INTRODUCTION:

Amlodipine besylate (AML) is chemically 3-ethyl 5-methyl (4RS)-2-[(2 aminoethoxy) methyl]-4-(2-chlorophenyl) 6- methyl-1, 4-dihydro pyridine-3, 5- dicarboxylate benzenesulphonate¹, is a Calcium channel blocker, used in the treatment of hypertension². It is official in IP, BP and USP, IP³, BP⁴ and USP⁵ describe HPLC method for its estimation. Literature survey reveals UV spectro-photometry⁶, RP-HPLC⁷, spectrophotometric⁸ method for simultaneous determination of AML with other drug and RP-HPLC⁹ method for simultaneous determination of AML with other drug methods for determination of AML in pharmaceutical dosage forms as well as in biological fluids. Indapamide (IND) is chemically 4-Chloro-N-[(2RS)-2-methyl-2, 3-dihydro-1Hindol- 1-yl]-3-sulpha moylbenzamide¹⁰ is a thiazide diuretics for the treatment of hypertension¹¹. Indapamide is official in IP¹², BP¹³ and USP¹⁴. IP, BP and USP describe HPLC method for its estimation.

Literature survey reveals LC-MS¹⁵, spectrophotometric¹⁶ and HPLC¹⁷ method for simultaneous estimation of INDA in whole human blood, RP-HPLC¹⁸ method for simultaneous estimation of INDA, LC-ESI-MS¹⁹ methods for the determination of INDA in human plasma. This combination is not official in any pharmacopoeia hence official and reported methods of analysis are not available for this combination. However, there is no work was reported for the simultaneous estimation of these drugs by UV spectrophotometry using Dual wavelength spectrophotometry, first order derivative spectrophotometry. Hence, in the present communication we propose fast, simple and accurate UV spectrophotometric method, without tedious extraction procedure, was developed for the simultaneous estimation of both drugs in tablet dosage form by multi-component mode of analysis.

MATERIALS AND METHODS:

Apparatus:

Double beam UV-visible spectrophotometer (Shimadzu, model 2450) having two matched quartz cells with 1 cm light path.

Reagents and solutions:

Amlodipine besylate (AML) and Indapamide (IND) were kindly supplied as a gift samples from Torrent research centre, Ahmedabad. The pharmaceutical formulation containing 5 mg AML and 1.5 mg IND in tablet dosage form was obtained from local market. AR grade methanol was procured from Merck Ltd., Mumbai, India.

Preparation of standard stock solution of IND:

Accurately weighed quantity of Indapamide 100 mg was transferred to 100-ml volumetric flask, dissolved and diluted up to mark with 100 ml methanol to give a stock solution having strength of 1mg/ml.

Preparation of working standard solution IND:

100 μg/ml of IND solution was prepared by diluting 10 ml of stock solution with methanol in 100 ml volumetric flask up to the mark.

Preparation of standard stock solution of AML:

Accurately weighed quantity of AML 100 mg was transferred into 100 ml volumetric flask, dissolved and diluted up to mark with 100 ml methanol to give a stock solution having strength of 1mg/ml.

Preparation of working standard solution of AML:

100 μg/ml of AML solution was prepared by diluting 10 ml of stock solution with methanol in 100 ml volumetric flask up to the mark.

Procedure for determination of wavelength:

0.6 ml of working standard stock solution of IND (100 μg/ml) and 2 ml of working standard stock solution of AML (100 μg/ml) were pipetted out into two separate 10 ml volumetric flask and volume was adjusted to the mark with methanol to get 6 μg/ml of IND and 20 μg/ml of AML. Each solution was scanned between 200-400 nm against methanol as a reagent blank. The first order derivative spectra of each solution were obtained. The zero crossing points were found to be 241 nm and 237.4 nm for IND and AML respectively.

Calibration curves for IND and AML:

Aliquots of 0.15, 0.3, 0.45, 0.6, 0.75 and 0.9 ml of working standard solution of IND (100 μg/ml) were transferred to a series of 10 ml volumetric flask. Aliquots of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of working standard solution of AML (100 μg/ml) were respectively transferred to the same above series of 10 ml volumetric flask. The volume was adjusted up to mark with methanol to get 1.5, 3, 4.5, 6, 7.5 and 9 μg/ml and 5, 10, 15, 20, 25 and 30 μg/ml of IND and AML respectively. Absorbance of each solution was measured at 237.4 nm and 241 nm for IND and AML respectively using first order derivative spectrophotometry. The graph of absorbance verses respective concentration was plotted for IND (at 237.4 nm) and AML (at 241 nm).

Validation of the method according to ICH guidelines²⁰

Parameters to be considered for the validation of method are:

Accuracy:

It was determined by calculating the recovery of IND and AML by standard addition method.

Precision:

Intraday/ Interday precision:

Mixed solutions containing 3-6μg/ml of IND and 10-20 μg/ml of AML was analyzed 3 times on the same day and on 3 different day using first order derivative spectrophotometry and % C.V. was calculated.

Robustness:

Mixed solutions containing 3-6μg/ml of IND and 10-20 μg/ml of AML was analyzed 3 times by changing volumetric flasks(10, 50 and 100 ml) using first order derivative spectrophotometry and % C.V. was calculated.

Ruggedness:

Mixed solutions containing 3-6μg/ml of IND and 10-20 μg/ml of AML was analyzed 3 times by different analyst using first order derivative spectrophotometry and % C.V. was calculated.

Simultaneous estimation of IND and AML in combined dosage form:

Twenty tablets were weighed and crushed to obtain a fine powder. An accurately weighed tablet powder equivalent to about 1.5 mg of IND and 5 mg of AML was transferred to 50 ml volumetric flask and the volume was made up to the mark using methanol as solvent. The solution was sonicated for 30 minutes. The solution was filtered through Whatman Filter No. 42 and first few ml of filtrate were discarded. Two ml of above solution was pipetted out in 10 ml volumetric flask and diluted to mark with methanol. The absorbance of the solution was measured using first order derivative spectrophotometry at 237.4 nm for IND and 241 nm for AML. The concentration of each drug was calculated using equation of straight line.

RESULTS AND DISCUSSION:

Spectrophotometric method was developed and validated for the simultaneous estimation of Indapamide and Amodipine besylate in combined tablet dosage form. In this method, linearity for was observed in the concentration range of 1.5 - 9 μg/ml for Indapamide and 5-30 μg/ml for Amlodipine besylate with correlation coefficient 0.99983 and 0.99966 for Indapamide and Amlodipine besylate respectively. The proposed method was successfully applied for the simultaneous estimation of both drugs in combined tablet dosage form. Assay value of Indapamide was found to be 99.72 % of labeled claim and that of Amlodipine besylate was found to be 100.28 % of labeled claim. Mean recovery were found to be 99.782% and 99.812% for IND and AML respectively. LOD and LOQ were found to be 0.116μg/ml and 0.306 μg /ml for IND respectively and 0.351 μg /ml and 0.927 μg/ml for AML respectively. Spectrophotometry methods are cost effective as it does not involve use of expensive solvents and clean up. All the parameters were found to be within limits as per ICH guidelines. So, Simple, sensitive, accurate, precise and rapid spectrophotometric methods were developed and validated for the simultaneous estimation of Indapamide and Amodipine besylate in combined tablet dosage form and can be applied in routine analysis.

Figure 1 Overlain first order spectra of IND (3 μg/ml) and (2) AML (10 μg/ml)

Figure2. Overlain first order calibration spectra of IND (1.5-9μg/ml) and AML (5-30 μg/ml)

Table1. Intraday/Interday precision data

Concentration (μg/ml)		%C.V. (n=3) (Intraday)		%C.V. (n=3) (Interday)
IND	AML	IND	AML	IND	AML
3	10	0.568	0.425	0.721	0.688
4.5	15	0.582	0.359	0.654	0.420
6	20	0.210	0.628	0.443	0.776

Table2. Accuracy data

Amount Of AML:IND in sample (μg)	Amount of Std AML Added (μg)	Amount of Std IND Added (μg)	Amount of AML found (μg) Mean ± S.D (n=3)	Amount of IND found (μg) Mean ± S.D (n=3)	%Recovery for AML	%Recovery for IND
10:3 (80%)	08	2.4	17.9515±0.1756	5.3581 ±0.0239	99.730	99.225
10:3 (100%)	10	3	19.8350±0.1562	5.9642 ±0.0408	99.175	99.403
10:3 (120%)	12	3.6	22.1165±0.1410	6.6474 ±0.0391	100.530	100.718

Table3. Robustness/Ruggedness data

Concentration (μg/ml)		%C.V. (n=3) (Robustness)		%C.V. (n=3) (Ruggedness)
IND	AML	IND	AML	IND	AML
3	10	0.570	0.344	0.568	0.741
4.5	15	0.524	0.282	0.379	0.326
6	20	0.422	0.394	0.554	0.581

Table4. LOD and LOQ data

Parameter	IND(μg/ml)	AML(μg/ml)
SD	0.0351064	0.092678
Mean slope	0.00121	0.00103
LOD (μg/ml)	0.116	0.306
LOQ (μg/ml)	0.351	0.927

Table5. Assay of combined marketed dosage form

Tablet

Label

Claim

Concentration Found

% Mean Assay (n=6)

IND:

AML

IND

AML

IND

AML

Amlodac-D

1.5:5

5.9835

20.0566

99.72

100.28

Table6. Summary of Validation parameters

S.No	Parameters	Result for IND	Result for AML
1	Linearity Range	1.5-9 μg/ml	5-30 μg/ml
2	Correlation coefficient(n=6)	0.99983	0.99966
3	Precision (C.V. (%)) Intraday precision (n=3) Interday precision (n=3)	0.454 0.606	0.471 0.628
4	Robustness (n=3)	0.408-0.542 %	0.425-0.568 %
5	Ruggedness (n=3)	0.541-0.717 %	0.423-0.712 %
6	Assay (% Found)	99.72	100.28
7	Accuracy (%recovery)	99.782	99.812
8	LOD (μg/ml)	0.116	0.306
9	LOQ (μg/ml)	0.351	0.927

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Received on 13.06.2014 Modified on 22.06.2014

Asian J. Research Chem. 7(7): July 2014; Page 615-618