INTRODUCTION:

Indapamide chemically is 4-chloro-N-(2methyl-2,3dihydroindole-1yl)-3sulfamoyl-benzamide.¹It is an antihypertensive agent and official in USP 2000.^2,3 Amlodipine besylate, chemically is (RS)-3ethyl5methyl2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4 dihydropyridine-3,5-dicarboxylate besylate. It is a calcium channel blocker and antianginal agent in the form of besylate salt.^{4, 5}Amlodipine besylate is official in British pharmacopoeia.Literature survey revealed that few spectrophotometric^{6, 7} and chromatographic^8,9,10, methods for determination of indapamide and amlodipine besylate alone or in combination with other drugs in various formulation are available.Till date, to the best of our knowledge, no HPLC method has been reported in the literature for estimation of indapamide and amlodipine besylate in combined dosage form. The objective of the present study was to develop a simple, accurate, precise and selective RP-HPLC method for estimation of indapamide and amlodipine besylate from tablet dosage form. The newly developed method was validated as per ICH guidelines to confirm the reproducibility and wide applicability of the method.¹¹

MATERIAL AND METHODS:

The liquid chromatographic system of Shimadzu with pump-SPD-20AD (LC-20A) having variable wavelength UV- Visible detector and auto injector (20 ml) was used. Chromatographic analysis was performed using LC solution software. A phenomenex C₁₈ (4.6 × 250 mm, 5µ) column was used. Reference standard of indapamide and amlodipine besylate was kindly supplied by Matrix Pvt. Ltd. Secundrabad, India and Supra Chemicals, Mumbai, India. The purity of indapamide and amlodipine besylate were found to be (100.86 %.) and (99.9%), respectively. Tablet of brands name Natrilam having combination of indapamide 1.5 mg and amlodipine besylate 5 mg purchased from local market. All chemicals and reagent used were of HPLC grade and analytical grade.

Preparation of mobile phase and stock solution:

A degassed mixture of acetonitrile, 0.05M ammonium acetate and triethylamine (65: 35:0.3) was filtered through 0.45 μm membrane filter and adjusted to pH 3 using dilute solution of 1% acetic acid.

Preparation of standard stock solution:

Accurately weighed quantity of indapamide (10 mg) and amlodipine besylate (10 mg) was transferred to separate volumetric flask, dissolved and diluted to the mark with methanol to obtained standard solution having concentration (100 μg/mL). Aliquot portion of standard stock solution of each drug was transfer to the 10 mL volumetric flask and diluted to the mark with mobile phase to prepare working standard solution 10 μg/mL of each drug.

Chromatographic condition:

The following chromatographic conditions were established.

Column : C₁₈(4.6 × 250 mm, 5µ)

Detection Wavelength : 238 nm

Injection Volume : 20 µL

Pressure : 2680psi

Flow rate : 1.0 mL/min

Temperature : Ambient

Mobile Phase : Acetonitrile: Buffer (0.05M Ammonium Acetate): triethylamine (65:35:0.3v/v/v ) pH 3, adjusted with dilute solution of 1% acetic acid

Calibration curves for indapamide and amlodipine besylate:

Appropriate aliquots of indapamide and amlodipine besylate working standard solutions were taken in different 10 mL volumetric flasks and volume was made up to the mark with mobile phase to obtained final concentrations of 1.5-30 μg/mL of indapamide and 5-50 μg/mL of amlodipine besylate. The solutions were injected using a 20 μL fixed loop system and chromatograms were recorded. Calibration curves were constructed by plotting peak area versus concentration of drug.

Figure.1.Calibration curves for indapamide.

Figure.2.Calibration curves for amlodipine besylate.

Analysis of marketed formulation:

Twenty tablets were accurately weighed and finely powdered. An accurately weighed quantity of tablet powder equivalent to about 1.5 mg of indapamide and 5 mg of amlodipine besylate was transferred to volumetric flask (50 mL). Sonicated with methanol (25 mL) for 15 minutes. The volume was made up to the mark with methanol and filtered through membrane filter (ultipore ‘Nsi’, Nylon 66, 0.45 μm pore size). Further dilution was done with mobile phase to get concentration of 3 mg/mL indapamide and 10 mg/mL of amlodipine besylate.

The chromatographic conditions were set as per the optimized parameters and mobile phase was allowed to equilibrate with stationary phase. After equilibration of stationary phase, five replicate injection of standard solution and each of five sample solutions were made separately and chromatograms were recorded.

Validation of Method:

Validation of proposed method was carried out for linearity and range, precision, accuracy, specificity and ruggedness.

Linearity and Range:

The linearity of the method was determined at six concentration levels ranging from 1.5-30 µg /mL for indapamide and 5-50 µg /mL for amlodipine besylate, respectively.

Accuracy:

Accuracy of proposed method was ascertained on the basis of recovery studies performed by standard addition method at different level of labeled claim (i.e. 80 to 120 %). The results are shown in Table1.

Table1. Data of accuracy study.

Drug	Level (%)	Amount added	Total amount found*	Recovery* (%±SD)	Average recovery (%±S..)
Indapamide	80	4	3.98	99.50	99.37± 0.148
	100	5	4.98	99.60
	120	6	5.99	99.03
Amlodipine besylate	80	13.2	13.18	100.08	99.86± 0.767
	100	16.5	16.61	100.67
	120	19.8	19.77	98.85

SD: Standard deviation, *Average of three determinations.

Precision:

Precision of an analytical method was expressed as SD and CV of series of measurement. It was ascertained by replicate estimation of both drugs by proposed methods. The results are presented in the Table 2.

Table 2. Data of drugs in laboratory mixture by RP-HPLCmethod

Drug	% Mean	± SD	CV
Indapamide	99.33	0.2356	0.23
Amlodipine besylate	99.98	0.2720	0.27

Specificity:

The specificity studies were carried out by attempting deliberate degradation of the tablet sample with exposure to stress conditions like acidic (0.1M HCL), alkaline (0.1M NaOH), oxide (3%H₂0₂) and heat (60ºC). The result of specificity studies were recorded in the Table 3.

Table 3.Data of specificity study

Ruggedness:

The studies were carried out for different parameters i.e. different elapsed times (interday, intraday) and different analysts. The results are shown in the Table 4.

Table 4. Data of ruggedness.

Drugs	Parameter	Intermediate Precision
Drugs	Parameter	Interday	Intraday	Different Analysts
Indapamide	% Mean ± SD	99.41 0.3015	98.90 0.09	98.81 0.2651
Indapamide	CV	0.30	0.091	0.26
Amlodipine besylate	% Mean ± SD	98.64 0.0413	98.34 0.342	99.23 0.1420
Amlodipine besylate	CV	0.04	0.34	0.14

Drug

Room

Temperature

Acid

Alkali

Oxide

Heat

Indapamide

100.42

99.21

99.03

68.69

96.62

Amlodipine besylate

98.72

98.44

97.28

97.80

99.64

RESULTS AND DISCUSSION:

A new RP-HPLC method was developed for the estimation of indapamide and amlodipine besylate in tablet dosage form and validated in accordance with ICH guidelines for the parameters like system suitability, linearity and range, precision (repeatability), intermediate precision (ruggedness), specificity and accuracy. The results obtained for each of the parameters lies well within the acceptance criteria. Hence the developed method was simple, specific, linear, precise, accurate, and rugged and could be extensively used for the estimation of indapamide and amlodipine besylate in tablet formulation.

Chromatogram of indapamide and amlodipine besylate shown in (figure 3). UV overlain spectra of indapamide and amlodipine besylate showed that both the drugs absorbed appreciably at 238 nm, so the same was selected as the detection wavelength during the studies (figure 4). System suitability parameters proved that the proposed method suits for the estimation of indapamide and amlodipine besylate presented in Table 5. After various trials performed chromatogram for indapamide and amlodipine besylate was found satisfactory on, C₁₈(4.6 × 250 mm, 5μ) column, using mobile phase composition of acetonitrile: buffer: triethylamine (65:35:0.3%). Drug peak was found to be symmetrical. Resolution of the proposed method was found to be satisfactory. Sensitivity of the method was good and also linearity was observed over a wide concentration range of 1.5-30 μg/ml for indapamide and 5-50 μg/ml for amlodipine besylate. Accuracy of the method was determined by recovery with spiked concentration of pure drug at three levels for indapamide and amlodipine besylate. Recovery of drug was well within the acceptance limits of 97-103%. Method was rugged as observed from insignificant variation in the results of analysis being performed by different analysts, in different days.

Table.5. system suitability parameter for the proposed method

Parameters	Indapamide	Amlodipine besylate
Area under Curve	1776801	232164
	1776803	232162
	1776802	232165
	1776806	232164
	1776801	232163
Mean	1776802	232163
±SD	1.998	1.37
CV	0.01	0.005
Height	1227105	183962
Theoretical (plates/column)	5123.28	4748.44
Tailing factor	1.10	1.21
Rt	5.718	6.876

Rt =Retention time, SD=Standard deviation.

CONCLUSION:

The proposed method was found to be suitable for simultaneous determination of indapamide and amlodipine besylate from bulk and in pharmaceutical dosage forms. The mobile phase is simple to prepare. The drugs recovered from laboratory mixture were good and they suggested non- interference of formulation excipients in the estimation. Hence, the method can be easily and conveniently adopted for routine analysis of indapamide and amlodipine besylate in combined dosage form.

ACKNOWLEDGMENTS:

Authors are grateful to Supra Chemicals Mumbai for providing gift sample of indapamide and Matrix Pvt. Ltd. Secundrabad for providing gift sample of amlodipine besylate.

REFERENCES:

1. The United State Pharmacopoeia, The National Formulary, United State Pharmacopoeial Convention, INC, Asian edition, Rockville, 2000:pp.331- 456.

2. The Martindale Pharmacopoeia, The Complete Drug Reference, 35^th edition, The Pharmaceutical Press, London, 2007:pp.759, 200,229.

3. The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals ,14^th edition, 2006:pp.2419,1924,6872.

4. Indian Pharmacopoeia, 3rd edition, Controller of Publication, New Delhi, 1996; Vol- I: p.199.

5. Tripathi K.D. Essential of medical Pharmacology, 6th edition. Jaypee brothers’ medical publishers Pvt.Ltd, New Delhi; 2008. Pp.539.

6. Modi D.K. et al. Development and validation of spectrophotometric method for simultaneous estimation of perindopril and indapamide in combine dosage form. International Journal of Pharm Tech Research, Jan-Mar. 2:2010: 411-416.

7. Wankhede S.B.et al. Spectrophotometric and HPLC method for simultaneous estimation of amlodipine besylate, losartan potassium and hydrochlorothiazide in tablet. Indian Journal of Pharmaceutical Sciences.72 (1): 2010:136-140

8. Patel A. et al. Method development, validation and stability study for simultaneous estimation of telmisartan and indapamide by reverse phase-high performance liquid chromatography in pure and marketed formulation. International Journal on Pharmaceutical and Biomedical Research .2(1): 2011:4-16.

9. Dewani M.G. et al. Simultaneous estimation of perindopril erbumine and indapamide in bulk drug and tablet dosage form by HPLC. Indian Journal of compressive Pharmacy. 2:2011: 1-4

10. Prasad Rao M.M. et al. RP-HPLC method of simultaneous estimation of amlodipine besylate and metoprolol in combine dosage form. International Journal of Pharma Research and Development. 2:2010: 69-76.

11. ICH, Validation of Analytical Procedure: Methodology, (Q2A). International Conference on Harmonization, Geneva, 1996.

Received on 20.04.2012 Modified on 02.06.2012

Asian J. Research Chem. 5(6): June, 2012; Page 738-741