1. INTRODUCTION:

Most hypertensive patients require more than one agent in order to achieve adequate blood pressure (BP) control. Fixed-dose combination antihypertensive treatments such as AT/LE have advantages over mono therapy including increased efficacy, reduced side effects and lower costs¹. Atenolol (AT) 1-3,(RS)-2-{4-[2-hydroxy-3-(propan-2-ylamino) propoxy] phenyl} acetamide is an analogue of acetamide, is a selective β1 receptor antagonist, a class of drugs used primarily in cardiovascular diseases. Atenolol is official drug in IP, BP and USP^2-4.

Lecarnidipine (LE) is chemically known as 1, 4-Dihydro-2, 6-dimethyl-4-(3- nitrophenyl)-3,5-pyridine dicarboxylic acid 2-[(3,3- diphenylpropyl)methylamino]-1,1-dimethylethyl methyl ester hydrochloride. Lercanidipine is a calcium channel blocker of the dihydropyridine class and is used in the treatment of hypertension⁵. Lercanidipine hydrochloride is official in Extra pharmacopeia Martindale⁶. Literature survey revealed HPLC^7-9, HPTLC¹⁰ and UV-Visible Spectro photometric methods^11-14 for the analysis of LE and AT as single component system or in combination with other drugs. One HPTLC¹⁵ Method and two analytical methods were reported for this combination of drugs by using UV Spectrophotometer^16-17.

For best of our knowledge, no method has been reported for simultaneous determination of Lecarnidipine hydrochloride and Atenolol by HPLC. Hence, an attempt has been made to develop new LC-UV method for its simultaneous estimation in pharmaceutical dosage form with good accuracy, precision and simplicity. The method proved to be simple since it does not contain a buffer system. The developed method is validated as per ICH guidelines¹⁸.

2. EXPERIMENTATION:

2.1 Equipment:

Chromatographic separation was performed on HPLC waters alliance 2695, having 2996 photo diode array detector and Rheodyne injector with 20µl loop volume. Empower software was applied for data collecting and processing.

2.2. Reagents and chemicals:

Acetonitrile HPLC grade were procured from E Merck (India) Ltd., Mumbai. Working standard of LE was provided by Glen mark pharmaceutical limited, Mumbai and AT was provided by Sun Pharmaceuticals Pvt. Ltd. Mumbai, India. Water HPLC grade was obtained from a Milli-Q RO water purification system. Tablet formulation manufactured by sun pharmaceutical industries (Lontesyl-AT) was purchased from local market containing AT(50mg) and LE (10mg) per tablet.

2.3Optimized chromatographic Condition:

A Phenomenex C₁₈ (250cm×4.6mm, 5µ) column was used as the stationary phase. The mobile phase comprised of methanol and water and in proportion of 75:25(v/v) with P^H adjusted to 4±0.2by using orthophosphoric acid. Injection volume was 20µl and run time was less than 5.00 min and flow rate 1.0ml/min. The column was maintained at ambient temperature and the eluent was detected at 257nm.

2.4 Standard preparation:

Standard stock solution (100µg/ml) of Atenolol and Lecarnidipine hydrochloride were prepared separately in mobile phase comprised of methanol and water and in proportion of 75:25(v/v) with P^H adjusted to 4±0.2 by using orthophosphoric acid The working standard solutions were prepared and further diluted in mobile phase to contain a mixture of Atenolol and Lecarnidipine hydrochloride in over the linearity range from60-160 µg/ml and 12-32µg/ml respectively.

2.5 Sample preparation:

Twenty tablets were weighed and finely powdered .A quantity of powder equivalent to 50 mg of AT and 10mg of LE was weighed and transferred to a 100 ml volumetric standard flask and added 50 ml of mobile phase. The sample was kept in an ultrasonic bath for 20 min and further diluted to 100ml by using mobile phase. Then it is filtered through 0.22µ membrane filter paper Two ml of this solution further diluted to 10 ml with same solvent to get 100µg/ml of AT and 20µg/ml of LE. 20µl of this solution was injected in to HPLC system and chromatograms were recorded. Concentrations of AT and LE in the tablet formulation were calculated by comparing area of the sample with that of standard. The percentage assay of individual drug was calculated and presented in Table 1.

3. RESULTS AND DISCUSSION:

The proposed HPLC method required fewer reagents and materials and it is simple and less time consuming. This method could be used in quality control test in Pharmaceutical industries. The chromatograms of standard and sample solution of AT and LE were shown in (Fig.1 and Fig 2). The chromatogram of placebo was shown in Fig.3.There was clear resolution between Atenolol and Lecarnidipine hydrochloride with retention time of 3.12 and 4.23 minutes respectively.

Table 1: Table for Assay

Tablet formulation

Drug

Amount present

(mg/tab)

Amount found* (mg/tab)

% label claim*

49.861

10.052

99.72%

100.53%

50.011

09.972

100.02%

99.72%

T1 and T2 are two different brands of tablet formulations. LE and AT denotes Lecarnidipine hydrochloride and Atenolol respectively.*each value is average of six determinations.

3.1. VALIDATION OF THE METHOD:

3.1.1. System suitability:

The column efficiency, resolution and peak symmetry were calculated for the standard solutions. (Table.2). The values obtained demonstrated the suitability of the system for the analysis of this drug combination and the system suitability parameters fall with in±3% standard deviation range during performance of the method. Here tailing factor for peaks of AT and LE was less than2% and resolution was satisfactory. The peaks obtained for AT and LE were sharp and have clear base line separation.

Fig 1: Typical Chromatogram of standard solution of Atenolol 100µg/ml and Lecarnidipine hydrochloride 20µg/ml

Fig 2: Typical Chromatogram of sample solution of Atenolol 100µg/ml and Lecarnidipine hydrochloride 20µg/ml

Fig 3: Typical Chromatogram of Placebo solution

Table No 2: System Suitability

S. No	Parameters	Atenolol	Lecarnidipine Hydrochloride
1	Capacity factor	1	1
2	Theoretical plate	2493	3720
3	Asymmetry of the peak	1.2	1.1
4	Retention time	3.12	4.19
5	Resolution	4.78

Table 3: Summary of analytical method validation

S. No	Parameters	Acceptance criteria	Atenolol	Lecarnidipine Hydrochloride
1	Linearity	r²=0.995 to1.0	0.9992	0.9991
2	Specificity	No interference with placebo	specific	specific
3	Accuracy(Recovery studies)	Recovery98.0-102.0%	99.97%	100.06%
4	Precision
	Intraday	RSD NMT 2.0%	0.1054	0.2560
	Interday	RSD NMT 2.0%	0.3124	0.3313
5	Robustness
	Change inflow rate	NMT±1%	0.4%	0.3%
	Change in mobile phase ratio	NMT±1%	0.2%	0.1%
	Change in p^H	NMT±1%	0.2%	0.3%
6	Limit of detection µg/ml	-----------------------	2µg/ml	5µg/ml
7	Limitof Quantification µg/ml	-----------------------	8µg/ml	20µg/ml

3.1.3Precision and Accuracy:

Recovery studies were carried out by applying the standard addition method. A known amount of standard AT and LE corresponding to 80%, 100%, and 120% of the label claim was added to pre analysed sample of tablet dosage form separately. The recovery studies were carried out six times, at each level of recovery. From the data obtained, recoveries of standard drugs were found to be accurate (Table.3.). The %RSD of interday and intraday precision obtained was less than2%for both the drugs. The intraday and interday precision of AT were found to be 0.1054 and 0.3124 and for LE 0.2560 and 0.3213 respectively. From the data obtained, the developed HPLC method was found to be precise and accurate.

Fig:4 Linearity curve of Lecarnidipine Hydrochloride

3.1.4 Specificity of the method:

The specificity of the method was checked for the interference of impurities in the analysis of a blank solution (without any sample) and then a drug solution of 20 μg/ml was injected into the column, under optimized chromatographic conditions, to demonstrate the separation of both LE and AT from any of the impurities, if present. As there was no interference of impurities and also no change in the retention time, the method was found to be specific and also confirmed with the results of analysis of formulation.

Fig:5 Linearity curve of Atenolol

3.1.5LOD and LOQ:

Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 ∂/S and 10 ∂/S, respectively as per ICH guidelines, where ∂ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3).The LOD for LE and AT was found to be 2µg/ml and 5µg/ml, respectively. The LOQ is the smallest concentration of the analyte which gives response that can be accurately quantified (signal to noise ratio of 10).The LOQ was 8µg/ml and 20µg/ml for CEF and OZ respectively. (Table 3).

3.1.6 Ruggedness and Robustness:

The ruggedness of the method was determined by carrying out the experiment on different instrument like Waters HPLC and Shimadzu HPLC by different operators using different columns of similar type like Phenomenex C18, Hypersil C18. Robustness of the method was determined by making slight changes in the experimental conditions such as the composition of the mobile phase, pH of the mobile phase, and flow rate of the mobile phase and the chromatographic characteristics were evaluated. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP-HPLC method developed, are rugged and robust.

4. CONCLUSION:

The developed RP-HPLC method for simultaneous determination of Atenolol and Lecarnidipine Hydrochloride can be used for routine analysis of both these components in combined dosage form.

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Received on 28.11.2010 Modified on 30.11.2010

Research J. Pharm. and Tech. 4(4): April 2011; Page 592-595