INTRODUCTION:

Atenolol, 2-(4-{2-hydroxy-3-[(propan-2-yl) amino] propoxy} phenyl) acetamide (Fig. 1), is a white crystalline powder, slightly soluble in water but freely soluble in alcohol, Acetonitrile and practically insoluble in ether. It is commonly used as Antihypertensive Agent. Atenolol competes with sympathomimetic neurotransmitters such as catecholamines for binding at β₁-adrenergic receptors in the heart and vascular smooth muscle, inhibiting sympathetic stimulation which results in a reduction in resting heart rate, cardiac output, systolic and diastolic blood pressure, and reflex orthostatic hypotension. It is official in I.P and B.P, which recommend a HPLC method for analysis.^[1,2]

Indapamide, 4-chloro-N-(2-methyl-2,3-dihydro-1H-indol-1-yl)-3-sulfamoylbenzamide (fig. 2) is a white to light crystalline powder, insoluble in water, freely soluble in alcohol, acetonitrile. It is commonly used as diuretic and in hypertension and edema due to congestive heart failure. It is official in I.P and B. P, which recommend a HPLC method for analysis. ^[1,2]

Fig. 1: Structure of Atenolol

Atenolol and Indapamide combination Tablet was introduced in market for reducing the hypertension by their synergistic effect. Literature survey reveals that many analytical methods are reported for determination of Atenolol ^[3-6]and Indapamide^[7-10] individually or with other combinations. But there is only one HPTLC method¹¹ for determination of both drugs simulatenously. However, no method is reported for simultaneous estimation of these two drugs by Reverse Phase HPLC during the last decade.

This paper reports validated an efficient RP-HPLC method for simultaneous determination of Atenolol and Indapamide. The proposed method is simple, accurate, reproducible and suitable for routine determination of Atenolol and Indapamide in combined dosage form. The method was validated in compliance with ICH guidelines.^[12,13]

Fig. 2: Structure of Indapamide

MATERIALS AND METHODS:

Atenolol and Indapamide of pharmaceutical grade were kindly supplied as gift samples by Sun Rise Pharma, Ahemdabad and Markson Pharmaceuticals, Goa respectively and were certified to contain 99.89% (w/w) and 99.77% (w/w) respectively, on dried basis. Methanol and water used were of HPLC grade and were purchased from RANKEM Pvt. Ltd. Mumbai, India. The Tablet formulation containing 50mg and 2.5mg of Atenolol and Indapamide was procured from local market and used for analysis of marketed formulation. The liquid chromatographic system was of Shimadzu (Japan) LC 20AD which consisted of following components: a isocartic pump, variable wavelength programmable UV/Vis detector UV-SPD-M 20A, autoinjector- SIL-20AC. The chromatographic analysis was performed using LC solution software on a Phenomenex C₁₈ Analytical Column (25×0.46 cm, i.d, 5 µm). In addition, an electronic balance (Shimadzu AX200), VI), a sonicator (Spectra Lab, model UCB 40), a hot air oven (Labhosp), Shimadzu UV-1700 UV-visible double beam spectrophotometer were used in this study.

Preparation of Mobile Phase and Stock Solutions:

825.0 ml of Methanol (HPLC grade), 675 ml of water (HPLC grade) were mixed and 1.5 ml of Ammonium Hydroxide Solution was added and the mixed solution was sonicated for 15 minutes and filtered through 0.22 μm membrane filter and used as mobile phase Stock solution were prepared by taking 50mg and 2.5 mg each of Atenolol and Indapamide . The weighed drugs were transferred to 10 ml volumetric flask. Volumes were made up to the mark with methanol to obtain a solution containing 5000 μg/ml and 250μg/ml of Atenolol and Indapamide respectively. The HPLC analysis was performed on reversed-phase high-performance liquid chromatographic system with isocratic elution mode using a mobile phase of methanol:water (55:45, v/v) with 0.1% v/v of Ammonium Hydroxide on Phenomenex C₁₈ Analytical Column (25×0.46 cm, i.d, 5 µm) with 1ml/min flow rate at 260nm using UV detector.

Calibration curves for Atenolol and Indapamide:

Tablet formulation contains Atenolol and Indapamide in a ratio of 20:1. Appropriate aliquots of Atenolol and Indapamide stock solutions were taken in different 10 ml volumetric flasks and diluted up to the mark with mobile phase to obtain final concentrations of 4.5-500 μg/ml and 0.225-25 μg/ml of Atenolol and Indapamide, respectively. The solutions were injected using an autoinjector and chromatograms were recorded for 20μl. (Figure.1).

Fig. 3: Chromatogram of mixture of Atenolol and Indapamide

Calibration curves were constructed by plotting average peak areas versus concentrations and regression equations were computed for both the drugs (Table 1).

Table 1: Linear regression data for calibration curves

Parameters (Units)

ATENOLOL

INDAPAMIDE

Linearity range (μg/ml)

r²±SD

Slope ±SD

Intercept ±SD

Average of SE of

estimation

4.5-500

0.9999±0.005

2058.60±0.0577

179.20±0.0869

0.7026

0.225-25

0.9999±0.0005

17749.81±0.1026

1354.587±0.1753

1.0734

Analysis of Marketed Formulations:

About 1860 mg of Tablet powder containing 50 mg of Atenolol and 2.5 mg of Indapamide was accurately weighed and transferred into a 10 ml volumetric flask containing 5 ml methanol, sonicated for 15-20 minutes for dissolving the drug and was then diluted up to the mark with same solvent to get final concentrations of 5000 μg/ml and 250 μg/ml of Atenolol and Indapamide. The above solution was filtered using Whatman filter paper No 1. Appropriate volume of the aliquot was transferred to a 10 ml volumetric flask and the volume was made upto the mark with mobile phase to obtain a solution containing 500 μg/ml of Atenolol and 2.5 μg/ml of Indapamide. Appropriate volume was transffered to the vail for injecting 20μl volume of above sample solution into HPLC and peak areas were measured under optimized chromatographic conditions. (Figure.4)

Fig. 4: Chromatogram of market formulation of Atenolol and Indapamide

METHOD VALIDATION:

The method of analysis was validated as per the recommendations of ICH ^[13] for the parameters like accuracy, linearity, precision, detection limit, quantitation limit and robustness.

The accuracy of the method was determined by calculating percentage recovery of Atenolol and Indapamide. For both the drugs, recovery studies were carried out by applying the method to drug sample to which known amount of Atenolol and Indapamide corresponding to 50, 100 and 120% of label claim had been added (standard addition method). At each level of the amount 3 determinations were performed and the results obtained were compared.

Intraday and interday precision study of Atenolol and Indapamide was carried out by estimating the corresponding responses 3 times on the same day and on 3 different days for the concentration of 500μg/ml and 25μg/ml of Atenolol and Indapamide, respectively.

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formulae: LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve.

System suitability tests are an integral part of chromatographic method which is used to verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the drug solution at the concentration level 500μg/ml and 25μg/ml for Atenolol and Indapamide, respectively to check the reproducibility of the system and the results are shown in Table 2.

For robustness evaluation of HPLC method a few parameters like change in Analyst, Flow rate, percentage of methanol and water in the mobile phase were deliberately changed to estimate the effect at three levels (-5, 0, +5) with respect to optimized parameters. Robustness of the method was done at the concentration level 500μg/ml and 25μg/ml for Atenolol and Indapamide respectively (Table.3 and Figure 5-7)

Table 2: Summary of validation and SST parameters

Parameters (Units)

ATENOLOL

INDAPAMIDE

Linearity range (μg/ml)

Correlation coefficient

LOD (μg/ml)

LOQ (μg/ml)

Recovery (%)

Precision (%RSD)

Interday (n=3)

Intraday (n=3)

Robustness

Retention Time ± allowable time (min.)

Resolution

Tailing Factor (asymmetry factor)

Capacity Factor

Similarity Factor

4.5-500

0.9999±0.0015

0.025

0.07

99.87

0.064

0.4083

Robust

7.52± 0.1

2.863

1.253

2.03

1.00

0.225-25

0.9999±0.0005

0.03

0.09

99.67

0.470

0.5723

Robust

8.99±0.2

3.081

1.002

2.52

0.999

*SST stands for system suitability test.

Fig. 5: Chromatogram of Mixture of Atenolol and Indapamide for Analyst-1

Fig. 6: Chromatogram of Mixture of Atenolol and Indapamide for Analyst-2

Fig 7: Chromatogram of Mixture of Atenolol and Indapamide for Analyst-3

RESULTS AND DISCUSSION:

The mobile phase consisting of methanol: water (60:40) with 0.1%v/v of Ammonium Hydroxide, at 1ml/min flow rate was optimised which gave two sharp, well-resolved peaks with minimum tailing factor for Atenolol and Indapamide. The retention times for Atenolol and Indapamide were7.5 min and 8.99 min, respectively. UV overlain spectra of both Atenolol and Indapamide showed

that both drugs absorbed appreciably at 260 nm, so this wavelength was selected as the detection wavelength. The calibration curve for ATENOLOL and INDAPAMIDE was found to be linear over the range of 4.5-500 μg/ml and 0.225-25 μg/ml, respectively. The data of regression analysis of the calibration curves is shown in Table 1.The proposed method was successfully applied to the determination of Atenolol and Indapamide in their Tablet dosage form. The results for the combination were comparable with the corresponding labelled amounts. The developed method was also found to be specific, since it was able to separate other excipients present in tablet from the two drugs.

The LOD for Atenolol and Indapamide were found to be 0.025μg/ml and 0.03μg/ml, respectively, while LOQ were 0. 07μg/ml and 0.09 μg/ml respectively. The results for validation and system suitability test parameters are summarized in Table 2. Insignificant differences in peak areas and less variability in retention times were observed.

In the proposed study, RP- HPLC method was developed for the simultaneous determination of Atenolol and Indapamide and validated as per ICH guidelines. Statistical analysis proved that method was accurate, precise and robust. The developed method was found to be simple, sensitive and selective for analysis of Atenolol and Indapamide in combination without any interference from the excipients. The method was successfully used for determination of drugs in a pharmaceutical formulation. Assay results for combined dosage form using proposed method showed 98.30 ±1.04 % of Atenolol and 100.40±1.09% of Indapamide.

ACKNOWLEDGEMENTS:

The authors thank sun rise pharmaceuticals and markson pharmaceuticals, goa, ahemdabad for providing atenolol and indapamide as gift samples for this work.

REFERENCES:

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Received on 10.01.2012 Modified on 12.02.2012

Asian J. Research Chem. 5(3): March 2012; Page 405-408

Analyst	Retention time		%Assay
Analyst	ATENOLOL	INDAPAMIDE	ATENOLOL (%)	INDAPAMIDE (%)
ANALYST –1	7.57	8.98	97.01	102.17
ANALYST- 2	7.58	9.00	99.07	98.79
ANALYST -3	7.58	8.98	98.89	101.32