted in the respective formula to obtain their respective concentrations. Results of tablet analysis are shown in Table No.2 the analysis procedure was repeated six times (n=6).

A set of two simultaneous equations as developed using these absorptivity coefficients as:

A₁ ay₂ – A₂ ay₁

Cx = ---------------------- . . . . . . . . . Eq. (i)

ax₁ay₂-ax₂ay₁

A₁ ax₂– A₂ ax₁

Cy = ---------------------- . .…………Eq. (ii)

a y₁ax₂ - ay₂ax₁

Where, C_xand C_y are concentrations of AT and LP respectively in μg/ml in sample solution. A1 and A2 are absorbances of the sample solution measured at 226 and 208 nm respectively. The absorbances (A₁ and A₂) of the sample solutions were recorded at 226 and 208 nm, respectively and concentration of both components were calculated using above mentioned equations (1 and 2)

Fig.no.3 Wavelength selected of Atenolol for Zero order method.

Method II (Area Under Curve Method):

For the selection of analytical wavelength solution of AT and LP (10 μg/ml each) were prepared separately by appropriate dilution of stock solution and scanned spectrum mode from 400 to 200 nm. Area under the curve in the range of 221-231nm (for AT) and 203-213nm (for LP) were selected for the analysis. The calibration curve for AT and LP were prepared in the concentration range of 6-36 μg/ml and 2-16 μg/ml at their respective AUC range. The calibration curve was plotted against concentration v/s area.

Table No:1 Optical Characteristics and Validation data of Atenolol and Losartan potassium.

Parameters	Atenolol		Losartan potassium
Parameters	Method I	Method II	Method I	Method II
Working λ (in methanol )	226	221-231	208	203-213
Beer Lamberts Law range	6-36 μg/ml	6-36 μg/ml	2-16 μg/ml	2-16 μg/ml
Molar absorptivity (lit/mole/cm)	5774.50	86268.933532	24816.89	396047.67433
Sandell’s sensitivity(mcg/sq cm/0.001)	0.046123	0.003087	0.018576	0.001164
LOD	1.23	3.31	0.7	1.32
LOQ	6.69	6.97	3.5	2.12
Slope	0.034329(at226), 0.021681(at 208)	0.323(at221-231) 0.245(at203-213)	0.091024(at208), 0.059202(at226)	0.859(at203-213) 0.856(at221-231)
Regression coefficient(r²)	0.999799(at 226) 0.999763(at 208)	0.999(221-231) 0.999 (203-213)	.999612(at 208) .999768(at226)	0.999(203-213) 0.996(221-231)

Table No:2 Result of analysis of tablet Formulation:

Methods	Drugs	Label claim	% label claim	S.D.^*	R.S.D.^*	%Recovery^*
I	AT	50	102.06	0.522	3.25	99.33
	LP	50	103.45	0.3912	1.224	99.52
II	AT	50	100	0.0031	0.67	99.02
	LP	50	100	0.00207	0.11	101.93

* indicates mean of six determinations.

For the estimation of drugs in the commercial formulations, ten tablets containing 50 mg of AT and 50 mg of LP were weighed and average weight was calculated. The tablets were crushed and powdered in glass mortar. The stock solutions were obtained 100 μg/ml AT and 100 μg/ml LP. This solution was then filtered through Whatman filter paper # 41. Further dilutions were made from this stock solution to get required concentration. Area of these solutions was measured at appropriate wavelengths, and values were substituted in the respective formula to obtain their respective concentrations. Results of tablet analysis are shown in Table No.2 the analysis procedure was repeated six times (n=6).

A set of two equation as developed using these absorptivity coefficients as:

₂₃₁

∫₂₂₁Adλ₁=k₁C₁+k₂C₂……..………………………………...3₁₃

∫₂₀₃Adλ₂=k₃C₁+k₄C₂……………………………….……….4

Where, area of curve between 221-231 nm and between 203-213 nm are represented by ∫Adλ₁and ∫Adλ₂ respectively, C₁ and C₂ are concentration of AT and LP respectively in μg/ml k₁, k₂ ,k₃,and k₄are constants.

Validation:^18-20

The methods were validated with respect to accuracy, linearity, Sensitivity and Limit of detection (LOD) and limit of quantification (LOQ).

Accuracy (Recovery Test):

To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percent recovery for AT and LP, by both the methods, was found in the range of 98.94-100.25 (Table No.2)

Fig.no.4 Wavelength range selected for AUC method of Atenolol

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of AT and LP. For both the method, the Beer-Lambert’s concentration range was found to be 6-36 μg/ml and 2-16 μg/ml for AT and LP respectively.

Sensitivity:

High Molar absorptivity and low Sandell’s sensitivity for the respective method reveals that all these methods are highly sensitive. (Table no.1)

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

The LOD and LOQ of AT and LP were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines. The LOD and LOQ were found to be as in Table no.1.

RESULTS AND DISCUSSION:

The methods discussed in the present work provide a convenient and accurate way for simultaneous analysis of AT and LP. In simultaneous equation method, wavelengths selected for analysis were 226 nm for AT and 208 nm for LP. In area under curve method, the area under curve in the range of 221-231 nm (for AT) and 203-213 nm (for LP) were selected for the analysis. In both the methods linearity were observed in the concentration range of 6-36 μg/ml and 2-16 μg/ml for AT and LP respectively. In method I, concentration of the individual drug present in the tablet sample solution was determined by solving the simultaneous equation at 226 nm and 208 nm using the respective absorptivity value. In method II, concentration of tablet sample solution were determine by solving two equation at the range of 221-231 nm and 203-213 nm using respective absorptivity value. Percent label claim for AT and LP in tablet analysis, by both the methods, was found in the range of 98.52 % to 100.43 %. S.D. and R.S.D. for six determinations of tablet sample, by both the methods, was found to be less than ± 2.0 indicating the precision of both the methods. Accuracy of proposed methods was ascertained by recovery studies and the results are expressed as % recovery. Percent recovery for AT and LP, by both the methods, was found in the range of 98.94% to 100.25 %.The results of validation parameters shown in table no. 1 are satisfactory, indicates the accuracy of proposed methods for estimation of AT and LP. These methods can be employed for routine analysis of these two drugs in combined tablet dosage form. The results obtained for tablets and recovery study is summarized in table no. 2.

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal Dr. S. B. Bhise, Govt. College of Pharmacy, Karad, Dist. Satara, Maharashtra for providing necessary facilities. The authors also thankful to Lupin Ltd Pune and Cipla Ltd Mumbai for providing gift samples of drugs Atenolol and Losartan Potassium respectively.

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Received on 23.06.2010 Modified on 05.07.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1050-1053