ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF IRBESARTAN BY DERIVATIVE SPECTROSCOPY (FIRST ORDER)

About Authors:
Gunjan Kalyani^*1, Vishal S. Deshmukh¹, Pranita Kashyap¹, Yogesh Vaishnav¹, Ram D. Bawankar
¹Shri Rawatpura Sarkar Institute of Pharmacy,
Kumhari, Durg, Chhattisgarh
*kalyani.gunjan@yahoo.in

Abstract
Irbesartan is chemically 2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl) phenyl] phenyl} methyl) -1,3-diazaspiro [4.4] non-1-en-4-one. Irbesartan is an Angiotensin II receptor antagonist effective in the treatment of   Hypertension.  It is also effective in the treatment of High blood pressure.  It is also effective when used alone or in combination with other drugs. Objective of the present study is to develop a simple, sensitive, accurate, precise and rapid first order derivative spectrophotometric method for the estimation of irbesartan in pure form. For the estimation of irbesartan, solvent system employed was 50% v/v aqueous ethanol and wavelength of detection (λ_det) was 237 nm. The linearity was obtained in the range 8 – 18 µg/ml, with a regression coefficient, R² = 1. The LOD & LOQ were found to be 0.5 µg/ml and 1.63 µg/ml respectively. Obtained results showed that there is minimum intra day and inter day variation. The developed method was validated and recovery studies were also carried out. Sample recovery using the above method was in good agreement with their respective labeled claims, thus suggesting the validity of the method and non-interference of formulation excipients in the estimation. First order derivative spectroscopy method is simple, rapid and reproducible and further it can be used for the analysis.

Reference Id: PHARMATUTOR-ART-1431

Introduction
Irbesartan is chemically 2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl) -1,3-diazaspiro [4.4] non-1-en-4-one. Irbesartan is an Angiotensin II receptor antagonist effective in the treatment of   Hypertension¹.  It is also effective in the treatment of High blood pressure¹.  It is also effective when used alone or in combination with other drugs. Irbesartan is a nonpeptide tetrazole derivative and an angiotensin II antagonist that selectively blocks the binding of angiotensin II to the AT1 receptor. In the renin-angiotensin system, angiotensin I is converted by angiotensin-converting enzyme (ACE) to form angiotensin II. Angiotensin II stimulates the adrenal cortex to synthesize and secrete aldosterone, which decreases the excretion of sodium and increases the excretion of potassium. Angiotensin II also acts as a vasoconstrictor in vascular smooth muscle¹. Irbesartan, by blocking the binding of angiotensin II to the AT1 receptor, promotes vasodilation and decreases the effects of aldosterone. The negative feedback regulation of angiotensin II on renin secretion is also inhibited, but the resulting rise in plasma renin concentrations and consequent rise in angiotensin II plasma concentrations do not counteract the blood pressure–lowering effect that occurs. The action of ARBs is different from ACE inhibitors, which block the conversion of angiotensin I to angiotensin II, meaning that the production of angiotensin II is not completely inhibited, as the hormone can be formed via other enzymes. Also, unlike ACE inhibitors, irbesartan and other ARBs do not interfere with response to bradykinins and substance P, which allows for the absence of adverse effects that are present in ACE inhibitors (eg. dry cough)¹.

Fig. 1: structure of irbesartan

UV spectrophotometry is generally preferred especially by small-scale industries as the cost of the equipment is less and the maintenance problems are minimal. The method of analysis is based on measuring the absorption of a monochromatic light by colorless compounds in the near ultraviolet path of spectrum (190-380nm). The fundamental principle of operation of spectrophotometer covering UV region consists in that light of definite interval of wavelength passes through a cell with solvent and falls on to the photoelectric cell that transforms the radiant energy into electrical energy.

Literature review suggested several analytical methods that have been reported for the estimation of Irbesartan in bulk or pharmaceutical formulations include High Performance Liquid Chromatography, and UV-Visible Spectrophotometry. Literature review also revealed that there is no First order Derivative Spectroscopic method. The objective of the work was to develop simple, accurate, precise and economic first order derivative Spectroscopic method to estimate the candesartan in bulk. The method should be simple, accurate, precise, reproducible and statistically valid.

Thus, the objectives of project:
I.            To develop a simple, precise, accurate method, less time consuming & economical derivative spectroscopic method.
II.            Under derivative spectroscopy, the development of First Order
derivative Method.
III.            Validation of developed method using common parameters:
a)      Linearity
b)      Precision
c)      Accuracy
d)     Sensitivity
e)      Limit of Detection (LOD)
f)       Limit of Quantification (LOQ)

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Materials and Methods

Drug
The standard sample of IBRESARTAN was obtained as gift sample from Dr. Reddy’s Laboratory Pvt. Ltd., Hyderabad, A.P., India. The irbesartan tablets were procured from local market.

Instrument specifications
UV Spectrophotometer, Shimadzu, model 1800.

Chemicals and reagents used
Ethanol obtained from local market, manufactured by Merck Pharmaceuticals.

Preparation of stock solution
The stock solution of candesartan is prepared by dissolving 100 mg of drug in 100 ml methanol in volumetric flask with continuous shaking; 0.08 ml of sample was withdrawn and diluted to 10 ml methanol to get 8 μg/ml of solution. The solution was than scanned in UV range between 200-400 nm UV-VIS Spectrophotometer, Shimadzu, Japan to determine the absorption maxima of the drug against blank as methanol.

Wavelength scanning and determination of absorption maximum
From the stock solution of Irbesartan, known concentration of 8μg/ml is prepared by suitable dilution with 50 % aqueous ethanol. Wavelength scanned for the maximum absorption of drug solution using UV-Visible spectrophotometer within the wavelength region of 200–400 nm against blank solvent. Convert the normal mode obtained spectra to first order derivative. The wavelength that shows the peak with a highest absorbance is considered as absorbance maximum of the drug. The result is presented in fig. 2.

Fig. 2: Wavelength scanning and determination of absorption maximum

Linearity studies
Stock solution was subsequently diluted with methanol to get 4μg/ml, 6μg/ml, 8μg/ml, 10μg/ml, 12μg/ml 14μg/ml, 16μg/ml, 18μg/ml 20μg/ml, 22μg/ml 24μg/ml. Convert the normal mode obtained spectra to first order derivative using software UV probe 2.34. The results are tabulated and the linearity curve was constructed by plotting concentration Vs. D¹ value. The result is presented in table 1 and fig. 3.

Fig. 3: standard graph of Irbesartan

Regression equation; y = 0.001x + 0.0012
R² = 1
X – Axis: Concentration
Y- Axis: D¹ value

Precision
The precision of method was ascertained; the percent relative standard deviation were calculated and presented.

Inter day and intraday studies for Irbesartan analytical method
The prepared stock solution was subsequently diluted to get 14 μg/ml. The resulting solution absorbance was measured at detection wavelength of 237 nm using double beam UV spectrophotometer against blank solvent. The findings was made at different time intervals in day times in a day and performed continuously for six days. Convert the normal mode obtained spectra to second order derivative. The results obtained were tabulated and studied for inter day and intraday variation. The results are tabulated in table 2(a) and 2(b).

Accuracy studies
The accuracy/recovery studies were carried out by adding a known amount of drug from the pre-analyzed tablet powder and percentage recoveries were calculated. Convert the normal mode obtained spectra to first order derivative. The reproducibility of estimation was determined by performing the tablet drug content of different samples. The results of accuracy studies were expressed in %. The result is presented in table 3.

Assay studies
The assay studies were carried out with the help of candesartan SMEDDS. The percentage purity was calculated. Convert the normal mode obtained spectra to first order derivative. The reproducibility of estimation was determined by performing the drug content of different samples. The results of assay studies were expressed in %. The result is presented in table 4.

Results and Discussion
Irbesartan is a novel, potent, highly selective non peptide Angiotensin II type 1 (AT1) receptor blocker which is administered orally. It is rapidly and completely hydrolyzed to the active moiety, during absorption from the gastrointestinal tract. The physico-chemical characteristic study of Irbesartan like melting point is 180°C nearer to the literature value 182°C in previous literature. The literature survey ascertains that HPLC analytical method is developed for Irbesartan, which is cost effective. In our laboratory we developed first order derivative spectroscopic method for the analysis of irbesartan. The known concentration of irbesartan is prepared and scanned for absorption maximum. The detection wavelength according to spectra is 237 nm. Different measured D¹ values at detection wavelength 237.0 nm is plotted as the curve as D¹ value versus concentration. Irbesartan obeys the beer’s law in the concentration range 8-18 μg/ml. Linearity study indicates the curve is linear in the range of 8 to 18 μg/ml. The linear regression equation is Y = 0.001x +0.012 with regression coefficient, R²= 1. The developed method is validated for repeatability, reproducible and the accuracy and precision. In the inter day and intraday study of standard graph, the % RSD is less than 2% indicating the developed method is reproducible. The different levels of standard concentration solutions are measured for D¹ value and actual concentration is calculated. The results showed that the amount recovered is 100% indicating the first order derivative spectroscopic method is accurate and precise.

TABLE 1: Linearity of Irbesartan

TABLE 2 (a) intraday precision

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TABLE 2 (b) Inter day precision

TABLE 3 Accuracy studies

Table 4 – Depicting the assay study

Conclusion
The developed analytical method for irbesartan by using first order derivative spectroscopy is found to simple, rapid and selective and the amount of drug recovered will be same as the label claimed and precise. It can be conveniently employed for the routine analysis and quantification of irbesartan.

Acknowledgements
Authors would sincerely thank Principal, staff & lab technicians SRIP, Kumhari, Durg, C.G. for their contribution in carrying forward the research work, for providing the necessary support, for his kind and valuable guidance, and for providing basic facilities in lab.

References
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