Download O A

1547
Journal of Applied Sciences Research, 8(3): 1547-1557, 2012
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLES
Application of Spectrophotometric and Chromatographic Methods for Stability
Indicating Determination of Leflunomide
1
D.S. Shokry, 2M. Abdel Kawy and 1S.A. Weshahy
1
2
Faculty of pharmaceutical sciences & pharmaceutical industries, Future University in Egypt.
Faculty of Pharmacy, Cairo University.
ABSTRACT
Leflunomide, an amide containing compound, pyrimidine synthesis inhibitor a leading drug in treatment of
moderate to severe rheumatoid arthritis. Four stability indicating methods are presented in this paper for the
selective determination of leflunomide in presence of its alkaline degradate. The resolution of the drug and its
alkaline degradate has been achieved using derivative spectrophotometry including second-, third- and fourth
derivatives. The derivative amplitudes are measured at 256.4 nm, 269.8 nm, 226.6 nm for D2, D3 and D4
respectively. The proposed methods were found to be linear over the range of (2.0-24.0 µgml-1). The fourth
method is based on the chromatographic separation on a C18 column using a mobile phase of 0.01 M aqueous
potassium dihydrogen phosphate adjusted at pH 3.5 with orthophosphoric acid, acetonitrile in a ratio of [30:70]
, a flow rate of 1 ml/min and UV detection at 262 nm. The proposed methods were validated with regard to
accuracy, precision, selectivity, robustness, application to pharmaceutical preparation and further validated by
applying standard addition technique.
Key words: Derivative spectrophotometry (DS); HPLC; Leflunomide; alkaline degradate.
Introduction
Leflunomide marketed under the brand name Arava® is a pyrimidine synthesis inhibitor (Dougados et al.,
2005). It is pharmacologically classified as a prodrug of the disease modifying antirheumatic drug (DMARD)
type (Pinto et al.,2006). It works as an immuno-modulatory drug suppressing the immune system (Bertolilni et
al., 1997). Also it was proven to have an anti-inflammatory effect causing a slow progression and relief of
symptoms of rheumatoid arthritis as well as psoriatic arthritis (Papageorgiou et al., 1997). Leflunomide was
approved by Food & Drug Administration (FDA) and in many countries in 1999 (Rozman, 2002; Bianco et al.,
2009).
It is chemically designated as 5-methyl-N-[4 (trifluromethyl) phenyl]-isoxazole-4-carboxamide (Budavari,
2001). Its molecular formula is C12H9F3N2O2 having molecular weight of 270.207 g/mole. Its structural formula
is:
Fig. 1:
Several spectrophotometric methods have been reported in literature for determination of leflunomide in
bulk or pharmaceutical preparations and in stability indicating determination of the drug including FIA
(Yeniceli et al., 2005), derivative (Abbas et al., 2006) & difference spectrophotometry (Abbas et al., 2006).
Reversed phase HPLC (Rao et al., 2008; Miron et al., 2006; Shui-Wang, 2005; Hong et al., 2005; Yeniceli et
al., 2006; Weng, 2005; Jiang et al., 2001; Zhu et al., 1999; Zhu et al., 2000; Chan et al., 2004; Duygu et al.,
2006; Molinaro et al., 2005; Vivien et al., 2004; Kher et al., 2011; Yadav et al., 2010; Sultana et al., 2011;
Zhang et al., 2002; Fairbanks et al., 1999; Dias et al., 1995; Schmidt et al., 2003) have been applied for
determination of the drug in pharmaceutical preparations in presence of related substances, its metabolites and
in biological fluids.
Corresponding Author: M. Abdel Kawy, Faculty of Pharmacy, Cairo University.
1548
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
Both spectrophotometry including derivative, difference spectrophotometry, colorimetric methods (Abbas
et al., 2006) as well as HPLC and TLC (Mohamed et al., 2006; Abbas et al., 2006) have been applied for
stability indicating determination of the studied drug in presence of its degradates.
Leflunomide is an amide containing compound, this means it is liable to hydrolysis. It was found that the
drug when being refluxed with sodium hydroxide, it undergoes alkaline hydrolysis causing complete cleavage of
the amide linkage. In this paper, four methods were proposed for the analysis of leflunomide in presence of its
alkaline degradate. Derivative spectrophotometry as a well established technique capable of enhancing the
resolution of overlapping bands, thus it was attempted for resolving the overlapped spectra of the drug and its
degradate without prior separation by applying second, third and fourth derivatives.
Another method applied was the chromatographic separation which was also successful in estimation of
leflunomide in bulk, pharmaceutical preparations as well as in synthetic mixtures.
II. Experimental:
II.1. Instrumentation:
A Shimadzu UV 1650 double beam spectrophotometer connected to a computer loaded with Shimadzu
software UV probe 2.10 was used [Hiroshima, Japan]. UV spectra were recorded using a 1 cm quartz cell; the scan
range was 200-350 nm with 0.2 nm intervals.
An Hplc unit equipped with a 20 μl loop injector, and an UV detector, the chromatographic column from
Agilent technologies, USA was ZORBAX Eclipse XDB-C18 (4.6x 150 mm i.d, 5 µm particle size). Data
acquisition was performed on agilent LC ChemStation software. All determinations were performed at ambient
temperature.
Ii.2. Samples And Reagents:
Ii.2.1. Samples:
II.2.1.1. Raw materials:
Leflunomide certified to contain 99.90% by the manufacturer method was kindly supplied by Eva
Pharmaceuticals (Cairo – Egypt).
II.2.1.2. Market Samples:
A commercial pharmaceutical formulation [Arthfree®] produced by [Eva Pharmaceuticals, Cairo, Egypt,
Batch no. 26177], each tablet is labeled to contain 20 mg of leflunomide was obtained from the local market.
II.2.2. Reagents:






All chemicals and reagents used throughout this work were of analytical grade.
Methyl alcohol used was of analytical spectroscopic grade [Sigma-Aldrich Co., USA].
Sodium hydroxide: [E. Merck, Durmstoolt, Germany], 4.0 M aqueous solution.
Hydrochloric acid: [BDH], 4.0 M aqueous solution.
Methanol used was HPLC grade [Sigma-Aldrich Co., USA].
Potassium dihydrogen phosphate [BDH, Poole; UK], orthophosphoric acid [BDH] used were analytical
grade.
Distilled water was used throughout the whole work.
II.3. Standard Solutions:
II.3.1. Standard Stock Solutions:
II.3.1.1. Standard Stock Solutions Of Leflunomide:
A portion equivalent to 100.0 mg of leflunomide is accurately weighted, transferred into 100.0 ml
volumetric flask and dissolved in a minimum amount of methanol, sonicated for 10 minutes, and the volume is
made up to the mark with the same solvent to give a final concentration of 1.0 mg ml-1.
1549
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
I I.3.1.2. Standard Stock Solution Of Alkaline Degradate:
It was prepared by mixing 100.0 mg of leflunomide with 4.0 M NaOH, followed by heating in a boiling water
bath at 100oC for 3 hours, the solution was then cooled and neutralized with a calculated volume of 4.0 M HCl and the
volume was made up to the mark in a 100 ml volumetric flask with methanol. Complete degradation was checked by
TLC using silica gel 60 F254 plates and a developing solvent system of chloroform: methanol in a ratio of (90:15).
II.3.2. Standard Working Solutions Of Leflunomide and Its Alkaline Degradate:
A portion equivalent to 10.0 mg of leflunomide & its alkaline degradate was transferred from the standard stock
solution into 100 ml volumetric flask and the volume was made up to the mark with methanol to give a standard
working solution of 100.0 gml-1.
II.4. Procedure:
II.4.1. Spectrophotometric Method:
Scanning Of The Absorption Spectra Of Leflunomide & Its Alkaline Degradate:
An aliquot equivalent to (50.0 μg) of leflunomide and its standard alkaline degradate was transferred from
their working standard solutions of concentrations (100.0 μg ml-1) separately into 10 ml volumetric flasks
respectively, completed to the volume with methanol. The absorption spectrum of each solution is recorded
against blank, similarly prepared without the drug.
II.4.2. Chromatographic Method:
Isocratic elution technique was utilized with the column maintained at room temperature. The mobile phase
used was a mixture of 10 mM potassium dihydrogen phosphate adjusted at pH [3.5] using orthophosphoric acid
and acetonitrile in a ratio [30:70].
The mobile phase was filtered through a 0.45 m membrane filtration system [Millipore Corp., Milford,
MA, USA] to remove any particulate matter then degassed by sonication for 20 minutes. The flow rate was 1
ml/min. samples of 20.0 l, were injected onto the column and the detector was set at 262 nm.
All the chromatographic determinations were performed 3 times at ambient temperature.
II.4.3. Method Validation:
II.4.3.1. Linearity
II.4.3.1.1. Spectrophotometric Method
Different aliquots (0.2-2.4 ml) of leflunomide were transferred from its working standard solution (100.0
μgm1-1) separately, into 10-ml volumetric flasks and diluted to the volume with methanol to give concentrations of
(2.0-24.0 μgm1-1), the zero order spectra were recorded using aforementioned instrument over the range of (200400 nm) using λ=0.2 nm. Then the second, third & fourth derivative of these spectra were computed using
λ=16.0 nm. & scaling factor = 100.0. The amplitudes were recorded at 256.4 nm, 269.8 nm, 226.6 nm
respectively. Calibration graph were constructed for concentrations over the range of (2.0-24.0 μgm1-1).
II.4.3.1.2. Chromatographic Method:
Different aliquots [0.5-4.0 ml] of leflunomide were transferred from their working standard solutions [100
g.ml-1] into 10 ml volumetric flasks and dilute to the volume with mobile phase. Each of those dilutions were
then chromatographed by injecting an aliquot of 20 l of each into the chromatographic system three times and
processed according to the method described in this work. The mean peak areas of three determinations of each
concentration were plotted against the same concentration and then the regression equations were computed.
II.4.3.2. Accuracy:
The previously mentioned procedures under linearity were applied for different concentrations of leflunomide.
The concentrations of the studied drug were calculated from its corresponding regression equations then the mean
percentage recoveries and standard deviations were calculated.
1550
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
II.4.3.3. Precision:
The previously mentioned procedure under linearity were repeated for the analysis of three different
concentrations of the drug for the evaluation of intraday (n=3) & interday (n=3) precision and the relative standard
deviations (RSD%) were calculated.
II.4.3.4. Analysis Of Laboratory Prepared Mixture:
This study was performed by adding known amounts of the alkaline degradate to a known concentration of
the intact drug. The resulting mixtures were assayed by adopting the calibration procedures under linearity. The
percentage recoveries were then calculated.
II.4.3.5. Application To Pharmaceutical Preparation:
Twenty tablets were accurately weighed, and finely ground in a mortar. A portion of the powder equivalent
to 20.0 mg of leflunomide was accurately weighed, sonicated for 15 minutes, centrifuged for 10 minutes, the
precipitate was then filtered through a micropore filter, washed into a 100 ml volumetric flask and the volume
was made up to the mark with the same solvent to provide a solution of concentration 200.0 μgml-1.
The previously prepared sample was further diluted to provide a solution of concentration 10.0 μgml-1, this
concentration was assayed by adopting the calibration procedures used under linearity and the results were
compared to the expected results and the percentage recoveries were calculated.
II.A.2.4.2.6. Validation By Standard Addition Technique:
This study was performed by adding amounts of working standard solution of the drug to a known
concentration of the commercial solution of leflunomide. The resulting mixtures were assayed by adapting the
calibration procedures using under linearity and the percentage recoveries were calculated.
Results And Discussion
For Spectrophotometric Method:
Many pharmaceutical compounds undergo degradation during storage or even during the different
processes of their manufacture. Several chemical and physical factors can lead to degradation of drugs (Henry
et al., 2001). Hydrolysis and oxidation are the most famous routes of chemical degradation of drugs (Florence
et al., 1998; Banker et al., 2002).
The main classes of drugs subjected to degradation are esters, amides and lactams. Leflunomide has an
amide linkage so trials were conducted for alkaline hydrolysis. It was found that the drug is liable to
degradation in strong basic medium. Sodium hydroxide was the hydrolyzing agent of choice according to the
ICH guidelines.
Figure (2) shows the UV absorption spectra of leflunomide and its alkaline degradate. As figure (2) shows,
the large overlap of the spectral bands of the two compounds prevents the selective determination of
leflunomide in a mixture of the intact drug and its degradate from their zero order spectra.
Since the derivative spectrophotometry combined with the zero crossing technique can be applied for the
determination of a substance in presence of another, by the selection of a wavelength where the contribution of
one compound (degradate) is zero or almost zero, while the other to be determined (intact drug) has a reasonable
value. On that basis, derivative spectrophotometry including D2, D3 and D4 has been applied in this paper as an
analytical method for selective determination of leflunomide in presence of its degradate.
Instrumental parameters affecting D2, D3 and D4 spectra were tested, namely Δλ interval and scaling factor.
The best results were obtained by using 16 nm as Δλ a scaling factor = 100 in order to diminish the error in
reading the signal. Figures (3-5) show that it can be determined at 256.4 nm, 269.8 and 226.6 nm for D2, D3 and
D4 respectively which correspond to the zero crossing points of its degradate.
Linear calibration curves were obtained in the range of 2.0-24.0 µg ml-1 by plotting the relationship between
peak amplitudes and their corresponding concentrations.
The accuracy of the proposed method for the assay of leflunomide in raw materials is shown in table (1).
The mean percentage recoveries were found to be 100.08±1.403, 99.83±1.155 and 98.61±0.667 at 256.4 nm,
269.8 and 226.6 nm respectively.
Several mixtures of leflunomide and its degradate were prepared and analyzed by the proposed methods,
the results proved that the proposed methods is selective and valid for stability testing of the drug in presence of
up to 100% of its degradate as shown in figures (3-5). The results are shown in table (2).
1551
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
The proposed methods were successfully applied for the analysis of the pharmaceutical preparation and the
validity of the methods was further assessed by applying the standard addition technique, the results are
summarized in table (3). No interference from the sample matrix was observed. The results were in good
agreement with the labeled content.
The results of assay validation for the calibration graphs obtained by applying the proposed derivative
methods are listed in table (4).
Fig. 2: Zero order absorption spectra of 5 μgml-1 of Leflunomide (a) and of 5 μgml-1 of its alkaline degradate (b)
using methanol as a blank.
Fig. 3: Second derivative absorption spectra of 10.0 μgml-1 of leflunomide (a) and of 10.0 μgml-1 of its alkaline
degradate (b).
Table 1: Accuracy of the proposed derivative spectrophotometric methods for the analysis of pure samples of Leflunomide at zero crossing points of its
alkaline degradate.
Found * (µgml-1)
Recovery %
D2 SpectroD3 SpectroD4 SpectroD4 SpectroD3 SpectroTaken
D2 Spectrophotometric
photometric
photometric
photometric
photometric
(µgml-1)
photometric method
method
method
method
method
method
At 256.4 nm
At 269.8 nm
At 226.6 nm
At 256.4 nm
At 269.8 nm
At 226.6 nm
4
4.05
4.01
3.93
101.25
100.23
98.27
8
8.14
8.11
7.94
101.75
101.42
99.31
12
11.81
12.01
11.77
98.42
100.05
98.09
16
15.86
15.76
15.68
99.13
98.48
98.02
20
19.97
19.79
19.87
99.85
98.95
99.35
Mean±SD
100.08± 1.403
99.83± 1.155
98.61± 0.667
*Average of three determinations.
1552
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
Fig. 4: Third derivative absorption spectra of of 10.0 μgml-1 of leflunomide (a) and of 10.0 μgml-1 of its alkaline
degradate (b).
Fig. 5: Fourth derivative absorption spectra of of 10.0 μgml-1 of leflunomide (a) and of 10.0 μgml-1 of its
alkaline degradate (b).
Table 2: Results obtained for the analysis of laboratory prepared mixtures containing different ratios of Leflunomide and its alkaline
degradate.
Recovery %*
3
Spectro
D4 SpectroD
Alkaline
Leflunomide
D2 Spectro-photometric
Sample No.
Photometric
Photometric
degradate
-1
(µgml )
method
method
method
(µgml-1)
At 256.4 nm
At 269.8
At 226.6 nm
1
10.00
2.00
99.79
101.80
98.14
2
10.00
4.00
99.32
98.47
98.43
3
10.00
6.00
101.67
99.96
99.01
4
10.00
8.00
101.2
101.45
101.91
5
10.00
10.00
102.14
99.32
99.88
Mean±SD
100.82±1.217
100.20±1.410
99.81±1.515
* Average of three determinations.
1553
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
Table 3: Quantitative determination of Leflunomide in pharmaceutical preparation and application of standard addition technique by the
proposed derivative spectrophotometric methods at the zero crossing points of the alkaline degradate.
Found % * ±SD
Pharmaceu
tical
preparation
Taken
(µgml1
)
D2
Spectrophotome
tric
method
At
256.4
nm
Arthfree®
tablets
claimed to
contain
100 mg
leflunomid
e/tablet
10.00
99.67
±1.908
Mean±SD
D3
Spectroph
otometric
method
At
269.8
nm
98.94
±1.427
Standard addition technique
D4
Spectrophotome
tric
At
226.6
nm
99.02
±0.936
Pure found** (µgml-1)
D2
Spectrophotome
tric
method
D3
Spectroph
otometric
method
At 256.4
nm
At 269.8
nm
3.00
5.00
7.00
12.95
14.98
16.91
9.00
18.96
Pure
added
(µgml-1)
Recovery %
12.93
15.00
16.97
At
226.6
nm
12.84
14.93
17.05
D2
Spectrophotom
etric
method
At
256.4
nm
99.26
100.27
99.11
18.78
19.06
D4
Spectrophotom
etric
101.05
102.10
101.03
D4
Spectro
photom
etric
At
226.6
nm
98.02
100.54
102.04
99.91
98.71
101.75
99.64±
0.546
100.72±
1.432
99.75±
0.449
D3
Spectrophoto
metric
method
At 269.8nm
*Average of ten determinations.
**Average of five determinations.
Table 4: Results of assay validation parameters of the proposed derivative spectrophotometric methods for the determination of Leflunomide
at the zero crossing points of the corresponding alkaline degradate.
Parameters
D2 Spectro-photometric
D3 SpectroD4 Spectro-photometric method
method
photometric method
At 256.4 nm
At 269.8
At 226.6 nm
Range
2 – 24 µgml-1
Linearity
Slope
0.0213
0.0014
0.0007
Intercept
-0.0061
4x10-5
0.0004
Correlation coefficient (r)
0.9999
0.9999
1
Standard error of the slope
0.000105521
6.32932x10-6
7.32495x10-5
Confidence limit of the slope
0.0209886640.001396690.0006882090.021531166
0.00142923
0.000691975
Standard error of the intercept
0.001654074
9.92139x10-5
1.4821x10-5
Confidence limit of the
-0.010387911-0.0002184930.000377793-0.000436824
intercept
(-0.001884044)
0.000291581
Standard error of estimation
0.002119
0.000127128
1.47125x10-5
Accuracy (mean±SD)
100.08±1.403
99.83±1.155
98.61±0.667
Selectivity
100.82
100.2
99.48
Precision (RSD%)
Repeatability*
0.694
0.621
0.450
Intermediate* precision
0.747
0.858
0.621
*The intra-day and inter-day relative standard deviations of the average of concentrations 2.0,14.0, 24.0 μgml-1 for D2,D3,D4.
For HPLC Method:
The aim of the present study was to develop a chromatographic system capable of eluting and resolving
leflunomide and its degradate and that complies with the general requirements for the system suitability.
Optimum conditions which are necessary for the quantitative analysis of the drug with maximum sensitivity
including the detection wavelength, the type and the quality of the organic modifier, and the pH of the mobile
phase were established by a number of preliminary experiments. Optimum conditions were fixed by varying one
parameter at a time and fixing other parameters constant and observing its effect on the response factor and also
on the peak resolution.
In preliminary experiments, the studied drug and degradation product were subjected to separation by
reversed phase HPLC using a RP-C18, 10μm, 150 x 4.6 mm) and acetonitrile- water as an eluent, a broad peak of
leflunomide that was not well defined was obtained, water was replaced by buffers as ammonium acetate and
potassium dihydrogen phosphate to control pH. The best buffer was aqueous potassium dihydrogen phosphate in
a concentration of 10 mM. In another attempt, replacing acetonitrile with methanol caused longer retention time,
excessive peak tailing and bad peak shape. Regarding the pH values, acidic pH was found optimum for peak
shape and retention time. Thus a mixture of 10 mM potassium dihydrogen phosphate adjusted at pH (3.5) using
orthophosphoric acid and acetonitrile in a ratio of 30:70 at ambient temperature was found optimum. A
wavelength of 262 nm was found best for detection of the investigated drug in presence of its degradate.
System suitability tests were carried out according to the United States Pharmacopoeia (USP) on the
chromatogram of freshly prepared solutions of the drug and its degradation product to check various parameters.
1554
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
The parameters include standard deviation of the retention times, capacity factor, selectivity factor, resolution
and tailing factor for repetitive injections and are shown in table (5). Typically at least two of these criteria are
required to demonstrate system suitability. As shown in table (5), the method enabled good resolution factors of
adjacent peaks.
The results obtained from the system suitability tests are in agreement with the USP requirements. Peak
areas were plotted against corresponding concentrations in the concentration range of 5.0-40.0 μgml-1
The mean percentage recoveries ± SD of pure samples were found to be 100.07±0.933 which confirms that
the proposed method can be successfully applied for the analysis of the studied drug in pure powder form. The
results are summarized in table (6).
In order to demonstrate the validity and the applicability of the proposed HPLC methods, recovery tests
were carried out by analyzing synthetic mixtures of different composition ratios of leflunomide and its
degradate, satisfactory results were obtained as shown in table (7).
The utility of the proposed method was verified by the means of replicate estimations of the marketed
product and the validity of the method was further confirmed by applying the standard addition technique,
satisfactory results were obtained indicating that there is no interference from the excipients used in the formulation
of tablets as shown in table (8).
The results of the assay validation parameters including slope, intercept, correlation coefficient, standard
deviation of the slope, standard deviation of the intercept are compiled in table (9).
Fig. 6: Scanning profile of HPLC chromatogram of 20 µgml-1 of leflunomide using ultraviolet detection.
Fig. 7: Scanning profile of HPLC chromatogram of leflunomide (a) and its alkaline degradate (b), each of (10.0
µgml-1) using ultraviolet detection.
1555
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
Table 5: The system suitability test results of the developed high performance liquid chromatographic method for determination of
leflunomide in presence of its alkaline degradate.
Number of
System suitability Retention time
Capacity factor Selectivity Factor
Theoritical Plates Resolution (RS)
Symmetry
parameters
(min)
(K')
()
(N)
Leflunomide
3.33±0.026
1.985
10.623
13232
22
0.89
Table 6: Accuracy of the proposed high performance liquid chromatographic method for the analysis of pure samples of Leflunomide.
Taken (µgml-1)
Found*
Recovery %
5
5.06
101.18
10
10.03
100.27
15
14.80
98.67
20
19.83
99.14
25
25.34
101.34
30
30.01
100.02
35
35.13
100.37
40
39.81
99.53
Mean ±SD
100.07±0.933
* Average of five determinations.
Table 7: Results obtained for the analysis of laboratory prepared mixture containing different ratios of Leflunomide with its alkaline
degradate by the proposed high performance liquid chromatographic method.
Sample no.
Leflunomide (µgml-1)
Alkaline degradate (µgml-1)
Recovery* %
1
20.00
4.00
100.26
2
20.00
8.00
101.33
3
20.00
12.00
101.02
4
20.00
16.00
100.06
5
20.00
20.00
101.82
Mean±SD
100.9±0.735
* Average of five determinations.
Table 8: Quantitative determination of Leflunomide in pharmaceutical preparation and application of standard addition technique by the
proposed high performance liquid chromatographic method.
Pharamceutical
Taken
Found*%± SD
Standard addition technique
preparation
(µgml-1)
(µgml-1)
Pure added (µgml-1)
Pure found*
Recovery %
Arthfree® tablets
10.00
19.97
100.2
claimed to contain
15.00
24.96
100.07
100mg
10.00
99.50±0.274
20.00
30.02
100.35
leflunomide/tablet
25.00
35.42
101.88
Mean±SD
* Average of five determinations.
100.63±0.844
Table 9: Results of assay validation parameters obtained by applying the proposed high performance liquid chromatographic method for
determination of Leflunomide.
Parameters
HPLC method
Range
5-40 µg ml-1
Linearity
Slope
30.428
Intercept
-6.818
Correlation coefficient (r)
0.9998
Standard error of the slope
0.18696029
Confidence limit of the slope
29.97032099-30.88527168
Standard error of the intercept
4.720515929
Confidence limit of the intercept
-18.3686826-4.732690098
Standard error of estimation
6.0582058
Accuracy (mean±SD)
99.94±0.210
Selectivity
99.98±0.978
Precision (RSD%)
Repeatability
1.084
Intermediate precision
1.238
* The intra-day and inter-day relative standard deviations of the average of concentrations 5.0, 20.0, 40.0 μgml-1 for the proposed HPLC
method.
Conclusion:
In order to improve the selectivity of the determination of leflunomide in presence of its alkaline degradate
as well as in pure form and in pharmaceutical preparations, both derivative spectrophotometry from 2nd to 4th
order and HPLC were proposed. The proposed methods were found simple, inexpensine, easy, time saving, stability
indicating and suitable for quality control purposes.
1556
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
References
Abbas, S.S., L.I. Bebawy, L.A. Fattah, H.H. Raafat, 2006. Spectrophotometric stability indicating methods for
the determination of leflunomide in the presence of its degradates; Journal of AOAC International, 98(6):
1524-1531.
Abbas, S.S., L.I. Bebawy, L.A. Fattah, H.H. Refaat, 2006. Bull.; The Faculty of pharmacy, Cairo University,
44(2): 51-61.
Banker, G.S., C.T. Rhades, 2002. "Modern Pharmaceutics", 4th ed., Marcel Dekker, Inc.,
Bianco, D., R.M. Hardy, 2009. Treatment of Evans' syndrome with human intravenous immunoglobulin and
leflunomide in a diabetic dog; J.Am.Anim.Hosp.Assoc., 45: 147-150.
Chan, V., B.G. Charles, S.E. Tett, 2004. Rapid determination of active leflunomide metabolite A77 1726 in
human plasma high performance liquid chromatography; J.Chromatogr. B., 803: 331-335.
Dias, V.C., J. Lucien, D.F. Legatt & R.W. Ytschoff, 1995. Measurement of the active leflunomide metabolite
(A77 1726) by reverse-phase high-performance liquid chromatography; Ther. Drug. Monit., 17: 84-88.
Dougados, M., P. Emery, E.M. Lemmel, C.A. Zerbini, S. Brin, P. Van Riel, 2005. When a DMARD fails,
should patient switch to sulfasalazine to continuing leflunomide 2005; Annals of the Rheum. Diseases,
64(1): 44-51.
Duygu, Y., D.A. Dilek, T. Muzaffer, 2006. Determination of leflunomide in tablets by high performance liquid
chromatography; J.Pharm. Biomed. Anal., 40: 197-201.
Fairbanks, L.D., E.A. Carrey, K. Ruckemann, R. Swaminathan, B. Kirschbaum & H.A. Simmonds, 1999.
Determination of leflunomide in pharmaceutical tablets by flow- injection analysis; J.Chromatogr.,732:
487-493.
Florence, A.T., D. Attwood, 1998. "Physicochemical Principles of Pharmacy", 3rd ed., London: Mcmillan
Press.
G.Bertolilni, M., M. Aquino, C. Biffi, F. d'Atri, F. Di Pierro, P. Ferrario, F. Mascagni, A. Somenzi, F. Zaliani,
Leoni, 1997. A new rational hypothesis for the pharmacophore of the active metabolite of leflunomide, a
potent immunosuppressive drug; J. Med. Chem., 40: 2011-2016.
Henry, M.B., O.L. Charles, R.L. Wait, 2001. "Physical and Technical Pharmacy", New York, Toronto, London:
Mc Graw-Hill Book Co., Inc.,
Hong, L., Z. Ma, L. Tu, 2005. Determination of leflunomide in tablets by high performance liquid
chromatography: Yiyao Daobao, 24(6): 526-527.
Jiang, S., L. Kang, G. Yang, Z. Li, Y.Z. Wang, 2001. Determination of leflunomide and related compounds by
reversed phase high performance liquid chromatography; Diqunfenxi Huaxue, 29(1): 113.
Kher, G.J., V.R. Ram, K.L. Dubal, A.H. Bapodara and H.S. Joshi, 2011. Validation of a stability-indicating
method for assay of leflunomide in tablets and for determination of content uniformity; International
Journal of Chem. Tech. Research, 3(2): 523-530.
Miron, D.S., C. Soldatelli, E.E.S. Schapoval, 2006. HPLC with diode-array detection for determination of
leflunomide in tablets; Chromatographia, 63(5-6): 283-287.
Mohamed, M.S., N.G. Mohamed, N.M. El Kousy, 2006. Stability study of drug containing amine group by
chromatographic methods; J. Drug Research, 27(1-2): 75-81.
Molinaro, M., C. Carazzone, D. Barbano, F. Abbiati, M. Alessiani, M. Regazzi, 2005. Assessment of an LC-MS
method for plasma quantification of the new immunosuppressant FK778 through comparison with HPLCUV; Transplantation Proceedings, 37(6): 2722-2727.
Papageorgiou, C., M. Zurini, H. Weber, X. Burer, 1997. Leflunomide's bioactive metabolite has the minimal
structural requirements for the efficient inhibition of human dihydrooratate dehydrogenase; Bio Org.
Chem., 25: 233-238.
Pinto, P., M. Dougados, 2006. Leflunomide in clinical practice; Acta Rheumatologica Portuguesa, 31(3): 215224.
Rao, S.V., K.K. Sunada, N.M. Rao, A.A. Rao, I. Maheswari, G. Srinubabu, 2008. Development and validation
of LC method for the determination of leflunomide in pharmaceutical formulations using an experimental
design; African Journal of pure and applied Chemistry, 2(2): 010-017.
Rozman, B., 2002. Clinical pharmacokinetics of leflunomide; Clin.Pharmacokinet., 41: 421-430.
Schmidt, A., B. Schwind, M. Gilich, K. Brune & B. Hinz, 2003. Simultaneous determination of leflunomide and
its active metabolite, A77 1726, in human plasma by high-performance liquid chromatography; Biomed.
Chromatogr., 17: 276-281.
Shui-Wang, W., 2005. Determination of 3-methyl isomer and related substances in leflunomide by HPLC;
Yaoxue Jinzhan, 29(8): 374-377.
Sultana, N., S.A. Muhammad, M.M. Khan, M. Nawaz, 2011. Development and Validation of a Liquid
Chromatographic Method for the Determination of leflunomide: Application to in vitro Drug Metal
Interactions; Chinese Journal of Chemistry, 29(9): 1933-1938.
1557
J. Appl. Sci. Res., 8(3): 1547-1557, 2012
The Merck Index, 2001. 13th Ed., S. Budavari (Ed.). Whitehouse Station, N.J., p: 971.
Vivien, C., G. Bruce, Charles & E.T. Susan, 2004. Rapid determination of the active leflunomide metabolite
A77 1726 in human plasma by high-performance liquid chromatography.; J.Chromatogr. B., 803(2): 331335.
Weng, S., 2005. Determination of leflunomide in tablets by HPLC; Zhongguo Yaowue Zazhi, (Beijing China),
40(2): 142-144.
Yadav, B., K. Nagariya and R. Sonaje Gajanan, 2010. RP-HPLC stability indicating method development and
validation for the estimation of leflunomide and related substance in solid oral formulations; Journal of
Global Pharma Technology, 2(6): 40-45.
Yeniceli, D., D. Dogrukol-AK, M. Tuncel, 2006. Determination of leflunomide in tablets by high performance
liquid chromatography; J. Pharm. Biomed. Anal., 40(1): 197-201.
Yeniceli, D., D.A.K. Dogrukol, M. Tuncel, 2005. Determination of leflunomide in pharmaceutical tablets by
flow injection analysis, 2005; J.Liq.Chromatogr. Relat. Technol., 28: 1693-1701.
Zhang, Q.L., W.M.L. Pang, H. Chen, J. Cherrington, K. Lipson, L. Antonian and L.K. Shawver, 2002.
Application of LC/MS/MS in the quantitation of SU101 and SU0020 uptake by 3T3/PDGFr cells; J.Pharm.
Biomed. Anal., 28: 701-709.
Zhu, Q., J. Hu, H. Sun, 1999. Determination of leflunomide by HPLC; Zhongguo Yaoxue Zazhi, (Beijing),
34(9): 617-619.
Zhu, Q., J. Hu, H. Sun, H. Song, 2000. Determination of leflunomide and A 77-1726 by RP-HPLC; Yaowu
Fenki Zazhi, 20(5): 299-301.