Spectrophotometric determination of tranexamic acid and mefenamic acid in

Spectrophotometric determination of tranexamic acid and mefenamic acid in

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Int J Pharm Biomed Res 2011, 2(1), 26-29
International Journal of
PHARMACEUTICAL
AND BIOMEDICAL
RESEARCH
ISSN No: 0976-0350
Research article
Spectrophotometric determination of tranexamic acid and mefenamic acid in
tablet dosage form using derivatization technique
N. Subramanian*, T. Devipriyadharshini, K. Venkateshwaran, P. Chandrasekar
Laboratory for Lipid Based Systems, Department of Pharmaceutical Technology, Anna University of Technology, Tiruchirappalli- 620 024, Tamilnadu, India.
Received: 25 Jan 2011 / Revised: 29 Jan 2011 / Accepted: 03 Feb 2011 / Online publication: 20 Feb 2011
ABSTRACT
A new effective UV spectrophotometric method has been developed for the simultaneous estimation of tranexamic acid
and mefenamic acid in combined tablet dosage form based on derivatization technique for making tranexamic acid UV
detectable employing reaction of ninhydrin with primary amino group of tranexamic acid in the presence of basic medium
via oxidation, deamination followed by condensation to form ruhemann purple product, quantitative determination is
performed by measuring the absorbance at two different wavelengths 571 nm and 335 nm. Beer’s law is obeyed in the
concentration ranging from 8- 40 μg/mL for tranexamic acid and 4- 20 μg/mL for mefenamic acid. The proposed method is
validated for linearity, recovery, precision, robustness and specificity by statistical evaluation as per ICH guidelines.
Tranexamic acid and mefenamic acid calibration curves are linear with correlation coefficient of 0.9983 and 0.9991.
Recovery is between 98.0% - 101.0% for both tranexamic acid and mefenamic acid. The developed method can be used for
routine quality control analysis of titled drugs combination in tablet formulation.
Key words: Mefenamic acid, Tranexamic acid, Ninhydrin, Method validation, Simultaneous equation method
1. INTRODUCTION
Tranexamic acid [Fig.1A], chemically known as trans- 4aminomethyl-cyclohexacarboxylic acid [1], is the most
potent antifibrinolytic agent that competitively inhibits
activation of plasminogen to plasmin (fibrinolysin), an
enzyme that degrades fibrin clot, fibrinogen, and other
plasma proteins [2]. It is used in treatment of pre and
postoperative conditions, bleeding disorders (menstrual blood
loss and bleeding during pregnancy) and during and
following tooth extraction of hemophilic patient [3, 4]. It also
acts as adjuvant drug for site specific pharmaco laser therapy
[3]. Tranexamic acid lacks π-electrons to behave as a
chromophore, to enable direct analysis and detectable by UVvisible absorption spectrophotometry, hence it is essential to
make derivatives of tranexamic acid [5] and its structure
shares similarity to aminoacids with its amino and carboxylic
acid groups.
*Corresponding Author. Tel: +91 431 2407978, Fax: +91 431 2407333
Email: [email protected]
©2011 PharmSciDirect Publications. All rights reserved.
Mefenamic acid [Fig.1B], chemically N-[(2, 3-dimethyl
phenyl) amino] benzoic acid [6], is the derivative of Nphenylanthranilic acid and official in USP, BP and IP [7]. It
has potent anti-inflammatory drug, analgesic and antipyretic
properties [6]. Mefenamic acid shows preferential inhibition
of cyclooxygenase- 2 there by inhibits the synthesis of
prostaglandins [8]. It is used in the treatment of primary
dysmenorrheal, osteoarthritis, nonarticular rheumatism,
healing of wounds, sport injuries [6], periodontitis,
antiphlogistic, rheumatoid arthritis [9] and other painful
musculoskeletal illnesses [10].
Trends in pharmaceutical analysis and compendial assay
methods reflects increasing replacement of time- honoured
classical methods of analysis with modern sophisticated
instrumental methods, like HPLC, LC- MS/MS. However,
due to high acquisition and maintenance cost of this
equipment makes adoption of an alternative and equivalent
UV method. The British Pharmacopoeia also gives allowance
for the use of an alternative method for any test or assay.
New tablet formulation in combination of tranexamic acid
500 mg and mefenamic acid 250 mg is commercially
available in Indian market for treatment of menorrhogia [4].
Few chromatographic and spectrophotometric methods are
N. Suubramanian et al.., Int J Pharm Bio
omed Res 2011, 2(1),
2
26-29
reeported for the determinnation of traanexamic aciid and
m
mefenamic
aciid individuallly or in com
mbination withh other
drrug. The repoorted methodss for the estim
mation of mefeenamic
accid was carrieed out by usinng different annalytical instruuments
like liquid chrromatographyy- tandem MS
S [11], HPLC
C [12],
coonductometerr [10], protonn NMR [10],, spectrophotoometer
[113], gas liquiid chromatogrraphy [9] andd spectrofluorrimeter
[110] where ass tranexamic acid was deetermined byy using
pootentiometer [14], spectrrofluorimeter [14], electrrospray
liquid
ioonization
m
mass
specctrophotometeer
[1],
chhromatographhy tandem maass spectrophootometer [11],, TLC
deensitometer [14]
[
and specctrophotometeer [4]. Howevver, so
faar no reporrt has been found for the simultaaneous
deetermination of tranexamiic acid and mefenamic acid
a
in
phharmaceuticall preparationss using UV sppectrophotomeeter up
too best of our knowledge.
k
Hence thee present stuudy aimed to
t develop an
a UV
sppectrophotom
metric method for the simuultaneous estim
mation
off tranexamic acid and mefenamic acid in combinedd tablet
doosage form baased on derivaatization technnique.
2.. MATERIAL
LS AND METHODS
2..1 Chemicals
was
obtainned
Zota
Tranexamiic
acid
from
Pharmaceuticalls, Chennai; mefenamic acid was obbtained
frrom Fourrts India Pvt. Ltd, Chennaai. Ninhydrinn was
puurchased from
m Merck, Mumbai.
M
Tableets were purrchased
frrom Indian market,
m
containning 500 mg of tranexamiic acid
annd 250 mg off mefenamic acid
a
per tableet. Methanol (HPLC
(
grrade) was purrchased from SD
S Fine Chem
micals Ltd, Muumbai.
2..2 Apparatus
Analysis was
w performeed on a Shim
madzu doublee beam
U
UV–visible
spectrophotom
meter equipped with 100 mm
m
matched
quartzz cell model UV
U 1800 (Japaan).
2..3 Standard preparation annd derivatizatiion
Standard stock
s
solutionns of tranexam
mic acid (25 µg/mL)
µ
annd mefenam
mic acid (12.5 µg/mL) were prepared by
trransferring 400 mg of traanexamic aciid and 20 mg
m of
m
mefenamic
aciid in to a 1000 mL volumettric flask conttaining
200 mL diluent (methanol: pH
p 7.2 phosphhate buffer, 70
7 : 30,
v//v). It was thhen sonicatedd for 15 minn. The solutioon was
diiluted up to voolume with diiluent. Series of working sttandard
soolutions were prepared. To the 1 mL worrking standardd 2 mL
off ninhydrin solution
s
(0.2 % in methannol) was addeed and
m
mixed
well [4]. The mixturee was warmedd for 20 min at
a 80°C
using constant temperature bath
b
[3] and then
t
cooled too room
teemperature finnally volume was made upp to the markk using
diiluent.
27
(A)
(B)
g.1. Chemical struucture of Tranexaamic acid (A) and
d Mefenamic acidd (B)
Fig
2.4
4 Sample prepparation and dderivatization
From the trriturates of 20 tablets an accurately
a
weeighed
po
ortion of pow
wder (45.0 m
mg) was transsferred to 1000 mL
vo
olumetric flaskk and mixed w
with 70 mL of diluent, sonicated
forr 15 min; the solution was made up to volume
v
with diluent
d
an
nd filtered throough 0.45 µm membrane fillter [15]. Aliqquot (1
mL
L) of the filtrrate was derivvatized using 2 mL of ninhhydrin
(0..2 % in metthanol) warm
med at 80°C for 20 min using
constant temperrature bath, ccooled, cooleed made up to
t the
maark using diluuent [16,17]. T
The absorban
nce of standarrd and
sam
mple was meeasured at dual mode (571
1 nm and 3355 nm)
ag
gainst ninhydriin blank.
2.5
5 Simultaneouus equations (M
(Multi-compon
nent method)
The wavellengths seleccted for solv
ving simultaaneous
eq
quation are 5771 nm and 3335 nm (Tablle 1). Absorpptivity
coefficients off both drugs were determ
mined at selected
waavelengths usiing simultaneoous equations 1 and 2. [7].
Cx
x = (A2 ay1 - A1 ay2)/ (ax22 ay1 - ax1 ay2) ------------- (1)
Cy
y = (A1 ax2 - A2ax1)/ (ax2 ay1 - ax1 ay2
2) --------------- (2)
wh
here, A1 and A2 are the abbsorbance of sample
s
solutioons at
56
68nm and 334nm resppectively. Cx
C
and Cyy are
concentrations of tranexam
mic acid and
d mefenamic acid
sam
mple solutionn. By substitutting the valuess of A1 and A2
A and
solving the twoo equations siimultaneously
y, the values of Cx
an
nd Cy can be
b obtained. Here, ax1 and ax2 are the
absorptivity coeefficient of traanexamic acid
d at 571nm annd 335
nm
m respectivelyy, ay1 and ay22 are the abso
orptivity coeffficient
off mefenamic accid at 571nm and 335 nm reespectively.
2.6
6 Method deveelopment
The solubillity of tranexaamic acid and mefenamic acid
a
in
diffferent buffeers like pH 5.5, 6.0, 7.2 and vaarious
combination of methanol andd buffer pH 7.2
7 was determ
mined.
Diiluents containning phosphatte buffer pH 7.2
7 and methaanol in
thee ratio of (330:70 v/v) ussed to record
d the spectruum of
traanexamic acidd and mefenaamic acid in the wavelenggth of
57
71nm for traneexamic acid annd 335nm for mefenamic accid.
N. Subramanian et al., Int J Pharm Biomed Res 2011, 2(1), 26-29
Table 1
Absorptivity for tranexamic acid and mefenamic acid
Concentration (μg/mL) Absorptivity
Tranexamic acid/
at 571 nm
at 335 nm
mefenamic acid
Tranexamic Mefenamic Tranexamic
acid
acid
acid
8/4
2.47
0.005
0.458
16/8
2.40
0.008
0.466
24/12
2.28
0.010
0.410
2.30
0.021
0.399
32/16
40/20
2.23
0.025
0.341
Mean
2.33
0.014
0.414
SD
0.000
0.000
0.0002
28
2.7.3 System and method precision
Mefenamic
acid
2.50
2.31
2.23
2.18
2.17
2.28
0.0001
Precision of an analytical procedure expresses the
closeness of agreement between a series of measurements
obtained from multiple sampling of same homogenous
sample under prescribed conditions. Method precision was
determined by analyzing the standard working solution at
five times [21]. System precision was determined by five
replicate measurements of working sample mixture at
analytical concentrations.
2.7.4 Ruggedness (Inter- intra day and analyst variations)
Table 2
Optical parameters for tranexamic acid and mefenamic acid
Parameters studied
Tranexamic acid
S. No
1
Beers law (μg/mL)
8- 40
2
Slope
0.023
Intercept
0.017
3
4
R2
0.999
λ max(nm)
571
5
Mefenamic acid
4- 20
0.031
0.012
0.998
335
The intra-day (repeatability), inter-day (intermediate
precision) variability and analyst to analyst variations were
determined using standard solutions [21]. These experiments
were repeated at different times on the same day to evaluate
intra-day variability, on second day to evaluate day- day
variability and by different analyst to evaluate analyst
variation.
2.7.5 Robustness
y = 0.031x + 0.012
R² = 0.998
1.2
MEFENAMIC ACID
1
TRANEXAMIC ACID
y = 0.023x + 0.017
R² = 0.999
Absorbance
0.8
Robustness of the method was determined by small
deliberate variations of the analytical method parameters
[19]. The condition studied is altered wavelength of 571±3
nm for tranexamic acid and 335±3 nm for mefenamic acid.
0.6
3. RESULTS AND DISCUSSION
0.4
The present UV spectrophotometric method has been
developed for the simultaneous estimation of tranexamic acid
and mefenamic acid in pharmaceutical formulation using
common conditions and to be used in routine analysis.
Tranexamic acid is soluble in water whereas mefenamic acid
is insoluble in water but soluble in alcohol. Mefenamic acid
is also shown pH dependent solubility (data not shown). The
common solvent for these drugs was selected by dissolving
the drugs in phosphate buffers of pH 5.5, 6.0 and 7.2 and in
various combination of methanol and phosphate buffer pH
7.2. The combination of methanol and phosphate buffer pH
7.2 in the ratio of 70:30 has shown highest solubility and
clear spectrum for the both drugs. Ninhydrin (0.2 % in
methanol) was used as derivatization agent for the tranexamic
acid. Cross wavelength study was performed to determine the
absorbance of the both drugs at 571nm and 335nm. The
absorptivity coefficient for the both drugs is determined by
using Beer’s law and the content of tranexamic acid and
mefenamic acid was calculated using simultaneous equations
3 and 4.
0.2
0
0
4
8
12
16
20
24
28
32
36
40
44
48
Concentration (mcg/mL)
Fig.2. Linearity of Tranexamic acid and Mefenamic acid
2.7 Method validation
2.7.1 Linearity
Linearity was determined by appropriate dilutions of
standard stock solutions [18] of tranexamic acid and
mefenamic acid ranging from 8 µg/mL to 40 µg/mL for
tranexamic acid and 4 µg/mL to 20 µg/mL for mefenamic
acid. The linearity of the working standard solution was
obtained by plotting the absorbance versus concentrations of
respective drugs shown in Fig.2.
2.7.2 Recovery
Recovery of the method was calculated by standard
addition at three levels like low, middle and high (80%,
100% and 120%) concentrations [19, 20] in triplicates.
Cx= (0.3744×0.014) - (0.5503×2.228) / (0.414×0.014) (2.233×2.28) ------------ (3)
Cy= (0.5503×0.414) - (0.3744×2.33) / (0.414×0.014) (2.233×2.28) ------------ (4)
N. Subramanian et al., Int J Pharm Biomed Res 2011, 2(1), 26-29
Table 3
Method validation parameters for tranexamic acid and mefenamic acid
Parameters
Tranexamic acid
RSD (%) of absorbance
Recovery
0.9
80
0.2
100
0.1
120
Precision
1.8
System precision
Method precision
0.2
Variations
Analyst-1 variation
0.9
Analyst-2 variation
0.4
Inter day variation
0.7
Intra day variation
1.0
Robustness (altered wavelength)
567nm/332nm
0.1
570nm/335nm
1.2
573nm/338nm
1.1
RSD- Relative standard deviation
The absorptivity coefficients of tranexamic acid at 571nm
and 335nm was found to be 2.33 and 0.414 and for
mefenamic acid was found to be 0.014 and 2.28. The optical
parameters are shown in Table 2 which shows an excellent
correlation between absorbance and concentration of each
drug within the concentration tested. The correlation
coefficient (R2) values for both the drugs were found to be
>0.996.
The mean percentage recoveries obtained for tranexamic
acid and mefenamic acid as shown in Table 3 are closer to
100% indicates non- interference from the excipients used in
the formulation. The % RSD of five replicate analysis for
method precision, system precision, intra- inter day and
analyst variations are found to be less than 1.0 %. The %
RSD for the deliberate change in the dual wavelength for
both drugs is found to be less than 2.0 %. The observed
results indicate the method to be specific, accurate, precise
and reproducible.
4. CONCLUSIONS
Proposed method has the advantage of simplicity,
convenience, precise and accurate. Ninhydrin provides a
simple reagent for spectrophotometric determination [22] of
tranexamic acid and mefenamic acid due to its high
sensitivity and low absorbance of ninhydrin blank. Moreover
the developed method does not involve any stringent reaction
conditions and offers advantage of color stability. Therefore
this method can be successfully applied for the determination
of tranexamic acid and mefenamic acid in tablet dosage form.
29
Recovered (%)
Mefenamic acid
RSD (%) of absorbance
Recovered (%)
99.2
98.7
98.2
1.0
0.7
0.7
99.4
99.5
100.1
2.0
0.7
0.7
0.1
1.1
1.3
0.8
0.7
1.1
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