DEVELOPMENT AND VALIDATION OF ANALYTICAL …



“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF DICLOFENAC SODIUM AND

CHLORZOXAZONE IN PHARMACEUTICAL FORMULATIONS”

DISSERTATION PROTOCOL

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

BY

P.V.V.N. SAI KUMAR

M.PHARM, PART-I

DEPARTMENT OF QUALITY ASSURANCE

NARGUND COLLEGE OF PHARMACY

BANGALORE-85

(2010-2012)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

|1. |NAME OF THE CANDIDATE |P.V.V.N. SAI KUMAR |

| |AND ADDRESS (IN BLOCK LETTERS) |NARGUND COLLEGE OF PHARMACY, |

| | |DATTATREYA NAGAR, II MAIN, |

| | |100 FEET RING ROAD, |

| | |BSK III STAGE, |

| | |BANGALORE-85, |

| | |KARNATAKA. |

|2. |NAME OF THE INSTITUTION |NARGUND COLLEGE OF PHARMACY, |

| | |DATTATREYA NAGAR, II MAIN, |

| | |100 FEET RING ROAD, |

| | |BSK III STAGE, |

| | |BANGLORE-85, |

| | |KARNATAKA. |

|3. |COURSE OF STUDY AND SUBJECT |MASTER OF PHARMACY IN |

| | |QUALITY ASSURANCE |

|4. |DATE OF ADMISSION OF COURSE | |

| | |1ST JULY 2010 |

|5. |TITLE OF TOPIC |“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS |

| | |ESTIMATION OF DICLOFENAC SODIUM AND CHLORZOXAZONE IN PHARMACEUTICAL |

| | |FORMULATIONS” |

| | | |

|6. |BRIEF RESUME OF THE INTENDED WORK: |

|6.1 |NEED FOR THE STUDY: |

| |Diclofenac sodium is a non-steroidal anti-inflammatory drug (NSAID) prescribed to reduce inflammation and as an analgesic |

| |in conditions such as arthritis. It can also be used to reduce menstrual pain, dysmenorrhea. The mechanism of action is |

| |inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (COX) [1]. |

| | |

| |Chlorzoxazone is a centrally acting muscle relaxant used to treat muscle spasm and the resulting pain. It acts on the spinal |

| |cord by depressing reflexes. It is used for the relief of pain and inflammation in rheumatoid arthritis, osteoarthritis. It |

| |works by blocking the action of cyclooxygenase [2]. |

| | |

| |The combination of Diclofenac sodium and Chlorzoxazone is used in post surgerical treatment for fast relief of pains. The term |

| |"muscle relaxant" is used to refer to two major therapeutic groups such as neuromuscular blockers and spasmolytics. |

| |Neuromuscular blockers act by interfering with transmission at the neuromuscular end plate and have no CNS activity. They are |

| |often used during surgical procedures and in intensive care and emergency medicine to cause paralysis. Spasmolytics, also known |

| |as "centrally-acting" muscle relaxants, are used to alleviate musculoskeletal pain and spasms and to reduce spasticity in a |

| |variety of neurological conditions [3]. |

| | |

| |There is no analytical method reported for simultaneous estimation of Diclofenac sodium and Chlorzoxazone. Hence, investigation |

| |of new analytical method is required for the quantitative estimation of Diclofenac sodium and Chlorzoxazone, in combination in |

| |pharmaceutical formulation. |

| | |

| |REVIEW OF LITERATURE : |

| |Diclofenac sodium IUPAC name: sodium 2-[2-(2, 6-dichlorophenyl) aminophenyl] acetate. It is also name as 2[(2, 6-dichloro |

| |phenyl) amino] benzene acetic acid sodium salt. Molecular formula is C14H10Cl2NNaO2 and molecular weight is 318.13. It is |

| |poorly soluble in water and acidic solution (pH 1-3 range) but is soluble in alkaline solution (pH 5-8 range) [1]. |

|6.2 | |

| |Chemical structure of Diclofenac sodium : |

| |[pic] |

| |Chlorzoxazone IUPAC name: 5-chloro-3H-benzooxazol-2-one. Molecular formula is C7H4ClNO2 molecular weight is 169.565g/mol. It |

| |is soluble in methanol, ethanol and isopropanol; sparingly soluble in water; freely soluble in aqueous solution of alkali |

| |hydroxides. Melting point is 191-1920C [2]. |

| | |

| |Chemical structure of chlorzoxazone: |

| |[pic] |

| |Jahanbakhsh G, Ali N and Sirous G worked on simultaneous spectrophotometric determination of Benzyl alcohol and Diclofenac |

| |sodium in pharmaceutical formulations by chemometrics method. It is possible to obtain a model adjusted to the concentration |

| |values of the mixtures used in the calibration range by a multivariate calibration method such as partial least squares (PLS) |

| |[4]. |

| | |

| |Sunil J, Sandeep J developed analytical methods for Diclofenac sodium and Mefenamic acid in tablet dosage form by atomic |

| |absorption spectrometry (AAS). These methods are based on formation of the metal complexes of Diclofenac sodium and Mefenamic |

| |acid with cupric chloride and cobaltous chloride. Both the |

| |drugs are indirectly estimated via determination of copper content in the formed complexes by AAS. The second method is based on|

| |the formation of pink colored complexes of both the drugs with cobaltous chloride. These metal complexes are extracted with |

| |dichloromethane and estimated via determination of cobalt content in the formed complexes after digestion with 0.1 M nitric acid|

| |by AAS [5]. |

| | |

| |Nagoji KEV, Vijayasrinivas S developed an RP-HPLC method for determination of Nimesulide and Diclofenac sodium using a mobile |

| |phase methanol and water in the ratio 50:50 at a flow rate of 1 ml/min on an isocratic HPLC system consisting of ODS C-18 RP |

| |column (4.6 mm x 25 mm x 5µm) at a wavelength of 230 nm. The Paracetamol was used as an internal standard [6]. |

| | |

| |Lohe RW, Suruse PB, Kale MK, Barethiya PR, Kasture AV and Lohe SW developed method for quantitative estimation of Diclofenac |

| |sodium and Rabeprazole in tablet dosage form using simultaneous equation and graphical absorbance ratio method the wave lengths|

| |used were 294nm and 281.2nm [7]. |

| | |

| |Choudhary B, Goyal A, Khokra S.L, Kaushik D worked on simultaneous estimation of Diclofenac sodium and Rabeprazole by HPLC in |

| |combined dosage form. A phenominex C18 column of length (250 x 7.5mm x 5 µm) and mobile phase was potassium dihydrogen phosphate|

| |buffer (pH – 7.5) and acetonitrile in the ratio 60:40. Retension time 9.20 min, 6.40 min and 11.87 min for Rabeprazole, |

| |Diclofenac sodium and internal standard Domiperidone respectively at a flow rate of 2ml/min [8]. |

| | |

| |Dowling G, Gallo P, Fabbrocino S, Serpe L worked on determination of Ibuprofen, Ketoprofen, Diclofenac sodium, Phenylbutazone |

| |in bovine milk by GC-tandem mass spectrometry. Milk samples were extracted with acetonitrile and sample extracts were purified |

| |on isolute™ C18 solid-phase extraction cartridges [9]. |

| | |

| |Pimenta AM, Araujo AN, Montenegro MCBSM developed two independent methods for determination of Diclofenac sodium, implemented |

| |simultaneously in an automated analytical system. The potentiometric detection was carried out with an ion-selective electrode |

| |based on a cyclodextrin while for the fluorimetric determinationthe sample was previously subject to in-line irradiation with UV|

| |light. Both methods were applied in the analysis of pharmaceutical formulations [10]. |

| | |

| |Shankar MB, Shah DA, Geetha M, Mehta FA, Mehta RS, Bhatt KK worked on simultaneous spectrophotometric estimation of Diclofenac |

| |sodium and Tizanidine in tablet dosage form. The wavelengths used were 296nm and 281.3nm for Q-absorbance equation whereas |

| |281.3nm and 317.1nm were used for simultaneous equation method using methanol as a solvent [11]. |

| | |

| |Shaik KA, Devkhile AB worked on simultaneous determination of Aceclofenac, Paracetamol, and Chlorzoxazone by RP-HPLC in |

| |pharmaceutical dosage Form. This method uses C18 (250 × 4.6 mm x 5 µm) analytical column. Mobile phase was acetonitrile and |

| |buffer (40:60, v/v), buffer containing 50mm orthophosporic acid; pH-6 at a flow rate of 1 ml/min the column at temperature 25°C |

| |detector wavelength used was 270 nm [12]. |

| | |

| |Puranik M, Hirudkar A, Wadher SJ, Yeole PG developed and validated of spectrophotometric method for simultaneous estimation of |

| |Chlorzoxazone and Tramdol HCl. The wavelengths used were 272.2nm and 248.3 nm for simultaneous equation where as 272.2nm and |

| |257.5 nm were used for absorption ratio method.The solvent used was 0.1N NaOH [13]. |

| | |

| |Anju G, Sandeep J worked on simultaneous estimation of Paracetamol, Chlorzoxazone and Diclofenac Sodium in pharmaceutical |

| |formulation by a novel HPLC method using C8 column with UV- Visible detection at 280 nm. Mobile phase used was acetonitrile – |

| |0.05 M ammonium dihydrogen ortho phosphate (60:40 v/v) at a flow rate of 1.5 ml/min using Diazepam as internal standard [14]. |

| | |

| |Venkatesh K, Vaidhyalingam, Yuvaraj G, Rajesh kumar N developed simultaneous estimation of Paracetamol, Chlorzoxazone and |

| |Aceclofenac in pharmaceutical formulation by HPLC method. The chromatographic separation was carried out on a C18 column using a|

| |mixture of acetonitrile-0.05M disodium hydrogen orthophosphate (65:35) as mobile phase with UV detection at 271 nm [15]. |

| | |

| |Pawar UD, Naik AV, Sulebhavikar AV, Datar TA, Mangaonkar KV developed an analytical method for simultaneous determination of |

| |Aceclofenac, Paracetamol and Chlorzoxazone by HPLC in tablet dosage form using an intensil C18 column, with |

| |mobile phase 10mM potassium dihydrogen phosphate (pH 5.55 with ammonia): acetonitrile in the ratio 60:40 (v/v) and UV detection |

| |was carried at 205 nm [16]. |

| | |

| |Wisanu T, Boonsom L, Chalermporn T and Theeraphan M worked on HPTLC method for the determination of Diclofenac Sodium in |

| |pharmaceutical formulations. The sample solutions were spotted automatically by means of Camag Linomat IV on a silica gel 60 |

| |F254 aluminium plate, using a mixture of toluene : ethyl acetate : glacial acetic acid (60:40:1, v/v/v) as mobile phase. The |

| |spot areas were quantified by densitometry at 282 nm [17]. |

| | |

| |Nagoji KEV, Kirankumar M, Rao MEB, Kannarao KV developed spectrophotometric method for simultaneous estimation of Nimesulide and|

| |Diclofenac sodium by using simultaneous equation method.The wavelengths used were 297 nm and 283 nm respectively in methanol, |

| |showing linearity in the concentration range of 5-40 μg/ml and 2.5-25 μg/ml respectively [18]. |

| | |

| |Garg G, Saraf S, Saraf S worked on simultaneous estimation of Aceclofenac, Paracetamol and Chlorzoxazone in tablets. The |

| |simultaneous estimation method is based on the additivity of absorbances. The wavelengths used were 276 nm, 282 nm, 248 nm in |

| |methanol. All the three drugs were obeyed Beers Lamberts law in the concentration range of 2-20 µg/ml [19]. |

| | |

| |Reddy PB worked on simultaneous estimation of Nimesulide and Chlorzoxazone in pharmaceutical formulations by a RP-HPLC |

| |method.The chromatography system used a reversed phase C18 column (150 mm x 4.6 mm x 5µm) and acetonitrile: methanol: |

| |phosphate buffer, in the ratio of 50:30:20: (pH adjusted to 4.50 with orthophosphoric acid) as a mobile phase at a flow rate of |

| |1.0 ml/min and detection at 280 nm [20]. |

| | |

| |OBJECTIVES OF THE STUDY: |

| |Review of literature revealed that there is no method for the simultaneous estimation of Diclofenac sodium and Chlorzoxazone in |

| |pharmaceutical dosage form. |

| |Hence the objectives of the present work are, |

| |To develop analytical methods to estimate Diclofenac sodium and Chlorzoxazone in combined form by UV-Visible spectrophotometer. |

| |To develop HPLC method to estimate Diclofenac sodium and Chlorzoxazone in combined dosage form. |

| |To validate the developed methods by ICH guidelines. |

| | |

| |MATERIALS AND METHODS: |

| |Diclofenac sodium, Chlorzoxazone, Diclofenac sodium, Paracetamol, Marketed formulation, Distilled water, Methanol, Ethanol, |

| |Isopropanol, Sodium hydroxide, Acetonitrile, Ortho phosphoric acid, Toulene, Ethyl acetate, Glacial acetic acid. |

| | |

| |SOURCE OF DATA: |

| |Data will be obtained from Science Direct, and other internet facilities, literature search and related articles from library of|

| |Nargund College of Pharmacy, Digital Library of RGUHS, Bangalore, etc. |

| | |

| |Journals |

| |Indian Journal of Pharmaceutical Sciences |

| |Journal of Chromatography B |

| |Journal of Pharmaceutical and Biomedical Analysis |

| |European Journal of Pharmaceutical Sciences |

| |Drug Development and Industrial Pharmacy |

| |Journal of Chemistry |

| |Journal of Planar Chromatography – Modern TLC |

| |Journal of Chinese Chemical Society |

| |Journal of Pharmacy Research |

| | |

| |Text Books and Pharmacopoeia |

| |Beckett AH, Stenlake JB, Practical Pharmaceutical Chemistry. 4th ed.,part Part II Delhi: CBS Publisher and Distributors; 1997 |

| |Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare, Published by The Indian Pharmacopoeia |

| |commission Gaziabad, Volume 2, 2007 |

| |Higuchi T And Brochman E, Hanseen H, Pharmaceutical Analysis, Delhi: CBS Publisher and Distributors; 2005 |

| |Sethi PD, Quantitative Analysis of Drugs in Pharmaceutical Formulation, |

| |3rd ed., Delhi: CBS Publisher and Distributors |

| |Mendham J, Denney RC, Barnes JD, Kthomas MJ, Vogel’s text Book of Quantitative Chemical Analysis, 6th ed. Pearson education Pvt |

| |Ltd; 2002 |

| | |

| |Internet Browsing |

| | |

| | |

| | |

| |helinet.jccc.in |

| |medline |

| | |

| |Method of Collection of Data (Including Sampling Procedures, If Any) |

|6.3 |Procurement of drugs sample and marketed formulations. |

| |Solubility determination of Diclofenac sodium and Chlorzoxazone in various solvents and buffers. |

| |Studying the spectra of both the drugs in UV–Visible region in different solvents/buffers and selecting the solvents for various|

| |analytical studies. |

| |Development of analytical methods by simultaneous equation, absorption ratio, derivative spectroscopy, etc for the estimation of|

| |Diclofenac sodium and Chlorzoxazone in combined form by UV-visible spectrophotometer. |

| |Development and optimization of HPLC method for estimation of Diclofenac sodium and Chlorzoxazone. |

| |Validation of all developed analytical methods as per ICH guidelines. |

| | |

| |Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, |

|7. |please mention briefly. |

| |- NOT APPLICABLE – |

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

| |- NOT APPLICABLE- |

| | |

|7.1 |LIST OF REFERENCES: |

| | sodium/ (access on Oct 27, 2010). |

| | (access on Oct 27, 2010). |

| | (access on Oct 27, 2010). |

| |Jahanbakhsh G, Ali N, Sirous G. Simultaneous spectrophotometric determination of benzyl alcohol and diclofenac in |

| |pharmaceutical formulations by chemometrics method. J Chin Chem Soc 2005; 52(5):1049-4. |

| |[] |

| |Sunil J, Sandeep J. Atomic absorption spectrometric method for estimation of diclofenac sodium and mefenamic acid. Int J Pharm |

| |Sci 2010 Jan-Mar; 2(1):45- 7. |

| |[ ] |

| |Nagoji  KEV, Vijayasrinivas S, kirankumar  M, Mathivanan N, Satish kumar M, Rao MEB. Simultaneous reverse phase HPLC |

| |estimation of nimesulide and diclofenac sodium. Indian J Pharm Sci 2003; 65: 407-9. |

| |[ ]  |

| |Lohe RW, Suruse PB, Kale MK, Barethiya PR, Kasture AV and SW Lohe. Spectrophotometric methods for simultaneous estimation of |

| |rabeprazole and diclofenac from combined tablet dosage form. Asian J Res 2008 Jul-Sep; |

| |1(1): 26-8. |

| |[] |

| |Chowdary B, Goyal A, Khokra SL, Kaushik D. Simultaneous estimation of diclofenac sodium and rabeprazole by HPLC in combined |

| |dosage form. Indian J Pharm Sci 2009 Apr-Jun; 1(1): 43-5. |

| |[] |

| |Dowling G, Gallo P, Fabbrocino S, Serpe L, Regan L. Determination of ibuprofen, ketoprofen, diclofenac and phenylbutazone in |

| |bovine milk by gas chromatography-tandem mass spectrometry. Food additives & contaminants 2008 Dec; 25(12): 1497-08. |

| |[] |

| |Pimenta AM, Araújo AN, Montenegro MCBSM. Simultaneous potentiometric and fluorimetric determination of diclofenac in a |

| |sequential injection analysis system. Analytica Chemica Acta 2002; 470: 185–4. |

| |[

| |arch&_coverDate=10%2F16%2F2002&_sk=995299997&view=c&wchp=dGLbVlW-zSkWb&md5=2e79a50535af7f30efc3eb9d92a8bfe2&ie=/sdarticle.pdf] |

| |Shankar MB, Shah DA, Geetha M, Mehta FA, Mehta RS, Bhatt KK. Simultaneous spectrophotometric determination of tizanidine and |

| |diclofenac in tablets. Indian J Pharm Sci 2004; 66: 332-6. |

| |[] |

| | Shaikh KA, Devkhile AB. Simultaneous determination of aceclofenac, paracetamol and chlorzoxazone by RP-HPLC in pharmaceutical |

| |dosage form. Journal of Chromatographic Science 2008 Aug; 46: 649-2. |

| |[] |

| |Puranik M, Hirudkar A, Wadher SJ, Yeole PG. Development and validation of spectrophotometric methods for simultaneous estimation|

| |of tramadol hydrochloride and chlorzoxazone in tablet dosage form. Indian J Pharm Sci |

| |2006; 68: 737-9. |

| |[] |

| |Anju G, Sandeep J. Simultaneous estimation of paracetamol, chlorzoxazone and diclofenac Sodium in pharmaceutical formulation by |

| |a novel HPLC Method. Acta Pharmaceutica Sciencia 2007; 49: 147-1. |

| |Venkatesh K, Vaidhyalingam, Yuvaraj G, Rajesh K. Simultaneous estimation of paracetamol, chlorzoxazone and aceclofenac in |

| |pharmaceutical formulation by HPLC method. Int J ChemTech Res 2009 Jul-Sep; 1(3): 457-0. |

| |Pawar UD, Naik AV, Sulebhavikar AV, Datar TA, Mangaonkar KV. Simultaneous determination of aceclofenac, paracetamol and |

| |chlorzoxazone by HPLC in tablet dosage form. J Chem 2009; 6(1): 289-4. |

| |Wisanu T, Boonsom L, Chalermporn T and Theeraphan M. HPLC method for the determination of diclofenac sodium in pharmaceutical |

| |formulations. Chiang Mai J Sci 2006; 33(1): 123-8. |

| |[] |

| |Nagoji KEV, Kiran kumar M, Rao MEB, Kannarao KV. Spectrophotometric method for simultaneous estimation of nimesulide and |

| |diclofenac sodium from combined dosage form. Indian J Pharma Sci 2003; 65: 404-6. |

|7.2 |[] |

| |Garg G, Saraf S, Saraf S. Simultaneous estimation of aceclofenac, paracetamol and chlorzoxazone in tablets. Indian J Pharm Sci |

| |2007; 69: 692-4. |

| |[] |

| |Reddy PB. Simultaneous estimation of nimesulide and chlorzoxazone in Pharmaceutical Formulations by a RP-HPLC Method. Int J |

| |ChemTech Res 2009, 1(2): 283-5. |

| |[] |

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|9. |Signature of the candidate | |

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| | |(P.V.V.N.SAI KUMAR) |

|10. |Remarks of the Guide |RECOMMENDED FOR THE DISSERTATION WORK. |

|11. |Name & Designation of | |

| |(in block letters) | |

| |Guide |DR. L.V.G. NARGUND |

| | |PROFESSOR, HEAD OF THE DEPARTMENT |

| | |OF PHARMACEUTICAL CHEMISTRY, |

| | |NARGUND COLLEGE OF PHARMACY, |

| | |BANGALORE. |

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| |11.2 Signature | |

| |11.3 Head of the department |DR. J. N. NARENDRA SARATH CHANDRA |

| | |PROFESSOR, HEAD OF THE DEPARTMENT |

| | |DEPARTMENT OF QUALITY ASSURANCE |

| | |NARGUND COLLEGE OF PHARMACY, |

| | |BANGALORE. |

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| |11.4 Signature | |

|12. | 12.1 Remarks of Principal |FORWARDED AND RECOMMENDED FOR FAVOURABLE CONSIDERATION. |

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| |12.2 Signature | |

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