INTRODUCTION:

Irbesartan (IRB) is chemically 2-Butyl-3-[p-(o-1H-tetrazol-5-ylphenyl) benzyl]-1, 3-diazaspiro [4.4] non-1-en-4-one¹ and is used as antihypertensive agent. Hydrochlorothiazide (HCTZ) is chemically 6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide²and is used as class of Diuretic compounds. Both of these drugs are available in combined tablet dosage form with the label claim of IRB 150 mg and HCTZ 12.5 mg per tablet. Literature survey of IRB and HCTZ revealed few methods based on chromatography^3-38,electrochemical methods³⁹, high performance thin layer chromatography ^{40, 41} and spectrophotometry ^42-52 either in single or in combined forms. The present work describes the development and validation of reverse phase high performance liquid chromatographic (RPHPLC) method, which can quantify these components simultaneously. Confirmation of the applicability of the developed method was validated according to the International Conference on Harmonization (ICH)⁵³ for the simultaneous determination of IRB and HCTZ in bulk and in tablet dosage form.

Figure1: Molecular structure of Hydrochlorothiazide

Figure2: Molecular structure of Irbesartan

MATERIALS AND METHODS⁵³:

Preparation of buffer:

Weigh accurately about 8.3g of sodium acetate anhydrous and transfer it into 1000 ml volumetric flask and made up to the mark with double distilled water. Mix the contents to dissolve. Adjust the pH of the above mixture to 3.5 ± 0.005 using ortho phosphoric acid.

Preparation of mobile phase:

Take Acetonitrile and buffer in the ratio of 55:45 (v/v). Mix the content, filter through 0.4 µ membrane filter and degas with ultrasonicator.

Standard stock Preparation:

Weigh and transfer accurately about 75.0 mg of Irbesartan and 6.3mg of Hydrochlorothiazide Working Standard into a 25 ml clean dry volumetric flask, add about 15 ml of methanol, sonic ate for 5 minutes, and dilute to volume with diluent (mobile phase).

Diluted Standard:

Pipette out 1ml from the standard stock solution, into a 25 ml clean dry volumetric flask, and dilute to the mark with 25 ml of diluent.

Sample preparation:

Weigh and powder about twenty tablets in a neat clean and dry motor and pestle grind and mix to uniform powder. weigh and transfer accurately about 0.2gm of the tablet powder into 25ml clean dry volumetric flask, add about 15ml of diluent (mobile phase) sonic ate for 5 minutes, and dilute to volume with mobile phase. filter through 0.4 µ membrane filter, from the filtrate pipette out 1ml of sample solution into a 25ml volumetric flask, make up the volume with diluent (mobile phase).

Chromatographic Conditions:

Freshly prepared acetonitrile and Buffer 55:45 (v/v) adjust PH 3.5 were filtered through 0.45µ membrane filter and sonicate before use. Flow rate of Mobile phase was maintained at 1.0 ml/min. the column temperature was ambient temperature. The detection was carried out at 260 nm. Injection Volume 20µL and total run time was 10 min. Column was 4.6 mm×250 mm C₁₈column, 5µ particle size.

System suitability parameters⁵³:

System suitability tests are an integral part of chromatographic method. They were used verify that the reproducibility of chromatographic system are adequate for analysis. To ascertain its effectiveness, System suitability tests were carried out on freshly prepared standard stock solution of Irbesartan and Hydrochlorothiazide. In addition to this standard deviation of Irbesartan and Hydrochlorothiazide standards were evaluated by injecting a mixed standard of both Irbesartan and Hydrochlorothiazide as internal standard five times at 15 min interval and the values were recorded. All the above parameters are shown in

Table -1 System suitability parameters

Parameters \Drugs	Irbesartan	Hydrochloro-thiazide
Tailing factor	1.22	1.24
Theoretical plates	4476.6	6733.6
Resolution	9.52
Relative standard deviation	0.401525	0.290616

Assay procedure:

Weigh the content of not lees then 20 tablets Irbesartan of and Hydrochlorothiazide to mortar and pestle, grind and mix to uniform powder. Transfer an accurately weighed portion of powder, equivalent about one tablet into 100 ml volumetric flask containing 40 ml of mobile phase. Mix thoroughly to dissolve and make up to volume with mobile phase. Filter the solution through whatmann filter paper. Pipette out 5 ml of the filtrate into 50 ml of volumetric flask make up volume and filter through 0.45 µ membrane filter. Chromatograph the standard preparation and record the chromatograms and measure the peak responses. The tailing factor for the principal peak is not more than 2.0 and the number of the theoretical plates is not less than 2500. The % RSD (Relative Standard Deviation) is not more then 2.0. Separately inject 20 µl of standard preparation and assay preparation in the chromatograph, record the chromatograms and measure the responses for the major peaks. The amount of drug present in tablet formulation was calculated as follows:

Assay of tablet dosage form:

Applicability of the proposed method of the simultaneous estimation of Irbesartan and Hydrochlorothiazide was studied by assay of commercial tablets Xarb-H label to contain Irbesartan 150mg and hydrochlorothiazide 12.5mg. The results indicate that the amount of each drug in the tablets is with in the requirements of 98-102%of the label claim.

Table.2

Drug	Amount		% label claim
Drug	Labeled	Measured	% label claim
Irbesartan	150mg	148.918mg	99.28
Hydrochlorothiazide	12.5mg	12.4mg	99.54

RESULTS AND DISCUSSION ^{54, 55}:

In order to develop simultaneous estimation of two components under isocratic conditions, the mixture of acetonitrile with buffer in different ratios were assayed as the mobile phase. A mixture of buffer and acetonitrile in different ratios were also tried for the assay of combined dosage forms. Finally a mixture of acetonitrile- sodium acetate buffer in ratio of 55:45(v/v), proved to be the effective mixture than the other mixture used for the separation. Then the flow rates tested includes 0.5, 0.8, 1.0, 1.5 and 2.0 ml, among these flow rates 1.0 ml was selected for the assay because better resolution of the peaks. System suitability test was applied to freshly prepare stock solution of Irbesartan and Hydrochlorothiazide, to check the parameters like Tailing factors, Resolution, Theoretical plates, Relative standard deviation as shown in Table-1. Linear correlation was obtained between peak areas and concentration of Hydrochlorothiazide and Irbesartan in the range of 5-200μg/ml and 1-400 μg/ml respectively. Data of the regression analysis are summarized in Table- 3,4. The developed method was studied for precision. The precision of the method was demonstrated by at least six determinations in method precision. Standard deviation and the results are given in Table-5,6. The accuracy of the proposed RP-HPLC method was expressed in terms of recovery. The recovery studies was carried out and given in terms of percentage recovery and given in Table -7,8. The proposed method was found to be simple, species, specific and highly accurate, required less time consumption for analysis and this can be employed for the routine analysis.

Table.3 Linearity of Irbesartan

Conc. (μg/ml)	Average area	Statistical Analysis
20	89.766	Slope	5.1189
40	189.629	y-Intercept	- 14.753
60	290.797	Limit of detection	2.3968396
80	390.12	Limit of quantification	7.2631503
100	503.213	Correlation coefficient (R²)	0.9989
120	589.214
140	717.977
160	796.858

Fig: 5 Linearity graph of Irbesartan

Table-4 Linearity of Hydrochlorothiazide

Conc (μg/ml)	Average area	Statistical Analysis
20	48.979	Slope	2.7995
40	104.243	y-Intercept	- 9.5735
60	157.375	Limit of detection	1.75143
80	206.11	Limit of quantification	5.30736
100	271.658	Correlation coefficient (R²)	0.9984
120	323.929
140	392.663
160	434.103

Fig: 6 Linearity graph of Hydrochlorothiazide

Table-5 Method Precision of Irbesartan

Concentration 100%	Injection	Rt Irbesartan	Peak Areas of Irbesartan
	1	4.823	503.122
	2	4.827	507.643
	3	4.827	506.702
	4	4.827	512.719
	5	4.823	513.217
Statistical Analysis	Mean	4.8254	508.5806
	SD	0.027979	4.26614
	% RSD	0.0203049	0.838833

Table-6 Method Precision of Hydrochlorothiazide

Concentration 100%	Injection	Hydrochlorothiazide Rt	Peak Areas of Hydrochlorothiazide
	1	2.98	277.226
	2	2.98	278.376
	3	2.983	278.346
	4	2.99	276.749
	5	2.993	275.545
Statistical Analysis	Mean	2.983	276.267
	SD	2.984833	277.0848
	% RSD	0.005419	1.134046

Table-7 Accuracy (Recovery):

Concentration of Irbesartan	Peak area	Amount found	%Recovery	Statistical Analysis
standard 80mcg	370.12
1	365.414	78.9828	98.728	MEAN	98.69433
2	367.578	79.45	99.313	SD	0.636168
3	362.873	78.433	98.042	%RSD	0.6445
standard 100mcg	503.213
1	505.837	100.52	100.52	MEAN	100.2517
2	501.921	99.74	99.74	SD	0.443293
3	505.704	100.495	100.495	%RSD	0.4422
standard 120mcg	589.214
1	587.038	119.59	99.66	MEAN	99.94867
2	589.958	120.15	100.126	SD	0.252161
3	589.556	120.06	100.06	%RSD	0.25229

Table-8 Accuracy (Recovery)

Concentration of Hydrochlorothiazide	Peak Area	Amount found	%Recovery	Statistical Analysis
standard 80mcg	2065.11
1	209.898	81.47	101.83	MEAN	99.99467
2	202.472	78.587	98.234	SD	1.799645
3	205.957	79.94	99.92	%RSD	0.0179925
standard 100mcg	271.658
1	274.849	101.116	101.12	MEAN	100.79
2	276.242	101.68	101.68	SD	1.091100
3	270.499	99.57	99.57	%RSD	0.010825
standard 120mcg	323.929
1	320.682	118.797	98.99	MEAN	99.61
2	323.64	119.89	99.91	SD	0.19226
3	323.717	119.92	99.93	%RSD	0.31005

CONCLUSION:

The developed RP-HPLC method was developed and validated for the simultaneous determination of Irbesartan and Hydrochlorothiazide in combined forms. The method was found to be simple, precise and rapid. The assay result obtained by this method is in fair agreement. This method can be used for the routine determination of Irbesartan and Hydrochlorothiazide in commercial formulations.

ACKNOWLEDGEMENTS:

I am very much thankful to Dr. M. chinna eswaraiah, Principal, Anurag Pharmacy College For his guidance, kind help and constant encouragement at every step during the progress of my work without which successful completion of this work would not have been possible. It is my pleasure to express my sincere thanks to Dr.P.RajeshwarReddy, correspondent and secretary, Amartya educational society, for his providing laboratory facilities and chemicals. I am also grateful to my friends for their kind help from time to time at each and every step of my project work.

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Received on 26.12.2011 Modified on 23.01.2012

Asian J. Research Chem. 5(4): April 2012; Page 472-476

INTRODUCTION:

Statistical

Statistical

Statistical Analysis

Statistical Analysis