RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

DEVELOPMENT OF NEW ANAYTICAL TECHNIQUIES AND THEIR VALIDATION FOR THE ESTIMATION OF ISONIAZID AND TOPIRAMATE IN BULK AND DOSAGE FORM

MASTER OF PHARMACY DISSERTATION PROTOCOL,

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE.

BY

RAVAL KASHYAP ASHVINBHAI

M.PHARM - I

Under The Guidance Of

Dr. E.V.S. Subrahmanyam,

M.PHARM, Ph D.

P. G. DEPARTMENT OF QUALITY ASSURANCE.

SRINIVAS COLLEGE OF PHARMACY, MANGALORE 574143.

2010 2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

1.0

NAME OF THE CANDIDATE

AND ADDRESS:

KASHYAP A. RAVAL

Srinivas College of Pharmacy

Valachil

Mangalore,

Karnataka.

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NAME OF THE INSTITUTE:

Srinivas College of Pharmacy

Valachil

Mangalore,

Karnataka.

3.0

COURSE OF THE STUDY AND SUBJECT:

Master of Pharmacy

(Quality Assurance)

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DATE OF ADMISSION TO THE COURSE:

31st MAY 2010

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TITLE OF THE TOPIC:

DEVELOPMENT OF NEW ANAYTICAL TECHNIQUIES AND THEIR VALIDATION FOR THE ESTIMATION OF ISONIAZID AND TOPIRAMATE IN BULK AND DOSAGE FORM

BANGALORE, KARNATAKA

ANNEXURE II

REGISTRATION OF SUBJECT FOR DISSERTATION

6.0

BRIEF RESUME OF THE INTENDED WORK:

NEED OF STUDY :

Analytical Method Development for Pharmaceutical Formulations:

The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs.

In brief, the reasons for the development of newer methods of drug analysis are:

The drug or drug combination may not be official in any pharmacopoeias,

A proper analytical procedure for the drug may not be available in the literature due to patent regulations,

Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients,

Analytical methods for the quantitation of the drug in biological fluids may not be available,

Analytical methods for a drug in combination with other drugs may not be available,

The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.

Analytical techniques that are generally used for drug analysis are biological and microbiological methods, radioactive methods, physical methods and miscellaneous techniques like conventional titrimetric, gravimetric and polarimetric methods.

Analytical method development provides the support to track the quality of the product from batch to batch. Estimation can be performed by the following two methods:

Titrimetric methods and

Instrumental methods.

Spectrophotometric Methods

Chromatographic Methods

Methods for analyzing drugs in dosage forms can be developed, provided one has knowledge about the nature of the sample, its molecular weight, polarity, ionic character and the solubility parameter. Method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase.

The following is a suggested method development scheme for a typical HPLC-UV

Related substance method.

1. To define the goals for method development (e.g. what is the intended use of the method), and to understand the chemistry of the analytes and the drug product.

2. To develop preliminary HPLC conditions to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments.

3. To develop a suitable sample preparation scheme for the drug product

4. To determine the appropriate standardization method and the use of relative response factors in calculations.

5. To identify the weaknesses of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples.

6. To complete method validation according to ICH guidelines as mentioned in

Q2 (R1).

6.1 BRIEF INTRODUCTION ABOUT ISONIAZID

It is an anti-tuberculosis drug.

STRUCTURE :

IUPAC NAME = Isonicotinohydrazide

FORMULA = C6H7N3O

MOL. WEIGHT = 137

PROTEIN BINDING = Very low (0-10%)

METABOLISM = liver, CYP450, 2C19, 3A4 inhibitor.

HALF LIFE = 0.5-1.6h fast acetylator, 2-5h slow acetylator.

EXCRETION = urine (primarily), feces.

SOLUBILITY = freely soluble in water , sparingly soluble in alcohol , very slightly soluble in ether

PHARMACOLOGY

Mechanism of action

Isoniazid is a prodrug and must be activated by a bacterial catalase-peroxidase enzyme called KatG. KatG couples the isonicotinic acyl with NADH to form isonicotinic acyl-NADH complex. This complex binds tightly to the enoyl-acyl carrier protein reductase known as InhA, thereby blocking the natural enoyl-AcpM substrate and the action of fatty acid synthase. This process inhibits the synthesis of mycolic acid, required for the mycobacterial cell wall. A range of radicals are produced by KatG activation of Isoniazid, including nitric oxide,[6] which has also been shown to be important in the action of another antimycobacterial prodrug PA-824.

Isoniazid is bactericidal to rapidly-dividing mycobacteria but is bacteriostatic if the mycobacterium is slow-growing. Isoniazid inhibits the P450 system.

Side effects

Adverse reactions include rash, abnormal liver function tests, hepatitis, sideroblastic anemia, high anion gap metabolic acidosis, peripheral neuropathy, mild central nervous system (CNS) effects, drug interactions resulting in increased phenytoin or disulfiram (Antabuse) levels and intractable seizures (status epilepticus).

Headache, poor concentration, weight-gain, poor memory, and depression have all been associated with isoniazid use. All patients and healthcare workers should be aware of these serious adverse effects, especially if suicidal thinking or behavior are suspected.

As p450 is required for porphyrin synthesis its deficiency leads to poor heam formation in early RBCs leads to sideroblastic anemia.

6.1.1 OBJECTIVES OF THE STUDY:

In the proposed work, attempt shall be made :

To develop a new instrumental method for estimation of ISONIAZID.

To develop a validated method according to ICH guidelines.

To apply validated method for the estimation of ISONIAZID in pharmaceutical formulation.

6.1.2 REVIEW OF LITERATURE:

A literature survey was carried out for the estimation of ISONIAZID in marketed dosage forms. It was found that very few methods have been reported for this drug. The collection of references are reproduced below:

Prodromos BP, Pantelis ET.1developed a sensitive colorimetric determination of microquantities of isoniazid, The method described is based on the reduction, under the optimum conditions of Fe (III) to Fe (II) by INH and then the Fe (II) resulted, is reacted with o-phenanthroline (f-Phen) to form the well-known highly stable orange-red coloured chelate complex [Fe (II)-(o-Phen)3]2+, the ferroin complex. The latter exhibits an absorption maximum at = 510 nm, with an apparent molar absorptivity of 2.28 104 1 mol1 cm1 (referred to drug analysed), while the Sandell's sensitivity was calculated as 6.0 ng cm2.

Safavi MA, Karmi A, Hormozi Nezhad MR, Kamali R and Saghir N.2 developed Sensitive indirect spectrophotometric determination of isoniazid, The procedure is based on the reaction of copper (II) with isoniazid in the presence of neocuproine (NC). In the presence of neocuproine, copper (II) is reduced easily by isoniazid to a Cu (I)neocuproine complex, which shows an absorption maximum at 454nm. By measuring the absorbance of the complex at this wavelength, isoniazid can be determined in the range 0.33.5gml1. This method was applied to the determination of isoniazid in pharmaceutical formulation and enabled the determination of the isoniazid in microgram quantities (0.33.5gml1).

Hoda Hassan Y, Abdel-Maboud Mohamed I, Fardos Mohamed A. 3 developed Utility of Certain -Acceptors for the Spectrophotometric, The interaction of isoniazid with three selected -acceptors in an aqueous-acetonitrile solution of pH 9.0-9.5 was found to give intensely colored products. The determination of products was carried out at 445 nm for chloranil, 450 nm for bromanil and around 750 nm for 7, 7, 8, 8-tetracyanoquinodimethane. The effect of several variables on the colour development was studied. The molecular ratios of the reactants have been established. The proposed methods have been applied successfully for analysis of isoniazid in pure samples and in a number of its pharmaceutical preparations with good accuracy and precision.

Liusheng Huang, Florence Marzan, Anura L, et. al.4 developed Development and validation of a hydrophilic interaction liquid chromatographytandem mass spectrometry method for determination of isoniazid in human plasma, An LCMS/MS method for the determination of isoniazid in human plasma was developed and validated. Human plasma aliquots of 100L were used for analysis. The assay used nialamide as the internal standard. The calibration curve concentration range was 5010,000ng/mL. Sample preparation utilized protein precipitation, and the supernatant was directly injected onto silica column without reconstitution.

Abdel-Hamid Ahme N, Samia El Gizawy M, Hussein El Subbagh I. 5 developed spectrophotometric determination of isoniazid using ethyl 8-quinolinoxy aceta, a colorimetric method based on the interaction of isoniazid and the newly synthesized ethyl 8-quinolinoxy acetate was developed. The absorbance was measured at 504 nm and the procedure was found to be sensitive for INH in the 10-4 m range. the method was applied to the analysis of the drug in pharmaceutical dosage forms and proved to be simple, demonstrating good accuracy and precision. Structural elucidation of the reagent 2 as well as the chromogen 4 has been achieved using 1h NMR and elemental analyses.

Garcia Bautista JA,Garcia Mateo Martinez Calatayud J.6 developed Spectrofluorimetric Determination of Iproniazid and Isoniazid in a FIA System Provided with a Solid-Phase reactor, A FIA assembly is proposed for the fluorimetric determination of iproniazid and isoniazid. The oxidation of both drugs is carried out by H2O2. The excess of reagent is destroyed and removed from the flow-injection manifold by means of a metallic copper reactor which acts as catalyst in the decomposition of H2O2 and a home made debubbler. The calibration graphs were linear up to 14 g ml-1 and 10 g ml-1 (n=6) for iproniazid and isoniazid, respectively, with limits of detection of 0.008 g ml-1 and 0.005 g ml-1. The sample throughput was 24 h-1 for both drugs. The influence of foreign compounds was studied and the procedure was applied to determination of iproniazid and isoniazid in pharmaceutical formulations.

Zhi-ming Zhou, Dao-yuan Zhao, Jing Wang, Wei-jun Zhao and Ming-min Yan 7 developed Study of cloud point extraction and high-performance liquid chromatographic determination of isoniazid based on the formation of isonicotinylhydrazon, ISoniazid reacted with p-dimethyl amino benzaldehyde (DABD) in the presence of trichloroacetic acid to give isonicotinylhydrazone (INZ) havingmax365nm. Cloud point extraction (CPE) is carried out to extract INH and IHZ in aqueous solutions using surfactant poly (ethylene glycol) 4000 (PEG4000), respectively. Langmuir model is used to study the adsorption behaviors of the two solutes on micelles of PEG4000. A linear correlation is found between variation of PEG4000 concentration required for feed concentration of the two solutes and used to predict PEG4000 concentration required for extracting INH and IHZ in CPE procedure. The results calculated show that, for a desired recovery level of 90%, only can IHZ be sufficiently extracted by PEG4000.

Amina El-Brashy M, Fawzia Ibrahimand A, Saadia El-Ashry M.8developed Colorimetric Determination of Some Important Hydrazine Derivatives, A simple and rapid colorimetric method for the determination of isoniazid, isocarboxazid, iproniazid phosphate, and phenelzine sulphate and phenylhydrazine hydrochloride is described. The method is based on the formation of ferroin, when the studied drugs react with a mixture of iron (III) and 1, 10-phenanthroline, and measurement of the absorbance at 512 nm. The procedure has been successfully applied to the assay of the pharmaceutical preparations of the studied drugs and the results are favorably comparable to the official methods.

Alexandrou NE, Vasilikiotis GS.9 Developed Infra-red spectra of isoniazid hydrazones, the infra-red spectra of a number of hydrazones, prepared from isoniazid and substituted benzaldehydes and acetophenones, have been examined. The influence of substituents on the carbonyl stretching frequency (Amide I band) is presented and discussed in terms of polar and steric effects.

El-Brashy, El-Ashry SM.10 developed colorimetric and titrimetric assay of isoniazid, two methods are proposed for the determination of isoniazid in pure form or in tablets. In the first method chlorpromazine hydrochloride, when treated with 2-iodoxybenzoic acid as an oxidant in 50% w/v o-phosphoric acid solution, is oxidized to chlorpromazine free radical which absorbs at 530 nm. The second method involves the titrimetric determination of isoniazid using N-bromophthalimide as a titrant. The end-point is determined either directly using methyl red or amaranth as indicator, or by a back titration method in which a known excess of N-bromophthalimide solution is added to isoniazid solution and then the residual unreacted reagent is determined eudiometrically.

6.2 BRIEF INTRODUCTION ABOUT TOPIRAMATE

Topiramate (brand name Topamax) is an anti-convulsant (antiepilepsy). It was originally produced by Ortho-McNeil Neurologics and Noramco. It was discovered in 1979 by Buuce E. Maryanoff and Joseph F. Gardocki during their research work at McNeil Pharmaceutical.

STRUCTURE

IUPAC NAME =2,3:4,5-Bis-0-(1-methylethylidene)-beta-D-fructopyranose sulfamate

CATEGORY = anticonvulsant (antiepilepsy)

MOL. WEIGHT= 339

FORMULA = C12H21NO8S

BIOAVAILABILITY = 80 %

HALF LIFE = 19- 23 HOURS

METABILISM = 30% hepatic, 70% is excreted unchanged.

EXCRETION = 70% renal (in urine) in unchanged form.

SOLUBILITY = soluble in water.

PHARMACOLOGY

Mechanism of action

Chemically, topiramate is a sulfamate-substituted monosaccharide, related to fructose, a rather unusual chemical structure for an anticonvulsant.

Topiramate is quickly absorbed after oral use. Most of the drug (70%) is excreted in the urine unchanged. The remainder is extensively metabolized by hydroxylation, hydrolysis, and glucuronidation. Six metabolites have been identified in humans, none of which constitutes more than 5% of an administered dose.

The exact mechanism of action is unknown, but four properties that may contribute to topiramate's antiepileptic and antimigraine efficacy include a blockage of voltage-dependent sodium channels, an augmentation of gamma-aminobutyrate acid activity at some subtypes of the GABA- A receptors, antagonism of AMPA/kainate subtype of the glutamate receptor, and inhibition of the carbonic anhydrase enzyme, particularly isozymes II and IV.

Side effects

A GlaxoSmithKline-sponsored Phase IV study suggested that cognitive side effects may be more common with topiramate than with lamotrigine. In studies of healthy volunteers, therapeutic doses of topiramate for bipolar disorder produced greater cognitive deficits than lamotrigine, including short term memory loss and word-finding difficulty.

The side-effects reported by > 10% of subjects in at least 1 clinical study Listed by prevalence:

Paresthesia (numbness & tingling) (23.7%)

Upper respiratory tract infection (17.5%)

Diarrhea (16.8%)

Nausea (15.4%)

Anorexia (loss of appetite) (13.3%)

Memory problems (11.2%)

The side-effects most frequently leading to discontinuation of therapy with topiramate were:

Psychomotor slowing (4.1%)

Memory problems (3.3%)

Fatigue (3.3%)

Confusion (3.2%)

Somnolence (3.2%)

6.2.1 OBJECTIVES OF THE STUDY:

In the proposed work, attempt shall be made :

To develop a new instrumental method for estimation of TOPIRAMATE.

To develop a validated method according to ICH guidelines.

To apply validated method for the estimation of TOPIRAMATE in pharmaceutical formulation.

6.1.2 REVIEW OF LITERATURE:

A literature survey was carried out for the estimation of TOPIRAMATE in marketed dosage forms. It was found that very few methods have been reported for this drug. The collection of references is reproduced below.

Bahrami GH, Mirzaeei SH, Kiani A.11 developed Sensitive analytical method for Topiramate in human serum by HPLC with pre-column fluorescent derivatization and its application in human pharmacokinetic studies, a sensitive and specific high performance liquid chromatographic method for quantitation of topiramate in human serum was developed using HPLC with fluorescence labeling reagent. Topiramate was extracted from human serum by dichloromethane and derivatized by reaction with 9-fluorenylmethyl chloroformate (FMOC-Cl) in the presence of borate buffer. Analysis was performed on a CN column with sodium phosphate buffer (pH 2.2) containing 1ml/l triethylamine and methanol (52:48 (v/v)) as mobile phase. Amantadine was used as internal standard. The standard curve was linear over the range 205000ng/ml of topiramate in human serum.

Arthur Micheel P, Chan KO Y, Guh HY.12 Developed Ion chromatography method and validation for the determination of sulfate and sulfamate ions in topiramate drug substance and finished product, a stability-indicating assay method has been developed for monitoring topiramate degradation in drug substance and finished product by quantifying sulfamate and sulfate ions. Topiramate in the solid state is stable under ambient conditions but can degrade under stress conditions .This method detects and quantities sulfamate and sulfate ions (the inorganic part of the decomposition) and in conjunction with an assay method for topiramate and its known organic degradation product provides total molar accountability. The chromatographic system consists of a sodium hydroxide gradient (225 mM) and an anion-exchange HPLC column and an anion suppressor. The analysis is complete in 30 min.

Maria Styslo-Zalasik and Weiyong Li.13 Developed Determination of topiramate and its degradation product in liquid oral solutions by high performance liquid chromatography with a chemiluminescent nitrogen detector, Topiramate is a sulfamate-substituted monosaccharide that is prescribed for the treatment of epilepsy. It has been a challenge to develop analytical methods for topiramate formulations because the compounds of interest do not have chromospheres that are active above 190nm and because of interference from excipients. This paper describes a simple, specific, precise, accurate, and sensitive method using a chemiluminescent nitrogen detector. The method has a validated linearity range of 324800ng of topiramate and excellent precision (system repeatability). The method has been successfully used for probe stability studies in support of early phase formulation development

Gholamreza Bahrami, Shahla Mirzaeei, Bahareh Mohammadi and Amir Kiani, 14 developed High performance liquid chromatographic determination of topiramate in human serum using UV detection; Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquidliquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5ml/min was used as mobile phase..

Mohammad A, Rezanour N, Ansari Dogaheh, Walker15 developed Development of a stability-indicating high performance liquid chromatographic method for the analysis of topiramate and dissolution rate testing in topiramate tablets, a stability-indicating high performance liquid chromatographic (HPLC) method was developed and validated for the quantitation and dissolution determination of topiramate in tablet dosage forms. An isocratic separation was achieved using a phenyl column with a flow rate of 1 mL/min using UV detection at 264 nm. Topiramate has low UV absorbtivity and was subjected to derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). The mobile phase for the separation consisted of acetonitrile: 50 mM sodium dihydrogen phosphate (NaH2PO4) containing 3 % v/v triethylamine (pH 2.8) in a 48:52 v/v ratio.

Rossi W.LIT. M.16 developed Determination of Sulfamate and Sulfate as Degradation Products in an Antiepileptic Drug Using Ion Chromatography and Indirect UV Detection. Topiramate is a potent antiepileptic drug currently in phase III clinical trials. Sulfamate and sulfate have been found to be two stoichiometrically formed degradation products in topiramate. An ion chromatographic method with indirect UV detection has been developed to assay sulfamate and/or sulfate in topiramate drug substance and formulated products. When used in combination with an HPLC assay method, this method is stability-indicating and can be used as a regulatory method.

Giancarlo la Marca, Sabrina Malvagia, Luca Filippi, et. al. 17 developed Rapid assay of topiramate in dried blood spots by a new liquid chromatography-tandem mass spectrometric method, Topiramate (TPM) is a new antiepileptic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D12-topiramate was linear in the concentration range of 0.01661.66mg/L (0.550mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.550mg/L.

Gholamreza Bahrami, Bahareh Mohammadi18 , develop A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent , new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquidliquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05M; pH 10.6). A mixture of sodium phosphate buffer (0.05M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (1504.6mm) column.

7. MATERIALS AND METHODS:

All experiments will be carried out in the Department of Quality Assurance. Srinivas college of Pharmacy, Valachil, Mangalore.

Pure sample of isoniazid and topiramte will be procured from Industries involved in bulk manufacture of this drug.

Dosage formulation will be procured from local market.

The methods will be developed and validated in Q.A. lab of Srinivas college of Pharmacy.0

The methods will be first developed, then Validated as per ICH guidelines, then the method shall be applied to the formulations.

UV spectrophotometer Shimadzu-UV1700 with spectral band width of 2nm and 10nm and matched quartz will be used for measuring absorbance for isoniazid solutions and topiramate solution.

We are supposed to use following type of reagents.

1) 1,10- phenanthroline

2) 3- methyl, 2- benzothiazolinehydrazone

3) 4- amino phenazene

4) p-dimethylamino benzaldehyde -orthophosphoric acid, 5)1, 10-phenanthroline

In combination ,

1) 3-methyl benzothiazoline hydrazine and ceric ammonium sulphate

2) 3-methyl 2-benzothiazoline hydrazine and ferric ammonium sulphate

Can be use as reagents for spectrophotometric method development.

7.1 Sources of data:

References from library Srinivas College of Pharmacy, Valachil, Mangalore.

www.pharmainfo.net.

www.google.com

www.sciencedirect.com

www.rxlist.com

www.pubmed.com

www.medline.com

www.Wikipedia.com

www.springerling.com

www.ncbi.nlm .nih.gov/pmc/articles.com

7.2 Does the study require any investigation to be conducted on patients or animals?

No

7.3 Has the ethical clearance been obtained from your institution in case of 7.2?

Not applicable

8.0

LIST OF REFERENCES:

1] Prodromos IB, Pantelis ET. A sensitive colorimetric determination of micro quantities of isoniazid. International Journal of Pharmaceutics 1989; 57(3): 235-39.

2) Safavi A,Karimi MA, Hormozi Nezhad MR, Kamali R, Saghir N. Sensitive indirect spectrophotometric determination of isoniazid. Spectrochimica Acta Part A: Molecular and Bimolecular Spectroscopy 2004; 60(4): 765-69.

3) Hoda Hassan Y, Abdel-Maboud Mohamed I, Fardos A. Utility of Certain -Acceptors for the Spectrophotometric. Journal of analytical letters 1990; 23(4): 617-25.

4) Liusheng Huang, Florence Marzan, Anura L. et. al. Development and validation of a hydrophilic interaction liquid chromatographytandem mass spectrometry method for determination of isoniazid in human plasma. Journal of Chromatography B analytical technologies of biomedical and life science 2008; 877(3): 285-90.

5) Abdel-Hamid Ahmed N, Samia El Gizawy M, Hussein El Subbagh I. Spectrophotometric Determination of Isoniazid Using Ethyl 8-Quinolinoxy Aceta. Journal of Analytical letters 1992; 25(1): 73-80.

6) Garcia Bautista A,Garcia Mateo JV, MartInez Calatayud J. Spectrofluorimetric Determination of Iproniazid and Isoniazid in a FIA System Provided with a Solid-Phase reactor. Journal of analytical letters 1998; 31(7): 1209-18.

7) Zhi-ming Zhou, Dao-yuan Zhao, Jing Wang, Wei-jun Zhao and Ming-min Yang, developed Study of cloud point extraction and high-performance liquid chromatographic determination of isoniazid based on the formation of isonicotinylhydrazon. College of Science, Nanjing Agricultural University, Nanjing, China 2008; 1216(1): 30-35.

8) Amina El-Brashy M, Fawzia Ibrahimand A, Saadia El-Ashry M. Colorimetric Determination of Some Important Hydrazine Derivatives. Chemistry and Materials Science Microchimica Acta 1991; 105 (4-5): 105-10.

9) Alexandrou NE, Vasilikiotis GS. Infra-red spectra of isoniazid hydrazones. Spectrochimica Acta part A: Molecular spectroscopy 1967; 23(3): 677-679.

10) El-Brashy, El-Ashry SM. colorimetric and titrimetric assay of isoniazid. Journal of Pharmaceutical and Biomedical Analysis 1992; 10(6): 421-26.

11) Bahrami G, Mirzaeei S, Kiani A. Sensitive analytical method for Topiramate in human serum by HPLC with pre-column fluorescent derivatization and its application in human pharmacokinetic studies. Journal of Chromatography B. Analytical Technologies in the Biomedical and Life science 2004; 813(1-2): 175- 80.

12) Arthur Micheel P, Chan Ko Y, Guh HY. Ion chromatography method and validation for the determination of sulfate and sulfamate ions in topiramate drug substance and finished product. Journal of Chromatography B: biomedical science and Application 1998; 701(1): 166-72.

13) Maria Styslo-Zalasik and Weiyong Li. Determination of topiramate and its degradation product in liquid oral solutions by high performance liquid chromatography with a chemiluminescent nitrogen detector. Journal of Pharmaceutical and Biomedical Analysis 2005; 37 (3): 529-34.

14) Gholamreza Bahrami, Shahla Mirzaeei, Bahareh Mohammadi and Amir Kiani. Developed High performance liquid chromatographic determination of topiramate in human serum using UV detection. Journal of Chromatography B 2005; 822 (1-2): 322-25.

15) Mohammadi A, Rezanour N, Ansari Dogaheh, Walker. Development of a stability-indicating high performance liquid chromatographic method for the analysis of topiramate and dissolution rate testing in topiramate tablets. Asian Journal of Chemistry 2010; 22(5): 3856-66.

16) Rossi W. LIT. M. Determination of Sulfamate and Sulfate as Degradation Products in an Antiepileptic Drug Using Ion Chromatography and Indirect UV Detection .Journal of liquid chromatography and related technologies 1995; 18 (5): 917- 23.

17) Giancarlo la Marca, Sabrina Malvagia, Luca Filippi, et. al. Rapid assay of topiramate in dried blood spots by a new liquid chromatography-tandem mass spectrometric method. Journal of Pharmaceutical and Biomedical Analysis 2008; 48 (5): 1392-96.

18) Gholamreza Bahrami, Bahareh Mohammadi. A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent. Journal of Chromatography B 2007; 850 (1-2):400-04.

9.0

Signature of the Candidate:

RAVAL KASHYAP A.

10.0

Remarks of the Guide:

T

The candidate is working under my direct supervision in srinivas college of pharmacy , valachil,

Mangalore.

11.0

Name & Designation :

11.1 Guide:

Dr. E.V.S. Subrahmanyam,

Professor and head ,

Dept of Quality Assurance

Srinivas college of pharmacy

Valachil, Mangalore.

11.2 Signature of Guide:

(Mrs. ROHINI Dr. E.V.S. Subrahmanyam. M.)

11.3 Co-Guide:

11.4 Signature of Co-Guide:

11.5 Head of the Department:

Dr. E.V.S. Subrahmanyam,

Professor and Head,

Dept of Quality Assurance

Srinivas college of pharmacy

Valachil, Mangalore.

11.6 Signature of HOD:

Dr. E.V.S. Subrahmanyam.

12.

12.1 Remark of the Principal:

Forwarded for approval

12.2 Signature of the Principal:

Dr.A.R.Sharbaraya

Principal,

Srinivas college of pharmacy,

Valachil,

Mangalore.

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