“SIMULTANEOUS ESTIMATION OF MEFENAMIC ACID AND
TRANEXAMIC ACID IN COMBINED TABLET DOSAGE FORM BY
RP –HPLC METHOD.”
M. PHARM DISSERTATION PROTOCOL
Submitted to
Rajiv Gandhi University of Health Sciences,
Bangalore, Karnataka.
By
Mr. PRAVIN TARACHAND VIKHE
B. Pharm.
Under the Guidance
Of
Dr. KISHORE SINGH. CHATRAPATI
M. Pharm. Ph.D.
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
RAJIV MEMORIAL EDUCATION SOCIETY’S COLLEGE OF
PHARMACY,
GULBARGA – 585102.
2011—2012
Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name & Address of
the candidate
2.
Name of the Institution R.M.E.S’s College of Pharmacy, Gulbarga, Karnataka.
3.
Course of study and
subject
4.
Date of Admission to
course
5.
Title of the Topic
Mr. PRAVIN TARACHAND VIKHE
AT-POST-LONI BK, TAL- RAHATA, DISTAHEMADNAGAR, MAHARASHTRA, PIN NO.413736.
Master of Pharmacy in
Pharmaceutical Chemistry.
8th JULY 2010.
“SIMULTENOUS ESTIMATION FOR
MEFENAMIC ACID AND TRANEXAMIC ACID IN
COMBINED TABLET DOSAGE FORM BY RPHPLC METHOD.”
6.
BRIEF RESUME OF THE INTENDED
6.1 INTRODUCTION:Most of the drugs in multicomponent 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.
a. 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.
b. 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. An aqueous mobile phase allows
the use of secondary solute chemical equilibrium to control retention and
selectivity. The polar compounds gets eluted first in this mode and nonpolar 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 C18 Octa
Decyl Silane, C8 Octasilane, C4 Tetrasilane.
c. Ion Exchange Chromatography: The stationary phase contains ionic
groups like NR3+ or SO3-+ which interact with the ionic groups of the
sample molecules. This is suitable for the separation of charged
molecules only.
d. 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.
e. Size Exclusion Chromatography: It separates molecules accordingly to
their molecular mass. Largest molecules are eluted first and the smallest
molecules last1,2.
Mefenamic acid is a synthetic anti- inflammatory agent.
Chemically it is a 2[(2,3-dimetyl- phenyl) amino] benzoic acid. It is
clinically useful in the treatment of inflammation. It is official in Indian
pharmacopoeia3. It is listed in the Merck Index & Martindale4,5.
Mefenamic acid
Tranexamic
acid
is
chemically
trans-4-aminomethyl-
cyclohexaecarboxylic acid6. It competitively inhibits activation of
plasminogen, there by reducing conversion of plasminogen to plasmin,
an enzyme that degrades fibrin clots, fibrinogen, & other plasma protein,
including the procoagulant facture V & VIII. It is used for controlling
abnormal bleeding in a number of disease7. Tranexamic acid is official in
British Pharmacopeia8.
Hence the objective of the work is to develop new RP-HPLC
method for its estimation in bulk & formulation with good accuracy,
simplicity, precision & economy.
Tranexamic acid
6.2 NEED FOR STUDY:An exhaustive literature survey on analytical methods led to
.
following observations,
1. No analytical methods were published for new drugs.
2. The reported methods for many drugs made use of complex or costly
methodology like LC-MS.
3. Some reported method did not make use of internal standards while some
used internal standards that were not easily available.
4. There is no consensus among authors about the validation criteria for
analytical methods.
Thus the above observations warrant the need for development and
validation of new methods for estimation of drugs.
Hence, we design the objective of work as follows,
6.3
OBJECTIVES OF THE STUDY:To establish simple, sensitive & precise method for the estimation of the
Mefenamic acid & Tranexamic acid from tablet dosage form.
--The objectives of the present work are-1. To develop analytical method for is Mefenamic acid & Tranexamic acid
in bulk and various pharmaceutical dosage forms by RP-HPLC method.
2. To validate all developed analytical methods as per ICH guidelines.
6.4 PLAN OF WORK :1. Literature survey for selection of drug and to gain theoretical
knowledge on analytical method development and validation.
2. Procurement and standardization of drugs.
3. Development and optimization of analytical method.
4. Description of final method for drug.
5. Validation of Developed Method As per ICH guideline.
6.5 REVIEW OF LITERATURE:
Review should be based on assimilation of data from various
sources for the purpose of establishing a new concept. A preliminary
survey of literature for suitable method development for newer drugs has
been made.
Review of literature reveals that extensive of work has been
carried out for the routine analysis of drugs and pharmaceuticals of in
recently marketed as well as existing formulations and bulk drugs.
Development of high performance liquid chromatographic method can
be successfully employed for the analysis of formulations. And the some
literature survey of the present study is under.
1) Niopas I., et al. (1994)9. A simple, rapid sensitive & reliable HPLC
method described for the determination of mefenamic acid &
indomethacin in human plasma. The chromatographic separation was
performed on a C8 column (250 x 4.6 mm ID) using 10mM phosphoric
acid –acetonitrile (40:60,v/v) as the mobile phase & both drug were
detected at 280 nm.
2) Yasunori Morimoto, et al. (2003)10. The preparation of mefenamic acid
(MH)- alkanolamine complexes was attempted to increase the
transdermal flux of MH. a lipophilic enhancer system consist of
isopropyl myristate
& ethanol (9:1; EI system) produced a marked
enhancement of MH flux from the alkanolamine complex through
hairless rat skin membrane.
3) Mohammad-Reza Rouini, et al. (2004)11.A simple, rapid & specific
method for analysis of mefenamic acid by a sensitive high –performance
liquid chromatography
is described the mefenamic acid peak was
separated from endogenous peak on a C8 column by a mobile phase of
acetonitrile & water (50:50,v/v, pH 3)& drug were detected as 280nm.
4) Ophelia O.P.Yin, et al. (2004)12. A new method for the determination of
tranexamic
in
human
plasma
using
high
performance
liquid
chromatography with tandem mass spectrometric detection was
described. Chromatographic separation was performed on an X trraTM
MSC18 column with mobile phase consisting of 10% acetonitrile in 2
mM ammonium acetate buffer at flow rate of 0.15 ml/min. detection was
performed in mass spectrometry in tranexamic acid is m/z 158—95.
5) Freek Ariese, et al. (2007)13. A high performance liquid chromatographyfluorescence method was developed for the quantification of tranexamic
acid in blood. Derivative were separated on a 2.1mm C18 column using
an acetate buffer/ acetonitrile gradient & drug were detected as 440 / 520
nm.
6) Pezza L., et al. (2007)14. The characteristics, performance & application
of a novel electrode, namely Pt/Hg/Hg2 (MF)2/Graphite, for mefenamate
ion are described. This electrode responds to MF with sensitivity of
(58.9) mVdecade-1over the range of 1.0 x 10-6 to 1.0 x 10-2 mol L-1 at pH
6.0 – 9.0. The potentiometric sensor was successfully applied to the
determination of mefenamic acid in pharmaceutical & human serum
sample.
7) Arayne Saeed M. et
al. (2008)15. Two simple & sensitive
spectrophotometric method in ultraviolet & visible region are described
for the determination of tranexamic acid in pure form and pharmaceutical
preparation. In first method TA reacted with ninhydrin at boiling
temperature measuring in absorbance at 575nm. In second method TA
was reacted with ferric chloride solution yellowish orange colored
chlomogen showed 375 nm.
8) Jyh - Myng Zen. et al .(2008)16. The method was successfully applied to
the analysis of the content of tranerxamic acid in cosmetic product and
proved to
be suitable for rapid
& reliable quality control.
Chromatographic separation was performed on a Hamilton PRP-X100
anion exchange column (150mm x 1.4mm i.d 10m particle size) with a
(80:15,v/v) mixture of 0.1 mol l-1 NaOH & acetonitrile as mobile phase
& flow rate of 0.9 ml mln-1 .
9) Stanislas Grassin Delyle. et al, (2010)17. The object this study was
developed a method for measurement of Tranexamic acid in human
serum using liquid chromatography coupled to ion-trap mass
spectrometry, protein precipitation with perchloric acid & internal
standard were separated on C8 column in isocratic eluted using mobile
phase constituted of formate buffer/ acetonitrile (95:5,v/v).
10) Kaushik Kumar vankar. et al, (2010)18. A simple, rapid, accurate,
precise, specific
&
economical
spectrophotometric method for
simultaneous estimation of Ethamsylate & Tranexamic acid in combined
tablet dosage form has been developed & detected were wavelength
299.0 & 286.2. mobile phase used in sodium dihydrogen phosphate &
methanol.
11) Popat b. Mohite. et al, (2010)19. A new, simple, accurate & sensitive UVspectrophotometric absorption correction method has been developed for
simultaneous determination of Mefenamic acid & Tranexamic acid in
combined pharmaceutical dosage form. The is based upon determination
of mefenamic acid at 352.0 & tranexamic acid at 284.8nm in aqueous
methanol(20%v/v).
7.MATERIALS AND METHODS:7.1. SOURCE OF DATA:-
a) Internet
b) Gulbarga University Library, Gulbarga
c) RGUHS ( Helinet )
d) International Pharmaceutical Abstracts
7.2 MATERIAL :Cyber lab HPLC Model High pressure system-2003 series
comprising of LC-P-100 pump & LC-UV-100 as UV-Visible detector,
Rheodyne manual Injector fitted with 100µl loop and cyber store
chromatography software.
Other instrument to be use UV visible Spectrophotometer of
pb labs, analytical balance, sonicator etc.
7.3 METHODS :Method development included the following stages;
1. Procurement and Standardization of drugs reference standards by
measurement of physical constants and spectroscopy techniques like IR
Spectroscopy. Accordingly this analysis and standardized is done.
2. Procurement of HPLC grade to chemical.
3. Selection and Optimization of chromatographic conditions.
4. The stationary and mobile phase will be select and optimized machine
trust resolution between drug and internal standards. Retention
wavelength will be select to improve specificity and sensitivity.
5. Calibration Experiment.
6. Validation: The method will be validated using the validation criteria
provided by U.S FDA & ICH on it guidance document. Validation
experiment will be perform using Quality Control (QC) sample.
7.4
Does the study require any investigation or invention to be conducted on
Patients or other humans or animals? If so please mention briefly.
NOT APPLICABLE.
7.5
Has ethical clearance been obtained from yours institution in case of 7.4
NOT APPLICABLE.
8. REFERENCES:1. Swarbrick James, and Boylan James C. Encyclopedia of Pharmaceutical
Technology, Marcel Dekker Inc, New York.1998; 1: 217-224.
2. Beckett A.H, Stenlake J.B, Practical Pharmaceutical Chemistry CBS
Publication and Distributors, 4thEdition. 1997; 2: 275-337.
3. Indian Pharmacopeia, Govt. of Indian Ministry Of Health & Family
Welfare, Publish By: The Indian Pharmacopeia Commission, Ghaziabad.
2007 ;1, 2 ,3: 156, 358, 1468.
4. Busavari S. The Merck Index: An Encyclopedia of Chemical, Drug &
Biological. Whitehouse Station (NJ): Merck Research Lab, Division of
Merck, 13th Edition. 2001:1152.
5. Budavari S. The Merck Index: An Encyclopedia of Chemical, Drug &
Biological Whitehouse Station (NJ): Merck Research Lab, Division of
Merck, 13th Edition. 2001: 854.
6. The Merck Index –An Encyclopedia of Chemical, Drug & Biological,
Merck & Company, USA,13th Edition.1989; 3757- 9648.
7. Martindale – The Complete Drug Reference, Pharmaceutical Press,
London, 29th Edition. 2002; 758, 1960.
8. British Pharmacopeia, Her Majestry’s Stationary Office London. 2004;
1: 758 – 1960.
9. I. Niopas, K.Mamzoridi, Determination of Indomethacin and Mefenamic
acid in plasma by High – Performance Liquid Chromatography, Journal
of Chromatography B. 1994; 656: 447 – 450.
10. Liang Fang, Yasunori Morimoto, Sachihiko Numajiri, Daissuke
Kobayashi, The use of complexation with alkanolamines to facilitate skin
permeation of mefenamic acid, International Journal of Pharmaceutics.
2003; 262: 13 – 22.
11. Mohammad- Reza Rouini, Ali Asadipour, Yalda Hoseinzadeh Ardakani,
Fakhredin Aghdasi, Liquid chromatography method for determination of
mefenamic acid in human serum, Journal of Chromatography B. 2004;
800 : 189- 192.
12. Qi Chang, Ophelia Q. P. Yin , Moses S.S Chow, Liquid chromatography
– tandem mass spectrometry method for the determination of
Tranexamic acid in human plasma, Journal of Chromatography B. 2004;
805: 275 – 280.
13. Jose Fernando Huertas- Perez, Michal Heger, Henk Dekker, Hans
krabbe, Jan Lankelma, Freek Ariese, Simple ,rapid & sensitive liquid
chromatography- Fluorescence method for the quantification of
tranexamic acid in blood, Journal of Chromatography A. 2007; 1157:
142- 150.
14. A. O. Santini, L. Pezza, H. R. Pezza, Development of a potentiometric
mefenamic ion sensor for the determination of mefenamic acid in
pharmaceutical & human blood serum, Sensor and Actuators B. 2007;
128: 117 – 123.
15. M.Saeed Arayne, Najma Sultana, Farhan Ahmed Siddiqui, Agha
Zeeshan Mirza, M. Hashim Zuberi, Spectrophotometric techniques to
determine tranexamic acid: Kinetic studies using ninhydrin & direct
measuring using ferric chloride, Journal of Molecular Structure. 2008;
891: 475 – 480.
16. Ying Shih, Kuan-Lin Wu, Jun-Wei Sue, Annamalai senthil Kumar, JyhMyng Zen, Determination of tranexamic acid in cosmetic product by
high – performance liquid chromatography coupled with barrel plating
nickel
electrode,
Journal
of
Pharmaceutical
and
Biomedical
Analysis.2008; 48: 1446 – 1450.
17. Stanislas Grassin Delyle, Emuri Abe, Anne Batisse, Benjamin Tremey,
Marc Fischler, Philippe Devillier, Jean Claude Alvarez, A validated
assay for the quantitative analysis of tranexamic acid in human serum by
liquid chromatography coupled with electrospray ionization mass,Clinica
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18. Roshan Issarani, Kaushik Kumar Vankar, Diptish Kumar Nayak,
Spectrophotometric
Methods
For
Simultaneous
Estimation
Of
Ethamsylate & Tranexamic Acid From Combined Tablet Dosage Form,
International Journal of Chem Tech Research. 2010; 2(1): 74 – 78.
19. Popat B. Mohite, Shantaram G. Khanage, Vaidhun H.Bhaskar,
Absorption correction method for Estimation of mefenamic acid &
tranexamic acid in combined tablet dosage form, International Journal of
Biomedical 7 Advance Research. 2010; 01[01].
20. B. G. Osborne, B. Demetriou, Detection of mefenamic acid & its
metabolites in urine by thin – layer chromatography, Journal of
chromatography. 1974; 90 : 405-407.
21. L. J. Dusci, L. P. Hackett, Gas – liquid chromatography determination of
mefenamic acid in human serum, Journal of chromatography. 1978; 161:
340-342.
22. Marc Hoylaerts, Henri Roger Lijnen, Desire Collen, Studies on the
mechanism of the Antifibrinolytic Action of Tranexamic Acid,
Biochimica et Biophysica Acta, 1981; 673: 75 – 85.
23. Paul M. Elworthy, S. A. Tsementzis, Donald Westhead, Determination
of plasma Tranexamic acid using cation-exchange high – performance
liquid
chromatography with
fluorescence
detection,
Journal
of
chromatography. 1985; 343: 109 – 117.
24. M. Abrahamsson, Determination of a prodrug of tranexamic acid in
whole blood by reversed- phase liquid chromatography after pre-column
derivatization with fluorescamine, Journal of Pharmaceutical &
Biomedical Analysis. 1986; 4(3): 399 – 406.
25. Sabina Zommer Urbanska, Halina Bojarowicz, Spectrophotometric
investigation
on
protolytic
equilibria
of
mefenamic
acid
and
determination by means of Fe (III) in methanol – aqueous media, Journal
of Pharmaceutical & Biomedical Analysis. 1986; 4(4): 475 – 481.
26. Nina Maron, Gerry Wright, Application of photodiode array UV
detection in the development of stability – indicating LC method:
determination of mefenamic acid, Journal of pharmaceutical &
biomedical Analysis. 1990; 8(1): 101 – 105.
27. Erdal
Dinc,
Cem
Yucesoy,
Feyyaz
Onur,
Simultaneous
spectrophotometric determination of mefenamic acid & paracetamol in a
pharmaceutical
preparation
using
ratio
spectra
derivative
spectrophotometry & chemometric method, Journal of Pharmaceutical &
Biomedical Analysis. 2002; 28: 1091 – 1100.
28. Sanae Muraoka, Toshiaki Miura, Inactivation of creatine kinase during
the interaction of mefenamic acid with horseradish peroxidase and
hydrogen peroxide: participation by the mefenamic acid radical, Life
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IL Farmaco 58. 2003; 58: 397 – 401.
Signature of Candidate
9.
10.
Remark of the guide:- The above 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.
Name and Designation of
(in block letters)
11.1 Guide
Dr. KISHORE SINGH. CHATRAPATI
M.PHARM PH.D
DEPARTMENT OF PHARMACEUTICAL
CHEMISTRY, RMES’s COLEEGE OF
PHARMACY,
GULBARGA- 585102.
11.2 Signature of guide
11.3 Co-guide (if any)
Mr. SOMNATH D. BHINGE
M.Pharm(Ph.D)
11.4 Signature of co-guide
11.5 Head of the Department
Dr. KISHORE SINGH. CHATRAPATI
M.Pharm Ph.D.
11.6 Signature
12.
12.1 Remarks of the
Chairman & Principal
The above mentioned information is correct
and I recommend the same for approval.
12.2 Signature
Dr. KISHORE SINGH. CHATRAPATI
M.Pharm Ph.D.