M. Pharm. Dissertation Protocol Submitted To s2

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF DICLOFENAC SODIUM IN SOLID DOSAGE FORM BY USING HPLC.

M. Pharm. Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore –560041.

Mr. MANISH DWIVEDI B.Pharm

Under the Guidance of

SRI. C.S.HALLIKERIM. Pharm,

LECTURER

DEPT. OF PHARMACEUTICAL CHEMISTRY,

Department of Pharmaceutical Chemistry

SET’s College of Pharmacy,

S. R. Nagar, Dharwad,

Karnataka –580002.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE –II

PROFORMA FOR REGISTRATION OF SUBJECT DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS / Mr. MANISH DWIVEDI
DEPT. OF PHARMA CHEMISTRY
SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD- 580002.
2. / NAME OF THE INSTITUTION / SET’s COLLEGE OF PHARMACY
S. R. NAGAR,
DHARWAD- 580002.
3. / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN
PHARMACEUTICAL CHEMISTRY
4. / DATE OF ADMISSION TO THE COURSE / JULY – 2013
5. / TITLE OF THE TOPIC
“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF DICLOFENAC
SODIUM IN SOLID DOSAGE FORM BY USING HPLC”
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 menstrualpain, dysmenorrhea. The mechanism of action is inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (COX). 1
A high performance liquid chromatography method is described for the determination of diclofenac sodium, its related compounds and degradation products in commercial sources of raw materials and solid dosage forms. This method is specific, accurate and stability indicating. The method employs a reverse-phase octylsilane (C18) column with a mobile phase composed of acetonitrile/methanol/pic B-6 (25:25:50) and detection at 229 nm. The method resolves six principal related compounds with quantitation in the range 0.3-1.5%. Assay recoveries by spiking commercial formulations with diclofenac sodium were 99.64 ± 1.30%. Drug content in several commercial formulations are reported. Accelerated stability tests were conducted on raw materials and drug products and 1-(2,6-dichlorophenyl)-2-indolin-2-one was identified for the first time as a degradation product in solid dosage forms which are stressed under humidity and heat.
Validation of analytical method: - Validation of analytical method is a process by which is established by laboratory studies, Industrial knowledge etc. It is the process of verifying the suitable method which is applicable for purpose or not. Method validation is an important element of quality control. Validation helps provide assurance that a measurement will be reliable.
Different guidelines like ICH, WHO etc. suggest that analytical methods used for the analysis must be stability indicating and validated. There are different parameters that need to be evaluated for the purpose of validation of developed method such as, accuracy, precision, specificity, LOD (LOSS ON DRYING), robustness, linearity, and range.
Various chromatographic methods such as HPLC, HPTLC, and GC etc. are used because it fulfills requirement of various guidelines. So the interest of work is emphasized on the HPLC technique, because in the modern pharmaceutical industry, high performance liquid chromatography (HPLC) is the major and integral analytical technique applied in all stages of drug discovery, development, and production.2
Most of the drugs in multicomponent and combination dosage form can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision, and ease of automation in these methods. HPLC method eliminates tedious extraction and isolation procedures. There are different modes of separation in HPLC. They are Normal Phase Mode, Reversed Phase Mode, Reversed Phase Ion Phase Chromatography, Affinity Chromatography and Size Exclusion Chromatography.3
· Normal Phase Mode: In these the stationary phase is polar and the mobile phase is non-polar in nature. In these techniques, non-polar compounds travel faster and are eluted first. This is because of the lower affinity between the non-polar compounds and the stationary phase.
· Reversed Phase Mode: The stationary phase is Non-polar Hydrophobic packing with Octyl or Octa decyl functional group bounded to silica gel and the mobile phase is polar solvent. The polar compounds gets eluted first in this mode and non-polar compounds are retained for longer times. As most of the Pharmaceutical drugs are polar in nature, they are not retained for longer times and hence elute faster. The different columns used are Octa Decyl Silane (ODS) C18, Octasilane (OS) C8, Tetrasilane C4, etc.
· Ion Exchange Chromatography: The stationary phase contains ionic groups like NR3+ which interact with the ionic groups of the sample molecules. This is suitable for the separation of charged molecules only.
· Affinity Chromatography: In these techniques highly specific biochemical interactions are used for separation. The stationary phase contains specific group of molecules which can adsorb the sample if certain steric and charge related conditions are satisfied.
· Size Exclusion Chromatography: It separates molecules accordingly to their molecular mass. Largest molecules are eluted first and the smallest molecules last.
Hence it was thought of interest to develop a simple, rapid, precise, accurate and economic HPLC method for determination of diclofenac sodium. The aim of this study is to develop a more reliable, selective, precise and accurate HPLC method and validating it for the determination of drugs.
6.2 Review of literature:
Extensive literature survey was carried out in libraries of SET’s College of Pharmacy Dharwad and KLE’s College of Pharmacy Belgaum. Karnataka University, Dharwad and by visiting various web sites through internet the relevant data has been collected.
Literature review showed following importance
Ø Diclofenac sodium IUPAC name: sodium2-[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 (pH1-3 range) but is soluble in alkaline solution (pH 5-8 range).[1]
Ø Chemical structure of Diclofenac sodium :

Kamble D et al.,have reported the development and validation of rp-hplc method for simultaneous estimation of eperisone hydrochloride and diclofenac sodium in bulk and pharmaceutical dosage form.4,5A simple, selective, precise and accurate Reverse Phase High Pressure Liquid Chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of eperisone hydrochloride and diclofenac sodium in marketed sustained release(SR) capsules.,6,7
Heydari R et al. ,have reported the Simultaneous determination of EDTA, sorbic acid, and diclofenac
Sodium in pharmaceutical preparations using high-performance liquid chromatography. Separation was achieved on a C (18) column (10 cm×4.6 mm) using gradient elution. The mobile phase consisted of acetonitrile-ammonium dihydrogen phosphate buffer solution (0.01 M, pH=2.5, containing 0.8% tetra-n-butyl ammonium hydroxide). The detector wavelength was set at 254 nm.8
Terasa Kubala, et.al.have reported the A Specific Stability Indicating Hplc Method to Determine Diclofenac Sodium in Raw Materials and Pharmaceutical Solid Dosage Forms. The method employs a reverse-phase octylsilane (C18) column with a mobile phase composed of acetonitrile/methanol/pic B-6 (25:25:50) and detection at 229 nm.9
Panda SS, et.al., have reported the New Stability-Indicating RP-HPLC Method for Determination of Diclofenac Potassium and Metaxalone from their Combined Dosage Form. A simple, precise and accurate isocratic RP-HPLC stability-indicating assay method has been developed to determine diclofenac potassium and metaxalone in their combined dosage forms.10
Isocratic separation was achieved on a Hibar-C (18), Lichrosphere-100(®) (250 mm × 4.6 mm), Particle size 5 μm) column at room temperature in isocratic mode, the mobile phase consists of methanol: water (80:20, v/v) at a flow rate of 1.0 ml/min, the injection volume was 20 μl and UV detection was carried out at 280nm.
Shaalan RA, et.al., have reported the Validated Stability-Indicating HPLC-DAD Method for the Simultaneous Determination of Diclofenac Sodium and Diflunisal in their Combined Dosage Form.11
A simple, rapid, and highly selective HPLC-DAD method was developed for the simultaneous determination of diclofenac sodium (DIC) and diflunisal (DIF) in pure form and in their combined formulation. Effective chromatographic separation was achieved using a Zorbax SB-C8 (4.6×250 mm, 5 μm particle size) column with a mobile phase composed of 0.05 M phosphoric acid, acetonitrile, and methanol in the ratio of 40:48:12 (by volume).12,13
Ashvin H Munjapara, et.al.,have reported the development and validation of analytical methods for simultaneous estimation of diclofenac sodium and tolperisone hydrochloride in combined Pharmaceutical formulation.14
A simple and sensitive spectrophotometric method has been developed for simultaneous Determination of Tolperisone hydrochloride and Diclofenac sodium in a combined pharmaceutical dosage form. In the proposed method, the absorbances were measured at 261.0 nm and 276.0 nm corresponding to the absorbance maxima of Tolperisone hydrochloride and Diclofenac sodium in Dist. Water respectively.
Nasir F, et.al., have reported the Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV.15
Sunil R. Dhaneshwar, et.al., have reported the Validated HPLC Method for Simultaneous estimation of Diclofenac Sodium and Misoprostol in Bulk Drug and Formulation.16
Ronald J., et.al. have reported the Analysis of Diclofenac and Four of Its Metabolites in Human Urine by HPLC. An HPLC method for the determination of diclofenac (DCF) and four of its metabolites (3′-hydroxydiclofenac,4′-hydroxydiclofenac,5-hydroxydiclofenac, and 3′-hydroxy-4′-methoxydiclofenac) in human urine is described.17
Korodi T, et.al.have reported the Evaluation of a short stability-indicating HPLC method for diclofenac sodium gels. The method employs a RP-LiChrospher select B (C8) column with a mobile phase containing methanol/water (63:37, v/v) and detection at 220 nm.18
Objectives of Study:
A) The primary objective of this study is to develop HPLC method and validate for determination of diclofenac sodium is as follows:-
1. Information of sample and different separating goals.
2. Need for the basic HPLC procedure, sample, pre-treatment, column, mobile phase etc.
3. Selection of detector and detectors setting.
4. Selection of LC method, preliminary run & best estimate separation.
5. To optimized different terms and conditions.
6. Check for problems or requirement for the specific/special procedure.
7.1Recovered & purified material.
7.2 Quantative calibration.
7.3Qualitative calibration.
8. Validated method for release to the laboratory.
B) The secondary objective is to validate developed HPLC method
· Specificity
· Accuracy
· Precision
· Repeatability
· Intermediate precision
· Linearity
· Limit of detection
· Limit of quantification
· Robustness
· System suitability test.
Materials and methods:
7.1Source and Collection of data:
Ø Chemical Abstracts.
Ø Journals
Ø Library of SET’S college of pharmacy
Ø Bioorganic and Medicinal Chemistry Letters.
Ø World Wide Web.
Ø J-Gate@ Helinet etc.
Ø Industries
Ø Research publication
7.2 Method of collection of data :
Data collected from the following stepwise experimental procedure proposed in the study.
1) Development of analytical method for estimation of DICLOFENAC SODIUM by HPLC.
· By performing experiment in the laboratory from the information obtained from the above source.
· Perform trials with different stationary phase and mobile phase in different phase with suitable column and sample concentration.
· Determine parameters of HPLC i.e., Retention, degree of separation, column efficiency etc.
2) Validation of the above developed HPLC method’s Steps.
Ø Develop a validation protocol or operating procedure for the validation.
Ø Define the application, purpose, and scope of the method.
Ø Define the performance parameters and acceptance criteria.
Ø Define validation experiments.
Ø Verify relevant performance characteristics of equipment.
Ø Qualify materials standards and reagents.
Ø Perform prevalidation experiments.
Ø Adjust method parameters and acceptance criteria if necessary.
Ø Perform full internal and external validation experiments.
Ø Develop SOPs for executing the method in the routine.
Ø Define criteria for revalidation.
Ø Define type and frequency of system suitability tests and/or analytical quality control (AQC) checks for the routine.
Ø Document validation experiments and results in the validation report.
7.3 Does the study require any investigation or interventions to be conducted on
patients or other humans/animals? If so please describe briefly.
------No.------
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
------Not applicable.------
LIST OF REFERENCES:
1) http://www.en.wikipedia.org/wiki/Diclofenac sodium/ (access on Oct 27, 2010).
2)Dunge A, Sharda N, Singh B, Singh S. Validated specific HPLC method for determination of zidovudine during stability studies. J Pharm.Bio.Anal. 2005;37(5):1109-14
3)Neves JD, Sarmento B, Amiji MM, Bahia MF. Development and validation of rapid reversed phase HPLC method for the determination of non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles. J Pharm. Bio. Anal. 2010 Jun 5;52(2):167-72
4)D. KambleNeelam, S. Mahajan Supriya, S. Pradhan Nitin, S. Prabhune Swarup, and S. Reddy Sajjalavarlakshmi, Int. Jur. of pharma., chem and bio sci, ijpcbs2013, 3(4), 1286-92
5)Roelofs PD, Deyo RA and Koes BW. Non- steroidal anti-inflammatory drugs for low back pain: an updated Cochrane review. 2008; 33:1766-74.
6) Malanga GA and Nadler SF. Non-steroidal anti- inflammatory treatment of muscle spasm. Medical, clinical proce.and practices. 1999; 74:1135- 48.
7) Cabitza P and Randellu P. Safety and efficacy of eperisone in patients with low back pain a double blind randomized study.Europe. Review for medic. and pharmaco.Sci. 2008; 12:229-35.
8) Heydari R, Shamsipur M, Naleini N. Simultaneous determination of EDTA, sorbic acid, and diclofenac sodium in pharmaceutical preparations using high-performance liquid chromatography. AAPS PharmSciTech. 2013 Jun; 14(2):764-9.
9) Kubala T, Gambhir Band Borst S I. A Specific Stability Indicating HPLC Method to Determine Diclofenac Sodium in Raw Materials and Pharmaceutical Solid Dosage Forms. 1993; 19: 749-57.
10) Panda SS, Patanaik D, Ravi Kumar BV. New Stability-Indicating RP-HPLC Method for Determination of Diclofenac Potassium and Metaxalone from their Combined Dosage Form.Sci Pharm.2012;80(1):127-37.
11)Shaalan RA, Belal TS. PubMed – NCBI Validated Stability-Indicating HPLC-DAD Method for the Simultaneous Determination of Diclofenac Sodium and Diflunisal in Their Combined Dosage Form, Sci Pharm. 2013 Mar 17;81(3):713-31
12)The British Pharmacopoeia. Her Majesty’s Stationery Office; London: 2010. 671–673, 686–687, 2606–2615.
13)Sweetman SC.Martindale-The Complete Drug Reference.Thirty-sixth editions.The Pharmaceutical Press; London, UK: 2009, 44–48.
14)Ashvin h munjapara, Nikunj N Bhadani, Hasmukh N Prajapati, development and validation of analytical methods for simultaneous estimation of tolperisone hydrochloride and diclofenac sodium in combined pharmaceutical formulation",inventi rapid: pharm analysis & quality assurance,2013, article id- “inventi/687/13”,2013.
15) Nasir F, Iqbal Z, Khan A, Ahmad L. Simultaneous determination of timolol maleate, rosuvastatin calcium anddiclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV. J Chromatogr B,Analyt. Technol. Biomed. Life Sci. 2011 Nov 15; 879(30):3434-43.
16) Sunil R. Dhaneshwar, Vidhya K. Bhusari.Asian J Phar Biol Res.2011; 1(1): 15-21
17) Ronald J. Sawchuk Jill A. Maloney ,Linda L. Cartier , Analysis of Diclofenac and Four of Its Metabolites in Human Urine by HPLC, Springer link May 1995, 12, Issue 5, pp 756-62.
18)Korodi T, Bukowski K, Lachmann B.Evaluation of a short stability-indicating HPLC method for diclofenac sodium gels.Pharmazie.2012 Dec; 67(12):980-3.
9. / SIGNATURE OF THE STUDENT
10. / REMARK OF THE GUIDE
The above mentioned information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance.
11. / 11.1 NAME AND DESIGNATION OF THE GUIDE
11.2 SIGNATURE / Sri. C.S.HALLIKERIM. Pharm
Lecturer
DEPT. OF PHARMA - CHEMISTRY,
SET’s COLLEGE OF PHARMACY, S.R.NAGAR, DHARWAD- 580002.
11.3 NAME AND DESIGNATION OF CO-GUIDE
11.4 SIGNATURE / ------
11.5 HEAD OF THE DEPARTMENT
11.6 SIGNATURE / Dr. S. D. JOSHI M. Pharm, PhD.
PROFESSOR AND HEAD
DEPT. OF PHARMA - CHEMISTRY,
SET’s COLLEGE OF PHARMACY, S.R.NAGAR, DHARWAD- 580002.
12. / 12.1 REMARK OF THE PRINCIPAL
12.2 SIGNATURE / The above mentioned information is correct and I recommend the same for approval.
Dr. V.H. Kulkarni M. Pharm, PhD.
PROFESSOR & PRINCIPAL,
Set’s College of Pharmacy,
S.R.NAGAR, DHARWAD- 580002.