RP-HPLC Method for Simultaneous Estimation of Amlodipine Besylate and Indapamide in Tablet Dosage Form

Rima N. Shah, Deesha B. Gandhi and Mehul M. Patel^*

Department of Pharmaceutical Chemistry and Pharmaceutical Analysis , Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, At and Post- Changa, Ta- Petlad, Dist.- Anand. 388421, Gujarat, India.

*Corresponding Author E-mail: mehulpatel.ph@ecchanga.ac.in

ABSTRACT:

A simple, selective, accurate Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method was developed and validated for the Simultaneous estimation of Amlodipine Besylate and Indapamide in combined tablet dosage form.Chromatographic separation achieved isocratically on a C₁₈Column utilizing a mobile phase of Acetonitrile:Water contains 0.2% of Triethyl amine and adjusting the pH to 4.0 with orthophosphoric acid (70:30, v/v, pH 4.0) at a flow rate of 0.8 ml/min with UV detection at 238nm. The retention time for Amlodipine Besylate and Indapamide was found to be 3.27 and 4.7 min respectively. Linearity was obtained in the concentration range of 20-120 µg/ml of Amlodipine Besylate and 6-36 µg/ml of Indapamide with a correlation coefficient of 0.9999 and 0.9996 respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation.This study aimed at developing and validating an HPLC method, being simple, accurate, robust and selective, and the proposed method can be used for the estimation of these drugs in combined dosage forms.

KEYWORDS: Simultaneous Estimation; RP-HPLC; Amlodipine Besylate; Indapamide

INTRODUCTION:

A combined fixed dose formulation containing Amlodipine besylate (ADB) and Indapamide (INDA) is available as tablet dosage form for treatment of hypertension and angina pectoris. ADB is chemically known as (4 R, S)-3ethyl-5-methyl,2-(2-amino-ethoxymethyl)-4(2-chlorophenyl)-1,4-dihydroxy-6-methylpyridine-3,5dicarboxylate monobenzene sulphonate. B.P¹and I.P² describes a Reversed Phase High Performance liquid chromatographic (RP HPLC) method for the determination of ADB in bulk and pharmaceutical formulations. The literature survey reveals numbers of methods are reported for the quantitative determination of ADB alone or in combination with other anti hypertensive drugs including Spectrophotometric^3-8and RP-HPLC^9-13,HPTLC¹⁴ and LC-MS¹⁵ methods. Indapamide (INDA), chemically is a 4-chloro-N-(2methyl-2, 3-dihydroindol-1-yl)-3-sulfamoyl benzamide used as an antihypertensive agents and as a diuretics, which inhibits the reabsorption of sodium and calcium at the beginning of distal convoluted tubules.

U.S.P¹⁶describes a Reversed Phase High Performance liquid chromatographic (RP HPLC) method for the determination of INDA in bulk and pharmaceutical formulations. The literature survey reveals that Spectrophotometric^17-19, RP-HPLC^20-23, LC-MS²⁴ methods are reported for the estimation of INDA. However, there is no evidence in literature for simultaneous determination of ADB and INDA. Hence present work describes RP-HPLC method for estimation these two drugs simultaneously from tablet dosage form.

EXPERIMENTAL:

Reagents and Chemicals

Acetonitrile HPLC grade was procured from Sigma Aldrich Pvt. Limited, Mumbai. Reference standards of ADB and INDA were procured from Zydus Cadila, Ahmadabad, India, and Dishman Pharma, Ahmadabad respectively as a gift samples. The tablets Amlodac-D of the said combination were purchased from a local pharmacy (The label claim for both contained 5 mg of Amlodipine Besylate and 1.5 mg of Indapamide).

Apparatus and Chromatographic Conditions

A High Performance Liquid Chromatograph system, with Rheodyne injection syringe with 20 µl loop volume and LC solutions data handling system (Shimadzu-LC 20) was used for the analysis. The data was recorded using LC 20 solutions software. The purity determination performed on a stainless steel column 150 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5µm diameter (Spinchotech C₁₈ , 5µ , 150 mm x 4.6 mm, ) with the mobile phase containing Acetonitrile:Water contains 0.2% of Triethyl amine and adjusting the pH to 4.0 with orthophosphoric acid (70:30, v/v, pH 4.0).Flow rate was kept at 0.8 ml/min and the elution was monitored at 238nm.

Preparation of Standard Stock Solution

Standard stock solution 1000 µg/ml of ADB and 300 µg/ml of INDA were prepared by dissolving 50mg of ADB and 15mg of INDA in 50 ml of Mixture of 70:30 0f Acetonitrile :Water. The solutions were suitably diluted with Acetonitrile:Water Mixture to get mixed standard solution containing 100µg/ml of ADB and 30µg/ml of INDA.

Preparation of Sample Solution

Twenty tablets (Amlodac-D) each containing 5mg of ADB and 1.5 mg of INDA were weighed, and powdered. Powder equivalent to 50 mg of ADB was weighed accurately and taken into 50 ml volumetric flask. The drugs were extracted into acetonitrile:Water(70:30) Mixture, volume was adjusted to 50ml, sonicated for 20min and then filtered through 0.45 µ membrane filter. From this solution, further dilutions were made using acetonitrile:Water(70:30) Mixture to get a final concentration of 40µg/ml of ADB and 12µg/ml of INDA. This solution was used for the estimation. The concentration of the drugs were calculated.

Linearity

Several aliquots of standard solutions of ADB and INDA were taken in different 10ml volumetric flasks and diluted up to the mark with acetonitrile:Water(70:30) Mixture such that the final concentration of ADB and INDA was 20-120 µg/ml and 6-36 µg/ml respectively. Evaluation of two drugs were performed with PDA detector at 238 nm, peak area recorded for all the peaks. This linearity was represented by a linear regression equation as follows.

Y_ADB= 43091x + 71889 (r² = 0.9999)

Y_INDA= 100370x + 41976 (r² = 0.9996)

RESULTS AND DISCUSSION:

Estimation of ADB and INDA in Tablet Dosage Form

The HPLC procedure was optimized with a view to develop accurate and stable assay method. Both the pure drugs ADB and INDA were run in different mobile phase compositions. The flow rate was also varied from 0.5 mL to 1 mL/min. ODS C₁₈ column with a mobile phase of mixture of Acetonitrile and water(0.2% Triethyl amine, pH adjusted 4.0 with orthophosphoric acid (70:30 v/v), delivered at a flow rate of 0.8 mL/min with a detection at 238 nm gave sharp and symmetrical peak with retention time 3.27 and 4.70 min for ADB and INDA respectively. The typical chromatogram of the sample is shown in Fig. 1.The assay procedures were repeated for six times and the percentage of individual drugs found in formulations, standard deviation, and % R.S.D were calculated and presented in Table 1.

Table 1. Analysis of Tablet Dosage Form*

Drug	Labeled claim(mg/tablet)	Amount found(mg/tablet)	% Label Claim*	Standard deviation	% R.S.D.
ADB	5	5.01	100.15	0.0354	0.7068
INDA	1.5	1.51	100.51	0.0078	0.5196

*Average of six determinations

Figure.1.Chromatogram of ADB and INDA

Method Validation

Precision Studies

The Intaraday and Interday precision were determined by analyzing a set of laboratory sample (n=3) with each

of the three concentration. Results of Precision Studies were shown in Table.2

Limit of Detection and Limit of Quantification

The limit of Detection (LOD) and limit of Quantification (LOQ) of the developed method were determined by standard deviation of response and slope of calibration curve (Table.2).

Robustness

The robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP-HPLC method developed, was robust (Table.2).

Solution Stability

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 6 h at room temperature. The result show that for both solutions, the retention time and peak area of ADB and INDA remained almost unchanged (% R.S.D. less than 2.0) and no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 6 h, which was sufficient to complete the whole analytical process (Table.2).

Table 2.Summary of Validation Parameters

Parameters	ADB	INDA
Recovery%	99.95-100.42	100.31-100.46
Precision(%R.S.D.) Intra-day (n=3) Inter-day (n=6)	0.17-1.01 0.45-1.38	0.33-0.71 0.40-0.75
Specificity	Specific	Specific
LOD (µg/ml)	1.00	0.38
LOQ (µg/ml)	3.04	1.14

System Suitability Studies

The column efficiency, resolution and tailing factor were calculated for the standard solutions. The values (Table-3) obtained demonstrated the suitability of the system for the analysis of this drug combination.

Table 3.System suitability parameters

Parameters	ADB	INDA
Retention Time(min)	3.27	4.07
Theoretical plates	4417	8130
Resolution factor	-	7.14
Tailing factor	1.48	1.32

Recovery Studies

To study the accuracy and reproducibility of the proposed method recovery experiments were carried out. A fixed amount of pre-analyzed sample was taken and standard drug was added at 50% and 100% and 150% levels. Each level was repeated three times. The contents of ADB and INDA per tablet found by proposed method is shown in Table 4. The lower values of SD of assay indicate the method is accurate. The mean recoveries of ADB and INDA were in range of 98% and 102% that shows there is no interference from excipients.

CONCLUSION:

The proposed method gives good resolution between ADB and INDA within short analysis time (< 10min). The method is very simple, rapid and no complicated sample preparation is needed. High percent of recovery shows the method is free from interference of excipients present in the formulations.

ACKNOWLEDGEMENT:

The authors are grateful to Zydus Cadila, Ahmadabad, India, and Dishman Pharma, Ahmadabad for providing gift samples of ADB and INDA respectively.

Table 4. Recovery Study

Level of % Recovery*	Standard added		% Mean Recovery		Standard deviation
Level of % Recovery*	ADB	INDA	ADB	INDA	ADB	INDA
50	20	6	100.11	100.31	0.4550	0.8038
100	40	12	99.95	100.46	0.3098	0.5637
150	60	18	100.42	100.44	1.0037	0.6194

*Average of Three determinations

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Received on 13.04.2012 Modified on 20.04.2012

Asian J. Research Chem. 5(5): May 2012; Page 633-636