Introduction:
Diclofenac Potassium chemically it is -[(2, 6-dichlorophenyl) amino] benzene acetic acid
monopotassium salt, with a molecular weight of 334.25 g/mol. The empirical formula is
C14H10C12KNO2. It comes under the category of Non-steroidal anti-inflammatory agent and
inhibits prostaglandin synthesis.It is official in British Pharmacopeia, United States
Pharmacopeia and European Pharmacopeia[1-5]. Febuxostat is a xanthine oxidase inhibitor.
The active ingredient in Febuxostat is 2-[3- cyano-4-(2-methylpropoxy) phenyl]-4methylthiazole- 5-carboxylic acid, with a molecular weight of 316.38 g/mol. The empirical
formula is C16H16N2O3S. It was recently approved by the European Medicines Agency on
February 21, 2008 and USFDA on Feb 13, 2009[6-9]. Febuxostat is not official in any
pharmacopoeia.The chemical structures of DKP & FEB shown.Fig.1&2. The combined
dosage forms of DKP and FEB are available in the market for the treatment of GOUT.
Deep literature survey reveals that, not a single analytical method is reported for the
determination of these drugs in combined dosage forms. The present
manuscript
describes simple, accurate, precise, rapid and economic spectrophotometric methods
for simultaneous estimation of DKP and FEB in tablet dosage form using 0.1 N NaOH as a
solvent.
MATERIALS AND METHODS
Chemicals and Reagents
DKP and FEB bulk powder was kindly gifted by A.P.M.C. Pharmacy College,
Himatnagar,
Gujarat,
India and Intas Pharmaceuticals, Ahmedabad, Gujarat, India.
Respectively the commercial fixed dose combination XANFEB DSR was procured from the
local market. All other chemicals used were analytical grade. 0.1 N NaOH and caliberated
glass wares were employed throughout the work.
Apparatus
A shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with spectral
width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell
was used to measure absorbance of all the solutions. A Reptech electronic weighing
analytical balance based on EMFC technology and a Toshcon ultrasonic bath (Toshniwal
process instrument pvt ltd.) was used in the study.
Preparation of standard stock solutions
An accurately weighed quantity of DKP (10 mg) and FEB (10 mg) were transferred to a
separate 100 ml volumetric flask and dissolved in 10 ml methanol and diluted to the mark
with 0.1 N NaOH to obtain standard solution having concentration of DKP (100 μg/ml) and
FEB (100 μg/ml).
Method 1 :
The standard solutions of DKP (10 μg/ml) and FEB (10 μg/ml) were scanned separately in
the UV range of 200-400 nm to determine λmax of both the drugs. The λmax of DKP and
FEB were found to be 275 nm and 314.5 nm respectively (Fig.3). Five standard solutions
having concentration 6, 12, 18, 24 and 30 μg/ml for DKP and 3, 6, 9, 12 and 15 μg/ml for
FEB were prepared in 0.1 N NaOH using the solutions having concentration 100 μg/ml.
The absorbance of resulting solutions was measured at 275 nm and 314.5 nm
and calibration curves were plotted at these wavelengths. The absorptivity coefficients of
these two drugs were determined using calibration curve equations. The concentration of
DKP and
FEB in
sample
solution was determined by solving the respective
simultaneous equations generated by using absorptivity coefficients and absorbance values
of DKP and FEB at these wavelengths. The absorbance and absorptivities values at the
particular wavelength were substituted in the following equations to obtain the
concentration [26].
𝐶𝑥 =
A2 ay1 – 𝐴1 𝑎𝑦2
Ax2 ay1 − ax1 ay2
. . . (1)
𝐶𝑦 =
A1 ax2 – 𝐴2 𝑎𝑥1
Ax2 ay1 − ax1 ay2
. . .(2)
Where,
A1, A2 –– absorbance of the mixture,
ax1 , ax2 – denotes absorptivities of the x at 275 nm and 314.5nm respectively,
ay1 , ay2 –– denotes absorptivities of Y at 275nm and 314.5 nm respectively,
CX = concentration of DKP.
CY = concentration of FEB.
Method 2:
The standard solutions of DKP (10 μg/ml) and FEB (10 μg/ml) were scanned in the UV
range of 200-400 nm to determine isoabsorptive point. The isoabsorptive point was found
to be 293nm (Figure.3).Five standard solutions having concentration 6, 12, 18, 24 and 30
μg/ml for DKP and 3, 6, 9, 12 and 15 μg/ml for FEB were prepared in 0.1 N NaOH using
the solutions having concentration 100 μg/ml. The absorbance of resulting solutions was
measured at 293 nm (isoabsorptive point) and 314.5 nm (λmax of FEB) and calibration
curves were plotted at these wavelengths. The absorptivity coefficients of these two drugs
were determined using calibration curve equations. The concentration of DKP and FEB in
sample solution was determined by solving the respective Q-analysis equations generated by
using absorptivity coefficients and absorbance values of DKP and FEB at these
wavelengths. The absorbance and absorptivities values at the particular wavelength were
substituted in the following equations to obtain the concentration [26].
For DKP
𝐶𝑥 =
𝑄𝑀 − 𝑄𝑦 𝐴1
×
𝑄𝑥 − 𝑄𝑦 𝑎𝑥1
. . . (3)
For FEB
𝐶𝑦 =
𝑄𝑀 − 𝑄𝑥 𝐴1
×
𝑄𝑦 − 𝑄𝑥 𝑎𝑥1
. . . (4)
Where,
𝑄M =
Absorbance of Sample at 314.5 nm
Absorbance of sample at 293 nm
𝑄X =
Absorptivity of DKP at 314.5 nm
Absorptivity of DKP at 293 nm
𝑄Y =
Absorptivity of FEB at 314.5 nm
Absorptivity of FEB at 293 nm
A1 = Absorbance of sample at isoabsorptive point,
ax1 = Absorptivities of DKP at isoabsorptive point.
Validation of the proposed method:
The proposed methods were validated according to the International Conference on
Harmonization (ICH) guidelines[27].
Linearity (Calibration curve)
The calibration curves were plotted over a concentration range of 6-30 μg/ml and 3-15
μg/ml for DKP and FEB respectively for Simultaneous equation and Q-analysis. Accurately
measured standard solutions of DKP ( 6, 12, 18, 24 & 30 ml) and FEB (3, 6, 9, 12 and 15
ml) were transferred to a series of 100 ml of volumetric flasks and diluted to the mark with
0.1 N NaOH for simultaneous equation Method. Accurately measured standard solutions of
DKP ( 4, 6, 8, 10 & 12 ml) and FEB (3, 6, 9, 12 and 15 ml) were transferred to a series of
100 ml of volumetric flasks and diluted to the mark with 0.1 N NaOH for Q-analysis
Method. The absorbances of the solutions were measured at 275 and 314.5 nm against
0.1 N NaOH
as blank for simultaneous equation Method. The absorbances of the
solutions were measured at 293 and 314.5 nm against 0.1 N NaOH as blank for Q-analysis
Method. The calibration curves were constructed
by plotting absorbances versus
concentrations and the regression equations were calculated.
Precision
The intraday and interday precision of the proposed methods was determined by analyzing
the corresponding responses 3 times on the same day and on 3 different days 3 different
concentrations of standard solutions of DKP and FEB for both methods.
Accuracy (recovery study)
The accuracy of the method was determined by calculating recovery of DKP and FEB by the
standard addition method. Known amounts of standard solutions of DKP and FEB were
added at 80, 100 and 120 % level to prequantified sample solutions of DKP and FEB.
(100 μg/ml for DKP and 40 μg/ml for FEB) The amounts of DKP and FEB were
estimated by applying obtained values to the respective regression line equations. The
experiment was repeated for five times for both methods.
Limit of detection and Limit of quantification
The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were
derived by calculating the signal-to- noise ratio (S/N) using the following equations
designated by International Conference on Harmonization (ICH) guidelines.
LOD = 3.3 × σ/S
LOQ = 10 × σ/S
Where, σ = the standard deviation of the Intercept of Caliberation curve and S = slope of the
calibration curve.
Analysis of DKP and FEB in combined Dosage Form (Tablet)
Twenty tablets were accurately weighed and average weight was calculated. The tablets
were triturated to a fine powder. An accurately weighed quantity of powder equivalent to
100 mg DKP & 40mg FEB was dissolved in 10 ml methanol and sonicated for 20 min and
volume was made up to 100ml. The solution was filtered through Whatman filter paper No
41 and aliquot portion of filtrate was diluted to produce solution having concentration of 10
μg/ml of DKP and 4 μg/ml of FEB. The absorbance of sample solution was measured at
selected wavelengths and the concentrations of the two drugs were estimated
using
equations (1) and (2) for simultaneous equation method and equations (3) and (4)
for absorbance ratio method. The analysis procedure was repeated six times and the results
are depicted in Table 2.
RESULTS AND DISCUSSION
The overlain spectra of DKP and FEB exhibit λ max of 275 nm and 314.5 nm for DKP and
FEB respectively which are quite separated from each other. Additionally one isoabsorptive
point was observed at 293nm. This wavelength was selected for simultaneous estimation of
DKP and FEB for Q value analysis and it is assumed to be sensitive wavelength. The criteria
for obtaining maximum precision by Simultaneous equation method were calculated and
found to be out side the range 0.1-2 and for Q-analysis ratios of absorbances at 2 different
Wavelengths were found to be constant. Standard calibration curves for DKP and FEB were
linear with correlation coefficients (r) values in the range of 0.9984 – 0.9997 at all the
selected wavelengths and the values were average of three readings with standard deviation
in the range of 0.0015 – 0.0045. The calibration curves were repeated three times in a day
and the average % RSD was found to be 0.86 for DKP and 0.70 for FEB; similarly the
method was repeated for three different days and average % RSD was found to be for 1.26
DKP and 1.18. for FEB . The accuracy of the methods was confirmed by recovery studies
ACKNOWLEDGEMENT
The authors are thankful to Intas Pharmaceutical, Ahmedabad,
Gujarat,
India for
providing DKP and FEB for research.
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TABLE-1 Regression Analysis Data and Summary of Validation Parameter of the Caliberation Curves
Sr
No
Parameters
Method 1
DKP
Method 2
FEB
DKP
FEB
1
Wavelength(nm)
275
314.5
275
314.5
293
314.5
293
314.5
2
Beer’s law limit
6-30
6-30
3-15
3-15
6-30
6-30
3-15
3-15
Regression
y=
y=
y=
y=
y=
y=
y=
y=
equation
0.031x
0.003x 0.016x-
0.063x
0.020x
0.003x
0.040x
0.063x
(y = mx + c)
-
-
-
-
-
-
-
0.011
0.004
0.031
0.001
0.004
0.020
0.031
(μg /ml)
3
0.014
4
Slope (m)
0.031
0.003
0.016
0.063
0.020
0.003
0.040
0.063
5
Intercept (c)
0.011
0.004
0.014
0.031
0.001
0.004
0.020
0.031
6
Correlation
0.9997
0.9985 0.9984
0.9991
0.9997
0.9985
0.9985
0.9991
coefficient(r2)
7
LOD (μg/ml)
0.25
0.80
0.02
0.16
0.23
0.80
0.12
0.16
8
LOQ (μg/ml)
0.77
2.43
0.07
0.49
0.70
2.43
0.38
0.49
Precision(% RSD)
9
Interday(n=3)
0.77
1.38
0.95
0.55
0.71
1.38
0.61
0.55
10
Intrady(n=9)
0.88
1.69
1.42
1.04
1.23
1.69
1.10
1.04
TABLE 2 : Results of recovery studies
Amount of pure drug added
(ml)
Level
of recovery
Simultaneous
equation
Method
QAbsorbance
method
%Recover
% recovery
DKP
FEB
(100ug/ml)
(100ug/ml)
80 %
18
7.2
99.915
99.803
99.551
99.848
100 %
20
8.0
99.169
99.917
99.850
99.833
120 %
22
8.8
99.739
99.504
99.827
99.493
Mean % recovery
99.60767
99.74133
99.74267
99.72467
SD
0.389956
0.213294
0.166386
0.200769
RSD
0.391492
0.213847
0.166815
0.201324
DKP
FEB
DKP
FEB
Fig.01 Chemical structure of Diclofenac Potassium.
Fig.02 Chemical structure of Febuxostat.
Fig.03 : Overlain Spectra of Diclofenac potassium and Febuxostat.