Download Research Paper Development of Stability Indicating Reverse Phase

572
International Journal of Pharmaceutical Sciences and Nanotechnology
International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 • Issue 2 • July - September 2009
Volume 2 • Issue 2 • July – September 2009
Research Paper
Development of Stability Indicating Reverse Phase HPLC Method for
Aripiprazole from Solid Dosage form
Pankaj Nerkar1*, Parag Gide2, Abhishek Chitnis3, Hitendra Mahajan4, Surendra Gattani5
1, 4, 5
Dept. of Pharmaceutics, R.C.Patel College of Pharmacy, Shirpur (Dhule) MS 425 405.
Mumbai Education Trusts College of Pharmacy, Nashik.
3
University of Houston, Texas.
2
ABSTRACT: Aripiprazole is a novel antipsychotic drug, generally used in schizophrenia and known to act as a partial
agonist at D2 and 5 –HT2A receptors. Since stability indicating method is required to discriminate between the intact analyte
and degradation products, the present work is aimed at performing purposeful degradation of aripiprazole by exposing it to
various pH conditions, oxidative conditions, dry heat and sunlight. Also it describes the final method of analysis of
aripiprazole from the tablets. The chromatographic analysis was carried out on HiQSil C8 column (250 X 4.6 mm, 5 µm),
mobile phase was Acetonitrile: Potassium Phosphate buffer (pH =3, 100 mM), (40:60 V/V); injection volume 100 µL,
detection was carried out at λmax 225nm. Flow rate was 1.0 ml/min. The developed stability indicating method for
aripiprazole was validated as per International Conference on Harmonization (ICH) guidelines.
KEYWORDS: Aripiprazole; Stability indicating method; Purposeful degradation; ICH
Introduction
Aripiprazole is a novel antipsychotic drug, which is
available as tablets and in solution for oral administration.
Aripiprazole exhibits high affinity for dopamine D4,
serotonin 5- HT2C, serotonin 5 – HT7, α1 – adrenergic
receptors. It has moderate affinity for cholinergic
muscarinic receptors. Aripiprazole functions as a partial
agonist at the dopamine D2 and serotonin 5- HT1 receptors
and as an antagonist at serotonin 5- HT2A receptor.
Literature survey reveals that there are two methods
reported for the estimation of aripiprazole from human
plasma and rat brain [Kubo M et al., 2005; Shimokawa Y
et al., 2055] and rat plasma but no method reported so far
for aripiprazole estimation in the presence of its
degradation products i.e. stability indicating.
The present study describes quantitative estimation of
aripiprazole in the presence of its degradation products.
This study also deals with the forced degradation of the
drug under various stress conditions like hydrolysis (acid,
base and neutral), oxidation, photolytic and thermal.
Materials and Method
Reference standard of aripiprazole was procured from M/S
Torrent Drugs and Chemicals, Ahmedabad and used
without further purification. The drug was procured as a
gift sample along with Certificate of Analysis. Chemicals
like sodium hydroxide, potassium hydroxide, potassium
dihydrogen orthophosphate, phosphoric acid, hydrogen
peroxide and solvents, like acetonitrile, and methanol were
purchased from S. D. Fine Chemicals Limited, Mumbai.
Equipment
The HPLC system (2000 series) consisted of PU-2080 plus
intelligent pump, UV 2075 plus intelligent detector and
Rheodyne type injector fitted with 100 μl capacity loop all
from JSCO Japan. The column used was HiQSil C8 (250 x
4.6 mm, 5 μm). The output signal was monitored and
processed using Borwin software.
Chromatographic Conditions [Snyder L. R. et al., 1997]
Aripiprazole (10 μg/ml) was
subjected to chromatographic analysis using mobile
phases of differing strength employing an injection volume
of 100μL, flow rate of 1mL/min, and detection was carried
at 225 nm.
Purposeful Degradation of Aripiprazole
[Singh S et al., 2000]
Aripiprazole was subjected to a variety of stress conditions
to effect degradation up to about 10 %. The decision tree
suggested by Bakshi and Singh [Snyder L. R. et al., 1997]
* For correspondence: P. Nerkar,
E-mail: [email protected]
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Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for…
was followed. The acid hydrolysis and base hydrolysis
were carried out by preparing four samples. The first was
degradation sample in which drug sample was mixed with
10 ml of 0.1 and 1 normal solution of HCl and NaOH in
RBF respectively. The same was then heated under reflux
on a heating mantle at boiling temperature of 80 0C for 2
and 4 hrs respectively. The second was zero time sample in
which drug (10 mg) was mixed with 10 ml of 0.1 and 1
normal solution of HCl and NaOH in RBF respectively and
prepared just before the analysis. The third stressed blank
sample comprising of 10 ml of 0.1 and 1 normal solution
of HCl and NaOH was heated in RBF on a heating mantle
under reflux at boiling temperature of 80 0C for 2 and 4 hrs
respectively. The fourth blank sample made of 10ml of 0.1
and 1 normal solution of HCl and NaOH respectively were
kept at normal conditions for 2 and 4hrs respectively. The
oxidative degradation, dry heat induced degradation,
photolytic degradation and neutral degradation were
carried out by preparing three samples, degradation
sample, zero time sample and blank sample as described
above. In each degradation study the variables
concentration, temperature and exposure time were
selected in such a way to effect a degradation of less than
10 %. For dry heat and photolytic degradation, methanol
was used as a blank sample. For neutral and oxidation
degradation 10 ml of water and 3 % H2O2 (V/V) was used
as blank sample. For oxidation degradation the degradation
sample made of the drug (10 mg) was kept with 3 % H2O2
(V/V) in dark condition for 8 hrs. For neutral degradation
the degradation sample was prepared by mixing drug (10
mg) with 10 ml of distilled water in RBF and heating the
RBF on heating mantle for 4 hrs.
Calibration Experiments [Kiser M. M. et al., 2004;
Ermer J. et al., 2005; Nagaraja N. V. et al., 1999]
A 100 µg/ml solution of aripiprazole was prepared by
dissolving 10 mg of aripiprazole in methanol to make 100
ml. From this, appropriate dilutions were prepared in a
mobile phase to get final concentration of 2.5-15 µg/ml.
These standard solutions were analyzed in three replicates
using the above mentioned chromatographic conditions.
Description of Final Method
Based on the results of optimization of chromatographic
conditions and calibration experiments, the final method
for analysis of aripiprazole in bulk and a tablet formulation
was described.
Method Validation [Ermer J. et al., 2005;
Nagaraja N. V. et al., 1999; (ICH) 1994; (ICH) 1996]
Accuracy and Precision
Accuracy and precision were evaluated at three levels by
determining % recovery and % relative standard deviation
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(% R.S.D.) respectively for the results of analysis of
aripiprazole from tablets containing 75, 100 and 125 % of
the labeled amount of aripiprazole.
Twenty placebo tablets were weighed accurately, and
powdered. 25, 35 and 45 mg of aripiprazole was mixed
with 400 mg of powder of placebo tablets separately in
order to get mixtures containing 75, 100 and 125 % of the
label claim. The quantity of drug of powder equivalent to
one tablet (=117 mg) was weighed separately and analyzed
by the developed method. The analysis was performed in
triplicate at each level over three days. Percent relative
error (% R.E.) was used as measure of accuracy while %
R.S.D. which was calculated to determine precision.
A t-test [Bolton S. 1990] was performed where the null
hypothesis was that the experimental mean was not
significantly different from the nominal content of
aripiprazole in the tablets. The parameter t was calculated
as
± t = (x- μ)/ standard error
where x was the experimental mean and μ was the
nominal amount of aripiprazole in the tablet.
The calculated t value was compared with the tabulated
t value at 2 degrees of freedom for α = 0.05. Calculated t
values lesser than tabulated t values indicated lack of
significant difference and thereby acceptance of null
hypothesis and reflected the accuracy of the method.
Specificity
Twenty placebo tablets were weighed accurately, triturated
and mixed. A quantity equal to an average weight was
analyzed by the described method of analysis and the
chromatograms were observed for the interfering peaks at
the retention time of aripiprazole. The blank tablets
exposed to the degradation conditions were also analyzed
using optimized chromatographic conditions and resulting
chromatograms were inspected for interfering peaks at
retention time of aripiprazole. Specificity was also
indicated by the separation of aripiprazole from its
degradation products. The method was declared specific if
there were no interfering peaks at the retention time of
aripiprazole and if aripiprazole peak was well resolved
from the peaks of all possible degradation products.
Robustness
Small changes were made in the mobile phase composition
and effects of these changes were observed on the
chromatographic behavior of aripiprazole in presence of
degradation products and excipients. The effects on
retention time, resolution and peak shape were observed.
Analysis of Marketed Formulation
To determine the content of aripiprazole in marketed
tablets (label claim 10 mg/ tablet), 20 tablets were
weighed, and average weight was calculated. Tablets were
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International Journal of Pharmaceutical Sciences and Nanotechnology
triturated and powder equivalent to average weight was
weighed. The drug was extracted from the tablet powder
with 100 ml methanol. To ensure complete extraction, it
was sonicated for 15 min. One ml of supernatant was then
diluted up to 10 ml with mobile phase. The resulting
solution was injected in HPLC and drug peak area was
noted. Regression equation, peak area of the sample and
the amount of aripiprazole in the sample was calculated.
The amount of aripiprazole per tablet was thus found.
Results and Discussion
Optimization of Mobile Phase
When aripiprazole was subjected to chromatographic
analysis using mobile phases of differing strength it was
decided to use acetonitrile: potassium phosphate buffer
(pH 3, 100 mM)(40:60 V/V), since it gave adequate
resolution and good peak shape. (Fig. 1) (Table 1)
Volume 2 • Issue 2 • July - September 2009
Purposeful Degradation of Aripiprazole
Degradation was observed for aripiprazole during stress
conditions like acid, base, neutral, dry heat and oxidation
hydrolysis. (Fig. 2-6) The peak area of aripiprazole in
stressed sample when compared with the corresponding
peak of aripiprazole in zero hour sample revealed a
decrease in the area. Also there were no additional peaks at
the same retention time in zero sample as well as in blank
sample and in the room temperature sample, indicating that
the additional peaks were degradation products.
Degradation study of the photolytic stressed sample
also revealed the photostability of aripiprazole as there was
no change between the peak area of stressed sample and
the zero hour sample. (Fig .7) (Table 2).
Fig. 1 Representative chromatogram of aripiprazole in Acetonitrile: Potassium phosphate buffer (pH 3, 100
mM) (40:60 V/V). R.T. 9.28.
Table 1 The chromatographic behavior of aripiprazole in different mobile phases.
Mobile phase
Retention time (min)
Acetonitrile: Water (40:60 V/V)
21.1
Methanol: Potassium phosphate buffer (pH 3, 100 mM)(60:40 V/V)
7.7
Acetonitrile: Buffer (pH 2.4, 100M, potassium phosphate)(40:60 V/V)
5.8
Acetonitrile: Buffer (pH 3, 100 mM, potassium phosphate)(40:60 V/V)
9.2
Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for…
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Fig. 2 Representative chromatogram of acid treated aripiprazole (0.1 N HCl, refluxed for 2 hrs.)
Peak 1: Degradant one R. T. 2.85; Peak 2: Degradant two R.T. 3.80; Peak 3 Degradant three R.T. 5.42;
Peak 4: aripiprazole R.T. 9.3.
Fig. 3 Representative chromatogram showing base treated aripiprazole (1 N NaOH, refluxed for 4 Hrs.)
Peak 1: Degradant one, R.T. 3.83 min; Peak 2: Degradant two, R.T. 5.56 min; Peak 3: Degradant R.T. 7.08 min;
Peak 4: aripiprazole, R.T. 9.11min.
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International Journal of Pharmaceutical Sciences and Nanotechnology
Volume 2 • Issue 2 • July - September 2009
Fig 4 Representative chromatogram showing water treated aripiprazole. Peak 1 Degradant 1, R. T. 4.34;
Peak 2: Degradant 2, R.T. 5.38, Peak 3: aripiprazole R.T. 9.60; Peak 4: Degradant 3 R.T. 15.06
(water reflux for 4 hrs.).
Fig 5 Representative chromatogram of Oxidative degradation study of aripiprazole. Peak 1: Hydrogen
peroxide, R.T. 2.46; Peak 2: Aripiprazole R.T. 9.15; Peak 3: Degradant 1, R.T. 10.17(3% H2O2, for 8 hrs.).
Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for…
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Fig. 6 Representative chromatogram of dry heat degradation of aripiprazole. (800 C for 8 hrs) Peak 1:
Degradant 1, R.T. 7.34; Peak 2: aripiprazole, R.T. 9.08.
Fig 7 Representative chromatogram of degradation in photolytic condition of aripiprazole. (48 hrs in sunlight)
Peak 1: aripiprazole R.T. 9.28.
Table 2 Summary of degradation products of aripiprazole.
Conditions
Time (in Hrs)
Drug peak area
at zero Hrs
Drug peak area of
stressed sample
Retention time of
degradant (mins)
% Degradation
1 N HCl
2
4439205
4048554
2.8,3.8& 5.4
8.8
1 N NaOH
4
4488017
4039215
3.8,5.5&7.0
10
Water
4
4436390
4125842
4.3,5.3& 15.0
7
H2O2 , 3 %
12
4390908
3853633
10.1
5.8
Dry Heat 80 0C
4
4500788
4266747
7.3
5.2
Sun light
48
4475436
4470689
No Degradant
No Degradation
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International Journal of Pharmaceutical Sciences and Nanotechnology
Calibration Experiments
The data obtained in the calibration experiments when
subjected to linear-regression analysis showed a linear
relationship between peak areas and concentrations in the
range of 2.5 - 15 μg /ml (Fig. 8). A plot of residual
Volume 2 • Issue 2 • July - September 2009
obtained by plotting the difference between back
calculated areas and experimental areas against
concentration, showed a random scatter and the variance of
the peak areas was homogenous over the linear range
(Fig. 9) (Table 3).
8000000
y = 471996x + 11670
R2 = 0.9997
7000000
6000000
5000000
4000000
3000000
2000000
1000000
0
0
2
4
6
8
10
12
14
16
Fig 8 Calibration curve for aripiprazole.
2
1.5
1
0.5
0
0
2
4
6
8
10
12
14
16
-0.5
-1
-1.5
Fig 9 Residuals plot for aripiprazole.
Table 3 Linearity data for Aripiprazole.
Calibration
standard
Nominal
Concentration μg/ml
1
2
1
2.5
1192502
1184512
2
5
2385715
3
7.5
4
10
5
6
Equation
2
R
3
Mean peak area
(μV.sec)
Standard deviation
of peak area
1196216
1191077
5980.768
2346754
2356619
2363029
20256.09
3564122
3584134
3541082
3563113
21543.74
4749782
4788219
4730301
4756101
29471.48
12.5
5902146
5942557
5983318
5942674
40586.13
15
7199597
7081005
7042288
7107630
81964.62
Y = 471996X + 11670
0.9997
Peak Area of replicate (μV.sec)
Pankaj Nerkar et al. : Development of Stability Indicating Reverse Phase HPLC Method for…
Description of Final Method
Twenty tablets of aripiprazole (label claim 10 mg) were
weighed and powdered. Powder was mixed thoroughly and
quantity equivalent to 10 mg of aripiprazole was weighed
and mixed with 80 ml of methanol. The mixture was
sonicated for 10 min. and volume was made up to 100 ml
with methanol. 1 ml of the resulting solution was diluted to
10 ml with the mobile phase [i.e. Acetonitrile: Potassium
phosphate buffer (pH 3, 100 mM) (40:60 V/V)]. This final
dilution was then chromatographed at a detection
wavelength of 225 nm; the column used was HiQSil C8
column (250 X 4.6 mm, 5 µm); flow rate was 1 mL/min;
injection volume was 100 µL. The peak area was referred
to calibration equation y = 471996x + 11670 to get
concentration of aripiprazole from sample and thus the
content in tablets.
aripiprazole was well resolved from its degradation peaks,
indicating the specificity of the method.
Accuracy and Precision
The results of intra and inter-day variation of aripiprazole
at three different concentrations levels (75 %, 100 %, and
125 % of label claim) are depicted in (Table 4). The data
indicates that the maximum %relative error at 75%, 100%
and 125% was 2.89, 2.7 and -1.79 respectively while the
maximum % relative standard deviation was 2.25, 2.9 and
2.29 respectively indicating that the method has acceptable
accuracy and precision. Also, the calculated t-values were
lesser than the tabulated t-value of 4.3 for α = 0.05 at two
degrees of freedom. This indicated that the experimental
values were not significantly different from the nominal
which reflected the accuracy of the method.
Further, when the data given in Table 4 was subjected
to one way ANOVA to get estimates of within and
between day variability, the following observations were
made
Method Validation
Specificity
The HPLC chromatograms recorded for the matrix (blank
tablets exposed to the degradation conditions) showed no
peaks at the retention time of aripiprazole and also the
representative chromatograms of degradation samples in
various stress conditions (Figures 2-7) showed that
The calculated F value was lesser than the tabulated F2,6
(α = 0.01) of 10.92 indicating that the inter day variability
was not significantly different from the intra day
variability at 1% level of significance(Table 5).
Table 4 Results of accuracy and precision studies for Aripiprazole.
Day
1
2
3
579
Amount added
(in mg)
Amount found (in mg)
% relative
error
% RSD
Calculated tvalues
1
2
3
Mean
7.5
7.21
7.25
7.52
7.32
-2.31
2.25
-1.78
10
9.68
9.56
10.1
9.78
-2.2
2.9
-1.34
12.5
12.91
12.54
12.34
12.59
0.77
2.29
0.58
7.5
7.73
7.58
7.84
7.72
2.89
1.69
2.88
10
10.26
10.44
10.11
10.27
2.7
1.61
2.83
12.5
12.25
12.39
12.19
12.28
-1.79
0.84
-3.77
7.5
7.33
7.49
7.21
7.34
-2.1
1.91
-1.93
10
9.67
9.89
10.02
9.86
-1.4
1.79
-1.37
12.5
12.62
12.75
12.51
12.63
1.01
0.95
1.83
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International Journal of Pharmaceutical Sciences and Nanotechnology
Table 5 Lable claim at 75, 100 and 125 %.
Source
Degrees of freedom
SS
MS
F
At 75 % Lable claim.
Between Days
2
0.28
Within Days
6
0.12
Total
8
0.40
0.14
7
0.02
At 100% Lable claim
Between Days
2
0.414
Within Days
6
0.278
Total
8
0.692
0.207
4.5
0.046
At 125 % Lable claim
Between Days
2
0.226
Within Days
6
0.217
Total
8
0.443
0.113
3.1
0.036
Robustness
There was no significant change in the retention time of
aripiprazole and its degradation products after introducing
small changes in mobile phase composition indicating
robustness of the method.
Linearity and Range
These parameters were
experiments (Table 3).
From the purposeful degradation study it is clear that the
molecule is quite stable to sun light and it shows
degradation in acidic, basic, oxidative, dry heat and neutral
hydrolysis.
Acknowledgement
evaluated
in
calibration
Analysis of Marketed Formulation
The chromatograms of the drug samples extracted from
tablets did not show a change in the retention time. There
was no interference from the excipients, which are
commonly present in the tablets. The drug content was
found to be 102.66 % with a % RSD of 2.46 as shown in
(Table 6) Therefore it may be concluded that, degradation
of aripiprazole had not occurred in the marketed
formulations. The % low RSD value indicated the
suitability of the method for the routine analysis of
aripiprazole in pharmaceutical formulation.
Conclusion
Stability indicating reversed phase high performance liquid
chromatography method for aripiprazole is developed and
validated as per ICH guidelines. The analysis of
aripiprazole in the presence of degradants is possible now.
This method can also be used for further impurity profiling
of the drug. As the retention time of aripiprazole is 9.2
min, it can be used for the determination of aripiprazole
from the tablets too.
We are thankful to the Principal, Vilasrao Kadam, Bharati
Vidyapeeth’s College of Pharmacy, Navi Mumbai, for the
facilities.
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