HPTLC Method for the Simultaneous Estimation of Amlodipine Besylate and Indapamide in Tablet Formulation

 

A.K. Desai*, R.S. Chauhan, S.A. Shah, D.R. Shah

Maliba Pharmacy College, Bardoli, Gujarat, India -394350.

*Corresponding Author E-mail: akskmdesai@yahoo.com

 

 

 

1. INTRODUCTION:

Amlodipine besylate, 3-ethyl-5-methyl -2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl) - 6-methyl- 1, 4-dihydropyridine-3, 5-dicarboxylate, monobenzenesulphonate, belongs to the class of dihydropyridine derivative selective calcium-channel blockers used in the treatment of cardiovascular diseases. Indapamide, 3- (aminosulfonyl)-4-chloro-N-(2, 3-dihydro-2-methyl-1H-indol-1-yl)benzamide belongs to the class of non-thiazide indole derivative of chlorosulphonamide diuretics^1,2. The combined oral administration of amlodipine and indapamide has been found to be more effective than either of the drugs alone in the treatment of hypertension. Structures of amlodipine besylate and indapamide are shown in Figure 1.

 

AMLODIPINE                                                     INDAPAMIDE

Figure 1.Structure of amlodipine and indapamide

 

HPTLC is rapidly becoming a routine analytical technique due to its advantages of low operating costs, high sample throughput and minimum sample requirement. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC. Analytical methods reported for amlodipine in combination with other drugs are spectrofluorimetry ^{3, 4}, HPTLC ^{5, 6}, UV spectrophotometry ⁷ and RP-HPLC ⁸. Analytical methods reported for indapamide in combination with other drugs are UV ^{9, 10}, HPTLC ¹¹ and RP-HPLC ^{12, 13}. Literature survey showed that no HPTLC method has been reported for the simultaneous estimation of amlodipine and indapamide in marketed formulation. This paper reports a simple, precise, rapid and cost effective HPTLC method for the simultaneous estimation of amlodipine besylate and indapamide.

 

MATERIALS AND METHOD:

Materials

Pharmaceutical grade amlodipine and indapamide were received as generous gifts from Serdia Pharmaceuticals. Fixed dose combination tablets (NATRILAM, Serdia Pharmaceuticals) containing 5 mg amlodipine and 1.5 mg indapamide were procured from local market.

 

 

Figure 2: HPTLC chromatogram for standard amlodipine (Rf = 0.37) and indapamide (R­­­­­­­­­­­­­­­­­­­­f = 0.81)

 

All chemicals and reagents of analytical grade were purchased from S d Fine Chem. Ltd.

 

Method

The samples were spotted in the form of bands of width 8 mm with a 100 micro litre  (Hamilton, Switzerland) syringe on silica gel pre coated 60F254 aluminium plate, [20cm × 10cm with 250 μm thickness; E. Merck, Darmstadt, Germany)] using a sample applicator Camag Linomat V (Switzerland). A constant application rate of 150nl/s was used. The slit dimension was 4 mm × 0.30 mm and the scanning speed was 20 mm/s. The mobile phase consisted of dichloromethane: methanol: ammonia 8.5: 1.5: 0.1 (v/v/v). Linear ascending development was carried out in a 20 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase.

 

Standard solutions and calibration curves

Standard stock solutions of amlodipine and indapamide were prepared separately by dissolving 25 mg of each drug in 25 ml of methanol to get a solution containing 1000 μg/ml of each drug. Aliquots of 1.5 ml of amlodipine and 0.5 ml of indapamide were diluted to 10 ml with methanol to get a mixed standard solution of amlodipine (150 μg/ml) and indapamide (50 μg/ml). A calibration curve of concentration against their respective area for amlodipine and indapamide in different range were plotted. Linearity was observed in range 300 to 1500 ng/spot for amlodipine and between 100 to 500 ng/spot for indapamide.

RESULTS AND DISCUSSION:

HPTLC method development and optimization

Dichloromethane and methanol in the ratio of 8:2(v/v) was tried for separation of two drugs. To improve resolution, dichloromethane, methanol and glacial acetic acid in the ratio of 8.5:1.5:0.1 (v/v/v) was tried. Separation was achieved but tailing was observed. To the same mobile phase, 0.1 ml ammonia was added instead of glacial acetic acid to avoid tailing. The optimized mobile phase contained dichloromethane: methanol: ammonia in the ratio of 8.5: 1.5: 0.1(v/v/v). The analytes were monitored at 241 nm and Rf were found to be 0.37±0.2 and 0.81±0.2 for amlodipine and indapamide, respectively as depicted in Figure 2.

 

Validation of the developed method ^{(14, 15)}

The method was validated for linearity, accuracy, precision, LOD and LOQ.

 

Linearity:

The linearity was determined for amlodipine and indapamide in a mixture by plotting a calibration graph of peak area against their respective concentration. Amlodipine besylate showed linearity in the range 300 to 1500 ng/spot, whereas indapamide had a linearity range between 100 to 500 ng/spot. Calibration curve of amlodipine and indapamide is depicted in Figure 3 & 4 respectively. The linear regression equation for the two drugs is presented in table 1.

 

 

Figure 3: Calibration curve for amlodipine besylate.

 

Figure 4: Calibration curve for indapamide.

 

 

Table 1: Linear regression equations for amlodipine and indapamide

 

The 3D representation of calibration concentrations is described in Figure 5.

 

Figure 5: 3D representation of calibration concentrations for amlodipine (300-1500 ng) and indapamide (100-500 ng).

 

Table 2: Accuracy data (*average of three estimations)

AMLO:INDA (ng) in tablet	Spiked amount AMLO:INDA   (ng/spot)	Total AMLO:INDA    (ng/spot )	Recovery Of AMLO (%)	Recovery Of  INDA (%)
333.2:100	NIL	333.2:100	101.83	101.30
333.2:100	300:100	633.2:200	99.77	99.20
333.2:100	600:200	933.2:300	98.49	102.01
333.2:100	900:300	1233.2:400	100.64	99.86

 

 

Accuracy:

Accuracy of the developed method was determined by a recovery study at three concentration levels by replicate analysis (n=3). Standard drug solutions were added to a pre-analyzed sample solution, and percentage of total drug content was calculated. The results of the accuracy study are reported in Table 2.

 

Precision:

Precision was determined by studying the repeatability and intermediate precision. The standard deviation and relative standard deviation were calculated for the two drugs. Repeatability was determined by six estimations of AMLO:INDA 900:300 ng/µl and %RSD was calculated. Intermediate precision was determined by intraday and interday precision studies. In the intraday study, the concentrations of two drugs were determined on the same day. In the interday study, the drug content was determined on three different days. Precision data are described in the table 3 and 4.

 

Table 3: Intraday precision for estimation of AMLO and INDA

Concentration of                           AMLO:INDA (ng/ spot)	%RSD for Amlodipine (n=3)	%RSD for Indapamide (n=3)
300:100	1.36	1.84
600:200	1.18	0.54
900:300	1.15	0.19
1200:400	0.22	0.27
1500:500	0.86	1.34

 

Table 4: Inter day precision for estimation of AMLO and INDA

Concentration of                           AMLO:INDA (ng/ spot)	%RSD for Amlodipine (n=3)	%RSD for Indapamide (n=3)
300:100	2.21	2.18
600:200	1.51	1.93
900:300	0.85	0.85
1200:400	0.64	0.89
1500:500	0.69	0.79

Table 5: Assay of marketed formulation

TABLET	Drug	Label claim (mg/tablet)	Amount found (mg/tablet ±RSD)	Label claim (%)
NATRILAM	Amlodipine	5 mg	5.17 ± 0.59	103.40
	Indapamide	1.5 mg	1.51 ± 0.43	100.66

 

 

LOD and LOQ:

The LOQ was found to be 33.07 and 89.23 ng/spot for amlodipine and indapamide respectively. LOD was found to be  11.96 and 30.25 ng/spot for amlodipine and indapamide respectively.

 

Assay of marketed formulation

Twenty tablets containing the two drugs were weighed and powdered. Powder equivalent to 5 mg of amlodipine or 1.5 mg of indapamide was transferred to a 50 ml volumetric flask, dissolved and diluted to volume with methanol. The mixture was subjected to sonication for 5 min for complete extraction of the drugs and filtered. Ten μl of the solution was spotted on the HPTLC plate for analysis. The amount of amlodipine and indapamide per tablet was determined from the calibration curve. Assay of marketed formulation is described in table 5. The detailed summary of validation parameters are described in Table 6.

 

Table 6: Summary of validation parameters

	PARAMETERS	Results
		Amlodipine	Indapamide
1	Linearity range (ng/spot)	300-1500	100-500
2	Regression equation	y = 2.662x + (-73.73)	y = 9.879x + 470.9
3	Correlation coefficient	0.9995	0.9977
4	Precision(%RSD) Repeatability (n=6) Intraday (n=3) Interday (n=3)	0.30 0.22-1.36 0.64-2.21	0.87 0.19-1.84 0.79-2.18
5	Accuracy	98.49-102.05	99.2-102.01
6	LOQ(ng/spot)	33.07	89.23
7	LOD(ng/spot)	11.96	30.25

 

CONCLUSION:

The developed HPTLC method is simple, precise, accurate and reproducible and can be used for simultaneous determination of amlodipine besylate and indapamide in tablet dosage form.

 

ACKNOWLEDGEMENT:

The authors are grateful to Serdia Pharmaceuticals for providing a gift sample of amlodipine and indapamide and to Principal, Maliba Pharmacy College, Bardoli, India for providing necessary facilities to carry out the work.

 

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Received on 27.02.2012         Modified on 16.03.2012

Accepted on 05.04.2012         © AJRC All right reserved