METHOD DEVELOPMENT and validation OF IRBESARTAN, AN HYPERTENSIVE DRUG
a)Brief resume of the intended work:
6.1 Need for the study:
Hypertension (HTN) is a cardiacchronicmedical condition in which the systemic arterial blood pressure is elevated.
The antihypertensive agents are a class of drugs that are used to treat hypertension (elevated blood pressure).The reduction of the blood pressure by 5mmHg can decrease the risk of stroke by 34%, of ischaemic heart disease by 21%, and reduce the likelihood of dementia, heart failure, and mortality from cardiovascular disease.There are many classes of anti hypertensives, which lower blood pressure by different means; among the most important and most widely used are the thiazidediuretics, the ACE inhibitors, the calcium channel blockers, the beta blockers, and the angiotensin II receptor antagonists or ARBs.
Irbesartan, chemically it is 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 is an angiotensin II receptor antagonist. It has also been tested for use in the treatment of high blood pressure (hypertension). Literature survey reveals that LC, HPTLC, HPLC for determination of content uniformity and simultaneous estimation of Irbesartan is reportedbut there is no stability indicating high-performance liquid chromatography (HPLC) method for the determination of Irbesartan from its tablets, as its Pharmaceutical dosage form. The study focuses on validation of the assay by high performance liquid chromatography (HPLC).
Analytical method development and validation involve a series of activities that are ongoing during the life cycle of a drug product and drug substance.
Analytical quantitative estimation method development should be performed to the extent that it is sufficient for its intended purpose.Upon successful completion of method development, the quantitative estimation method will then be validated to show proof that it is suitable for its intended purpose. The scope and need of the present project is to develop and validate an analytical method forirbesartan to detect it quantitatively estimation as it is the major criteria for any drug to possses.
HPLC method is being used because it is the preferred method for development and validation in the recent trend, and validation of a drug is regulatory requirement and also it ensures that the product are fit for their intended use.
6.2 Drug profile:
Irbesartan:
IUPAC name : Systematic (IUPAC) name
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
Molecular formula : C25H28N6O
Molecular weight : 428.53 g/mol
Melting point : 180-181C
Category : Anti-Hypertensive drug.
Solubility : Irbesartan is slightly soluble in alcohol and methylene chloride and practically insoluble in water.
It is official in British pharmacopoeia.
6.4 Review of the literature:
Literature survey reveals that the International Conference on Harmonization (ICH) guideline entitled 'Stability Testing of New Drug Substances and Products' requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance. Susceptibility to oxidation is one of the required tests (ICH, 1993, 1996). The hydrolytic and the photolytic stability are also required. An ideal stability-indicating method is one that quantifies the drug per se and also resolves its degradation products. A very viable alternative for stability-indicating analysis of Irbesartan is HPLC. The aim of the present work was to develop an accurate, specific, reproducible, and stability indicating method for the determination of Irbesartan in the presence of its degradation products and related impurities as per ICH guideline.
Estimation of plasma irbesartan using HPTLC and HPLC. In this method irbesartan and internal standard were extracted as neutral complex with heptanes sulphonate and dry ethyl ether and evaporated to dryness. The residue was redissolved with the mobile phase and was injected on to a c18 column. The eluate was monitored at 224nm. HPTLC seperations were performed on coated plate chromatograms were developed using a mixture. Analyte was derivatised using acid , quantification was carried out using densiometre in flouresence mode at wavelength of 260nm and emission filter of 355nm. The response was linear from HPLC and HPTLC and quantification limit. The inter and intraday coefficient variation were <15%1.
Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by liquid chromatography. In this method chromatographic separation was achieved using supelcocil c18 and a mobile phase consisting of mixture irbesartan and hydrochlorothiazide were detected at 275nm and eluted 5.8 and 7.8min after injection. No endogenous substances were found to interfere . The method utilizes protein precipitation with acetonitrile as the only sample preparation involved prior to reverse phase HPLC. No internal standard were required. linearity and accuracy were determined. This method is being used in therapeutic drug monitoring2.
HPLC determination of irbesartan in human plasma: its application to pharmacokinetic studies. In this method sample prepation was accomplished through a simple deproteinisation procedure with acetonitrile containing losartan to a plasma sample.chromatographic separation was performed on a zorbax xcliplse XDB c18 coloumn at 40 degrees C. An isocratic mobile phase was run and the column eluent was monitored using a flouresence detector set at excitation and emission wave lengths 250nm and 370nm. This method was successfully applied for pharmacokinetic studies.The retention time for losartan and irbesartan were determined. This assay was linear over concentration range with lower limit of quantification. The coefficient of variation for this assay precision and accuracy were determined. The mean relative recoveries of losartan and irbesartan were 99.1% and 98.4%3.
Quantitation of irbesartan and major proteins in human plasma by mass spectrometry with time-of-flight analyzer. In this method simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was developed to analyze irbesartan. After simple micro-liquid–liquid extraction, irbesartan-containing supernatant was spotted on a target plate, mixed with matrix and then detected by MALDI-TOF MS within the clinically therapeutic range. After enzyme digestion, peptide mixtures were injected into nanoliquid chromatography (nanoLC) coupled with tandem mass spectrometry (MS–MS). Protein identification could be carried out simultaneously by peptide sequencing and database searching. Quantity change of proteins before and after administration of irbesartan could be detected by this method. Protein quantitation and identification are successful4.
Liquid chromatographic determination of irbesartan in human plasma. In this method irbesartan and losartan in human plasma were extracted using diethyl ether , dichloromethane followed by back extraction with sodium hydroxide. Neutralized samples were analysed using potassium dehydrogenate phosphate buffer and acetonitrile . Chromatographic separation was achieved on ODS-C-18 column using isocratic elution. The peak was detected using flouresence detector. The quatitation ranges with coefficient of variation and mean recoveries with coefficient of variation were determined. The precision and relative errors were determined. Stability of irbesartan in plasma was >89% with no evidence of degradation5.
Stability Indicating HPTLC Method for Determination of Irbesartan in Pharmaceutical Dosage Form. In this method the solvent system consists of toluene ,ethyl acetate, acetic acid. Densiometric analysis of irbesartan was carried out in absorbance mode of 305nm. This was found to give compact spots for irbesartan. No chromatographic interference from the tablet excipients was found. Irbesartan was subjected to acid alkali hydrolysis ,oxidation, dry heat, wet heat treatment, and photodegradation . the drug is susceptible to all these. The linearity was found to be in range. The method was validated for precision ,robustness and recovery. The method could effectively separate the drug from its degradation products 6.
Method development and validation of Irbesartan using LCMS/MS. In this method employed liquid liquid extraction. Samples containing irbesartan were chromatographed on hypersil gold column at a temperature of 40 degrees C. The isocratic mobile phase composition was a mixture of ammonium formate and methanol. The retention times in these chromatographic conditions was found to be 2.20 min. the developed LC/MS-MS method was found to be selective, simple, sensitive, accurate and linear for analysis of irbesartan in human plasma7.
Identification and characterization of degradation products of irbesartan using LC-MS/TOF, MSn , on line H/D exchange and LC-NMR. In this method irbesartan was subjected to hydrolytic, oxidative, photolytic and thermal stress conditions. The drug showed degradation only in acidic, basic and photoacidic conditions,while it was stable to other stress conditions. A total of three degradation products were formed which were separated on C-8 column employing a gradient HPLC method. Initially a complete mass fragmentation pathway of the drug was established with the help of MS/TOF, MSn and H/D exchange studies. The MS results helped to assign tentative structures to degradation products which were verified through H and 2D COSY LC-NMR experiments. The structures were justified by mechanism of their formation8.
RP-HPLC method development and validation of Valsartan tablet dosage form. The method was carried out using Thermohypersil ODS column with mobile phase comprised of water: acetonitrile: glacial acetic acid . The flow rate was set at 1.0 ml/min and effluent was detected at 273nm. The retention time of valsartan was found to be 4.6 minute. The method was validated for specificity, accuracy, precision, linearity, and limit of detection, limit of quantification, robustness and solubility stability. LOD and LOQ were found to be 2.72 μg/ml and 8.25 μg/ml respectively. The calibration curve was linear in the concentration range of 40-140 μg/ml with coefficient of correlation 0.9990. The percentage recovery for the valsartan was found to be 99.0-100.2 and the % RSD was found to be less than 2 %9.
Development and Validation of HPLC Method for Analysis of Some Antihypertensive Agents in their Pharmaceutical Dosage Forms. In this method HPLC method was developed and validated for the determination of Aliskerin, Ramipril, Valsartan and Hydrochlorothiazide in solid dosage forms. The quatitative determination of analytes were performed on a PUROSPHERE STAR RP 18e analytical column with buffer acetonitrile as mobile phase at a flow rate of 1.0 ml min-1. Detection was made by extracting PDA spectra at 215 nm respectively. During method validation , parameters such as precision, linearity, stability, robustness, ruggedness and specificity were evaluated, which remained in acceptable limits. The method was successfully applied to assess the assay of solid dosage formulations10.
6.3 Main objectives of the study:
Only few analytical methods have been reported for the quantitative estimation of Irbesartan, there is necessity for investigation of new analytical methods for quantitative estimation of Irbesartan in pharmaceutical dosage form. In view of the above fact, the following analytical methods are planned to develop with high sensitivity, accuracy and precision.
In the proposed work attempt shall be made to develop any of the following methods:
- Since the Irbesartan drug is slightly soluble in alcohol and methylene chloride and practically insoluble in water, a novel UV-spectrophotometric method can be developed for its quantitative estimation in pharmaceutical formulation.
- HPLC methods can be developed in different solvent systems for quantitative estimation of Irbesartan bulk drug and pharmaceutical formulation.
- Since the drug is thermostable, GC methods can be developed for quantitative estimation of Irbesartan in bulk drug and pharmaceutical formulation.
- The validation of the method used for quatification shall be carried out as per IP, BP, USP & ICH guidelines.
b) Materials and Methods
7.1Source of data:
- Internet – Science direct, Pubmed, DIS, Helinet, abstracts ,RGUHS.
- Indian Institute of Chemical Technology Library, Hyderabad, Andhra Pradesh. Journals and related articles from library of Krupanidhi College of Pharmacy.
Journals:
1)Indian drugs .
2)Indian journal of analytical chemistry and analyst.
3)Journal of pharmaceutical and biomedical analysis.
4)Journal of chromatography B: biomedical sciences and applications.
National and International E-Journals.
c) List of References:
1.Sane RT Francis M and Pawar S. Estimation of plasma irbesartan using HPTLC and HPLC. Indian Drug Manufacturers Association 2003; 40(2):104-10.
2.Nevin E. Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by liquid chromatography. J Chromatogr 2003; 784(A):195-201.
3.Bae SK, Kim MJ, Shim EJ, Cho DY, Shon JH, Liu KH, et al. HPLC determination of irbesartan in human plasma: its application to pharmacokinetic studies. Biomed Chromatogr 2009; 23(6):568-72.
4.Chi-yu Lu and Chia-Hsein Feng. Quantitation of irbesartan and major proteins in human plasma by mass spectrometry with time of flight analyzer. J Pharm Biomed Anal 2011; 54(1):100-105.
5.Ashok K. Shakya, Yusuf M. Al-Hiaric and Omran M.O. Alhamami. Liquid chromatographic determination of irbesartan in human plasma. J Chromatogr 2007; 848(2):245-50.
6.Mhaske Deepali Vijay, Dhaneshwar Sunil Rajaram and Kadam S.S. Stability Indicating HPTLC Method for Determination of Irbesartan in Pharmaceutical Dosage Form. Indian J Pharm Educ. Res 2007; 261-69.
7.Ganeshan M. Nanjudan S. Gomathi M. and Muralidharan S. Method Development and Validation of Irbesartan using LC-MS/MS; Application to Pharmacokinetic Studies. J Chem Pharm Res 2010; 740-46.
8. Ravi P. Archana sahu and Sarnjit singh. Identification and characterization of degradation products of irbesartan using LC-MS/TOF, MSn , on line H/D exchange and LC-NMR. J Pharm Biomed Anal 2010; 51:1037-46.
9.Bodela narendra reddy, Ustelamuri chenna reddy, Nargarjuna P and Dileep kumar CH. RP-HPLC method development and validation of Valsartan tabletdosage form. J Chem Pharm Res 2010; 2(4):878-86.
10.Shalini Pachauri , Sarvesh Paliwal , Kona.S.Srinivas, Yogendra Singh and Varun Jain. Development and Validation of HPLC Method of Analysis of Some Antihypertensive Agents in their Pharmaceutical Dosage Forms. J Pharm Sci Res 2010; 8:459-64.
11.Matsoukar J and Mavromostakas T. Structure elucidation and conformational properties of Irbesartan. Drug discovery and design 2002; 174-79.
12.ICH Guidelines 1993 and 1996
13.Rossi S, editor. Australian Medicines Handbook 2006. Adelaide: Australian Medicines Handbook; 2006.
14.Sweetman SC, Martindale.The complete drug reference. 33rd ed. London, Pharmaceutical press; 2002:537.
15.British Pharmacopoeia, Ph Eur monograph 1590. London, Medicines and Health care products Regulatory Agency (MHRA) 2005; 1:730.
16.O’Neil MJ, editor. The Merck Index- an encyclopedia of chemicals, drugs andbiologicals. 13th ed. New Jersey, Merck and co. Inc; 2001. 642. biologicals. 13th ed. New Jersey, Merck and co. Inc 2001; 642.
17. Retrieved on 22nd may 2011 at 2.30 pm.