Development and Validation of Analytical Methods for Estimationofciticoline in Bulk And

“DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR ESTIMATIONOFCITICOLINE IN BULK AND PHARMACEUTICAL FORMULATIONS”

DISSERTATION PROTOCOL

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

BY

SURANI SANJAYKUMAR SHAMJIBHAI

M.PHARM, PART-I

DEPARTMENT OF QUALITY ASSURANCE

NARGUNDCOLLEGE OF PHARMACY

BANGALORE-85

(2009-2011)

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / NAME OF THE CANDIDATE
AND ADDRESS (IN BLOCK LETTERS) / SURANI SANJAYKUMAR SHAMJIBHAI
NARGUNDCOLLEGE OF PHARMACY,
DATTATREYANAGAR, II MAIN,
100 FEET RING ROAD,
BSK III STAGE,
BANGALORE-85,
KARNATAKA.
2. /

NAME OF THE INSTITUTION

/ NARGUNDCOLLEGE OF PHARMACY,
DATTATREYANAGAR, II MAIN,
100 FEET RING ROAD,
BSK III STAGE,
BANGALORE-85,
KARNATAKA.
3. /

COURSE OF STUDY AND SUBJECT

/ MASTER OF PHARMACY IN
QUALITY ASSURANCE
4. / DATE OF ADMISSION OF COURSE / 1ST JUNE 2009.
5. /

TITLE OF TOPIC

/ “ DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR ESTIMATION OF CITICOLINE IN BULK AND PHARMACEUTICAL FORMULATIONS”
6.
6.1 / Brief resume of the intended work:
Need for the study:
Worldwide, stroke is the second leading cause of death, responsible for 4.4 million of the 50.5 million deaths each year. Citicoline has been investigated as a therapy in stroke patient1.
Citicoline is a complex molecule that functions as an intermediate in the biosynthesis of cell membrane phospholipids. Citicoline is also known as CDP-choline and cytidinediphosphatecholine (cytidine 5’-diphosphocholine). CDP-choline belongs to the group of biomolecules in living systems known as ‘nucleotides’ that play important role in cellular metabolism. CDP-choline is composed of ribose, pyrophosphate, cytosine (a nitrogenous base) and choline. Exogenous Citicoline research in animal experiments and human clinical trials provides evidence of its cholinergic and neuroprotective actions.
As a dietary supplement (energy drinks), Citicoline appears useful for improving both the structural integrity and functionality of the neuronal membrane that may assist in membrane repair. Animal and clinical studies indicate the potential of Citicoline to improve cognitive deficits, stroke rehabilitation, brain and spinal cord injuries, neurological diseases, age - related memory problem and eye conditions2.Sodium salt of Citicoline is available in the market in the form of tablets and injections.
There is no analytical method reported in journals to estimate Citicoline in pharmaceutical dosage form. Hence, there is need to develop simple, accurate, precise, sensitive analytical method for Citicoline in different dosage form.
6.2
6.3
7.
7.1
7.2
7.3
7.4
8. / Review of literature:
Chemically Citicoline is,
5'-O-[hydroxy({hydroxy[2-(trimethylammonio)ethoxy] phosphoryl}oxy)phosphoryl]cytidine3.
Molecular formula- C14H27N4O11P2+3, Molecular weight- 489.332 g/mol3
Solubility-Predicted water solubility 1000mg/ml at 25˚C and insoluble in ethanol4.
Chemical structure of Citicoline is3,

Richard J. Wurtman et al. worked on effect of oral CDP-Choline on plasma choline and uridine levels in humans. Plasma samples were deproteinized with methanol and fatty components were extracted into chloroform: methanol. A portion of the aqueous layer was lyophilized for subsequent HPLC analysis. Then the residue was redissolved in water, filtered and analysed by HPLC 5.
Mirakor V.A. et al. developed rapid and sensitive high performance liquid chromatography method for Citicoline in dosage form using C18 column and 50mM potassium dihydrogen orthophosphate : methanol (98:2) as mobile phase at a flow rate of 1ml/min, UV detection was made at 266nm6 .
Ed X. Wu et al. performed on proton magnetic resonance spectroscopy that revealed choline reduction in the visual cortex in an experimental model of chronic glaucoma by using cytidine 5’-diphosphocholine (CDP-choline, or citicoline), which is an intermediate in the generation of phosphatidylcholine (PtdCho) and acetylcholine (ACh) from choline (Cho), may improve visual cortical responses in glaucoma. Proton magnetic resonance spectroscopy has been increasingly utilized to investigate the metabolite distribution in selected volumes of the brain7.
Peter M.H. Heegaard et al. had developed a robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5’-diphosphocholine coupled dendrimers. The present paper described a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosphocholine. In this design synthetic globular polymers (dendrimers) were used as scaffolds for the multivalent display of phosphocholine molecules8.
Objectives of the study:
Literature review reveals that tremendous work has been done in pharmacological area butthere is hardly work found on analytical method development of Citicoline in bulk and pharmaceutical formulations.
The objectives of present work are,

1. To develop analytical method for Citicoline by UV-Visible spectrophotometer.
2. To develop HPLC method to estimate Citicoline in bulk and pharmaceutical formulation.
3. To validate developed analytical methods as per ICH guidelines.

Materials and Methods
Materials:
Citicoline, Citicoline tablets and injections, distilled water, hexane, ethylacetate, dichloromethane, methanol, acetonitrile, glacial acetic acid, acetone, propanol, hydrochloric acid, sodium hydroxide, hydrochloric acid and ethanol.
Source of data:
Data will be obtained from Pubmed, Science Direct, Medline, and other internet facilities, literature search and related articles from library of Nargund College of Pharmacy, Digital Library of RGUHS, Bangalore etc.
Journals and articles:

• Biochemical pharmacology.
• Experimental eye research.
• Journal of immunological methods.
• Indian Drugs.

Text Books:

• A.H. Beckett, J.B. Stenlake, Practical pharmaceutical chemistry. 4th ed., Delhi: CBSPublisher and distributors; 1997.
• P.D.Sethi, Quantitative analysis of drugs in pharmaceutical formulation 3rd ed.,Delhi: CBS publisher and distributors.
• T. Higuchi, E. Brochman, H. Hanseen, H. Hanseen, Pharmaceutical analysis, Delhi: CBSPublishers and distributors; 2005.
• J. Mendham, R. C. Denney, J. D. Barnes, M. J.Kthomas, Vogel’s Text Book of Quantitative chemical analysis, 6th ed., Pearson Education Pvt Ltd., 2002.

Internet Browsing.
Method of Collection of Data (Including Sampling Procedure, If Any)

1. Procurement of the drug sample and chemicals.
2. Determine the solubility of Citicoline in various solvents and buffers.
3. Scanning the solution in UV-Visible region and selecting the solvents for various analytical studies.
4. Preparation of standard calibration curve
5. Develop and validate analytical method for Citicoline by UV-Visible spectrophotometer using,

a)Standard absorptivity value.
b)Calibration graph.
c)Single point and double point standardization.

1. Develop HPLC method to estimate Citicoline in bulk and pharmaceutical formulation.
2. Validation of developed analytical method as per ICH guidelines.

Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please mention briefly.
NOT APPLICABLE
Has ethical clearance been obtained from your institution in case of 7.3?
NOT APPLICABLE
LIST OF ReferenceS:

1. Wurtman RJ, Regan M, Ulus I and Yu L.Effect of oral CDP-choline on plasma choline and uridine levels in Humans BiochemPharmacol

2000; 60: 989-92.

1. Mirakor VA, Vaidya VV, Baing MM and Joshi SS.Rapid and sensitive high performance liquid chromatography method for Citicoline in formulation dosage form. Indian drugs 2007; 44(9): 693-6.

1. Chan KC, So KF, Wu EX. Proton magnetic resonance spectroscopy revealed choline reduction in the visual cortex in an experimental model of chronic glaucoma. Exp Eye Res2009; 88: 65-70.

1. Heegaard P MH, Pedersen HG, Jensen AL, Boas U. A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5′-diphosphocholine coupled dendrimers. J Immunol Methods 2009 ; 343(2):112-8

9. / Signature of the candidate / (SURANI SANJAYKUMAR SHAMJIBHAI)
10. / Remarks of the Guide / RECOMMENDED FOR THE DISSERTATION WORK.
11. / Name & Designation of
(in block letters)
11.1Guide
11.2 Signature / MRS. RITU VIVEK KIMBAHUNE
ASSISTANT PROFESSOR
DEPARTMENT OF QUALITY ASSURANCE
NARGUNDCOLLEGE OF PHARMACY
MRS. RITU VIVEK KIMBAHUNE.
11.3 Head of the department
11.4 Signature / DR.J.N. NARENDRA SHARATHCHANDRA
PROFESSOR
HEAD OF THE DEPARTMENT
DEPARTMENT OF QUALITY ASSURANCE
NARGUNDCOLLEGE OF PHARMACY
Dr.J.N. NARENDRA SHARATHCHANDRA
12. / 12.1 Remarks of Principal
12.2 Signature / FORWARDED AND RECOMMENDED FOR FAVOURABLE CONSIDERATION.
PROF. M.S. HARISH