INTRODUCTION:

Irbesartan is used alone or in combination with other medication to treat high blood pressure. It also used to treat kidney disease caused by diabetes in patients with type 2 diabetes (condition in which the body does not use insulin normally and therefore cannot control the amount of sugar in the blood) and high blood pressure.⁽¹⁾ Irbesartan is in a class of medication called angiotensin II receptor antagonists. It works by blocking the action of certain natural substances that tighten the blood vessels, allowing the blood to flow more smoothly and the heart to pump more efficiently⁽²⁾.

Irbesartan is a nonpeptide tetrazole derivative and an angiotrnsin II antagonist that selectively block the binding of angiotensin II to the AT₁ receptor. In the rennin angiotensin system, aniotensin I is converted by angiotensin – converting enzyme (ACE) to form angiotensin II. ^(3-9)

MATERIAL AND METHOD ^(10,11):

Table 1: List of apparatus/ instruments used.

List of Instruments

Equipment	Company
MaXia 220lectronic balance	Shinko Denshi Co Ltd, Japan
UV 150-02, Visible double beam spectrophotometer	Shimadzu corporation, japan
Digital pH Meter	Global, Ltd. Model No. – PGB 100
sonicattor	Wenser
HPLC Binary Gradient System Model No. - 3000 series HPLC pump – P – 3000- M Reciprocating Column- Cosmosil C-18 (4.6 ID × 250 mm , Particle size 5µ) Detector – UV 3000	Analytical Technologies Ltd
Analytical Balance Model No. – PGB 100	Wenser

Table 2: List of chemical used.

List of Chemicals

Sr. No.	Reagents and Chemicals	Make	Details
1.	Water	In House Production	HPLC grade
2.	Methanol	Merck	HPLC grade

Table 3: List of API used.

Sr. No.	Name	Specification	Manufacturer/Supplier
1	Irbesartan	Working standard	Macleods Pharmaceutical Ltd Sarigram Gujarat

Preparation of mobile phase

Mixed a HPLC grade Methanol: Water with pH 3.0 (80:20) in volumetric flask. Filter through 0.45μ filter under vacuum filtration.

Diluent preparation:

Use mobile phase as diluent.

Standard Stock Solution:

Procedure:

Accurately weighed quantity of Irbesartan 10 mg individually dissolved in 10 ml volumetric flask using mobile phase and solution was sonicated for 20 minutes and volume is make up to the mark to get 1000 μg/ml and filtered through 0.45μm membrane filter. 1ml from each solution taken and dissolved in 10ml volumetric flask separately using mobile phase to get 100 μg/ml.

Preparations of working standard solution:

Procedure:

From the standard stock solution respectively Irbesartan solution taken and added in 10 ml volumetric flask and diluted up to the mark with mobile phase.

Preparation of Sample solution:

Procedure:

20 tablets were weighed and powdered, tablets powder equivalent to 10 mg of Irbesartan was transferred 100 ml volumetric flask, sufficient amount of mobile phase was added and dissolved by 20minutes ultrasonication. Then made the volume up to the mark with the mobile phase and filtered with 0.45µ filter paper. Pipette out respectively solution from above solution and diluted to 10 ml mobile phase and use for sample injection.

Selection of analytical wavelength:

Accurately weighed quantity of Irbesartan 10 mg dissolved in 100 ml volumetric flask using methanol and volume is make up to the mark to get 100 μg/ml. From this solution respectively solution was taken and added in 10 ml volumetric flask and diluted up to the mark using methanol. Solution was scanned using UV-Visible Spectrophotometer in the spectrum mode between the wavelength ranges of 400 nm to 200 nm. The wavelength selected was 209 nm.

Fig. 1. Wavelength of Irbesartan

Optimized chromatographic condition:

In the present study the validation of Irbesartan was achieved by using column Cosmosil C18, (250×4.6mm,5µ) with mobile phase consisting of mixture of methanol and Water (pH 3.0) in the ratio of 80:20 at a flow rate 1.0 ml/min with UV detection wavelength of 228nm at ambient temperature. The retention time for Irbesartan 3.257 min respectively.

RESULT AND DISCUSSION:

System Suitability:

HPLC system was optimized as per the chromatographic conditions. 20 µl of standard solutions of drugs were injected in triplicate into the chromatographic system. The chromatogram were recorded and measure the response for the major peak. system suitability parameter such as retention time, theoretical plate and asymmetry factor. then the %RSD of all parameter were calculated.

Table 4. System suitability parameters for Irbesartan

System suitability parameters	Irbesartan
Retention time	3.257 min
Theoretical plate no.	9234
Tailing factor	1.23
Resolution	0.0

Table 5 Assay of the Developed RP-HPLC Method

Assay of the Developed RP-HPLC Method:

Sr. No.	Concentration (µg/ml)	Area of Standard	Area of Sample	% Assay(w/v)
1	10	2327476	2129410	91.490%

Validation of the Developed Method:

A. Specificity:

Specificity is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix. It is a measure of the degree of interference from such things as other active ingredients, excipients, impurities and degradation products, ensuring that a peak response is due to a single component only i.e. no co-elution exist.

In practice, this can be done by spiking the drug substances or product with appropriate levels of impurities or excipients, and demonstrating that the assay result is unaffected by the presence of these extraneous materials. If impurities of degradation products to second well characterized procedure. These comparisons should include samples stored under relevant stress condition (e.g. light, heat, humidity, acid or base hydrolysis and oxidation).

Table 6 Specificity data of the Developed RP-HPLC Method

Drug	Area	Amount added(mg)	Amount recovered (mg)	% Recovery	SD	%RSD
Standard drug of Irbesartan	2867513	10	9.95	99.5	1.19	1.20
Standard drug of Irbesartan	2322410	10	9.94	99.4	1.19	1.20
Tablet sample of Irbesartan	2109074	10	9.92	99.2	1.70	1.72
Tablet sample of Irbesartan	2000558	10	9.99	99.9	1.70	1.72

B. Ruggedness:

The ruggedness of analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of conditions such as different laboratories, different instruments, different lots of reagents, different temperatures, different days, different analysts, etc. It is normally expressed as the lack of influence on test result of proportional and environmental variables of the analytical method.

Table 7 Ruggedness data of the Developed RP-HPLC Method

Drug	Area	Amount added(mg)	Amount recovered (mg)	% Recovery	SD	%RSD
Standard drug of Irbesartan	2327816	10	9.93	99.94	0.0424	0.0425
Standard drug of Irbesartan	2329450	10	10	100	0.0424	0.0425
Tablet sample of Irbesartan	2195713	10	9.85	99.85	0.0141	0.0142
Tablet sample of Irbesartan	1996358	10	9.90	99.87	0.0141	0.0142

C. Robustness:

The robustness of analytical method is the measure of its capacity, to remain unaffected by small but deliberate variation in method parameters and provides an indication of its reliability during normal usage. Experiments are performed by changing conditions such as temperature (± 5⁰C), buffer pH (± 0.5), and ionic strength of buffers, level of additives to mobile phase. The method must be robust enough to withstand slight change and allow routine analysis of sample.

Table 8 Result of change in wavelength for Irbesartan

Name of drug	Wavelength (nm)	Area	Mean	S.D	%RSD
Standard drug of Irbesartan	207	2327816	2328633	0.0424	0.0425
	209	2328633
	211	2329450
Tablet sample of Irbesartan	207	1996358	2096035	0.0141	0.0142
	209	2096035
	211	2195713

Table 9 Result of change in flow rate for Irbesartan

Name of drug	Flow rate (ml/min)	Area	Mean	S.D	%RSD
Standard drug of Irbesartan	0.9	2327816	2328633	0.0424	0.0425
	1.0	2328633
	1.1	2329450
Tablet sample of Irbesartan	0.9	1996358	2096035	0.0141	0.0142
	1.0	2096035
	1.1	2195713

D. Limit of Detection and Limit of Quantification

Limit of Detection:

The lowest conc. Of the analyte in the sample that the method can detect but not necessarily quantify under the stated experimental conditions simply indicates that the sample is below or above certain level. Limit test prescribed as percentage or as parts per million. The limit of detection will not only depend on the procedure of analysis but also on type of instrument.

Limit of Quantization:

The limit of quantization (LOQ) is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. It is expressed as the conc. of analyte (e.g. percentage, parts per billion) in the sample. The S/N ratio should not less than 10 and RSD ≤ 3%.

Limit of detection result for Irbesartan was found to be 0.074 µg/ml respectively and also limit of quantification result for Irbesartan was found to be 0.24µg/ml respectively and were within the limits.

CONCLUSION:

The developed RP-HPLC method offers several advantages such as rapidity, usage of simple mobile phase and easy sample preparation steps. From the present study, it can be concluded that the proposed method is simple, specific, sensitive, precise, accurate and reproducible. Results of validation parameters demonstrated that the analytical procedure is suitable or appropriate for its intended purpose. Further, improved sensitivity makes it and reliable specific for its intended use. Hence, this method can be applied for the analysis of pharmaceutical dosage forms and pure drug.

ACKNOWLEDGEMENT:

The authors express their gratitude to the Gajanan Maharaj College of Pharmacy, Aurangabad for providing all the facilities and Macleods Pharmaceutical Ltd Sarigram Gujarat for providing me the gift samples of Irbesartan.

REFERENCES:

1. Gandla K, Rao JS, Kumar JR, New Stability Indicating Validated RP-HPLC Method for Simultaneous Estimation of Irbesartan and Atorvastatin in Combined Tablet Dosage Forms. Ame. J. Pharm. Tech. Res., 2015; 5(4):380-390

2. Gandla K, Suthakaran R, Kandoji P, Development and Validation of RP-HPLC Method for Simultaneous Estimation of Irbesartan and Simvastatin in Tablet Dosage Form. Ame. J. Pharm. And Health Res., 2014; 2(10):199-205

3. Kalaiselvi P and Lalitha K G, Development and Validation of RP-HPLC Method For The Simultaneous Estimation of Chlorthalidone And Irbesarton In Pharmaceutical Dosage Form. Pharmacophore.2014; 5(2):279-286.

4. Galen W Ewing. Instrumental Methods of Chemical Analysis. 5^thed. McGraw Hill Book Company; 1985; 1-2.

5. Gawande Vandana. Pharmaceutical Analysis-1. 3^rded. Nirali Prakashan; 2017; 1.1-1.2.

6. Jeffery GH, Bassett J, Mendham J. Vogel’s Textbook of Quantitative Analysis. 5^thed. Pearson Education; 3-5.

7. Dr. Uttam Singh Baghel, Dr. Arora Progi. A Text book of Pharmaceutical Analysis- 1. S. Vikas and Company (Medical Publishers); 2017; 3-6.

8. Siddiqui Anees A, Siddiqui Seemi. Pharmaceutical Analysis. Volume-2. 3^rded. CBS Publisher and Distributors; 2014; 1-3.

9. Mendham J, Denney RC, Barnes JD, Thomas M. Vogel’s Textbook of Quantitative Analysis. 6^thed. Pearson Education; 2006; 36.

10. Dr. Rao D. Muralidhara, Dr. A V N Swamy. Instrumental Method of Analysis. CBS Publisher and Distributors; 7-11,272.

11. Gray N, Calvin M, Bhatia SC. Instrumental Method of Analysis. 1^sted. CBS Publishers and Distributors; 2009; 8-9, 126-128.

Received on 10.05.2020 Modified on 21.05.2020

Research J. Science and Tech. 2020; 12(2): 131-135.

DOI: 10.5958/2349-2988.2020.00016.9