Development of Rapid and Robust Stability-Indicating Method for Analysis of Alogliptin
Benzoate in Pharmaceutical Formulations Using Reverse Phase Liquid Chromatography
BRC Sekhar Reddy1, Nallagatla Vijaya Bhaskar Rao2, K. Saraswathi3
1. Department of Chemistry, Acharya Nagarjuna University, Andhra Pradesh, India.
2. Govt. Degree College, Alair, Nalgonda Dist, Andhra Pradesh, India.
3. SV University (Rete), Andhra Pradesh, India.
Abstract
This study describes the development and validation of stability indicating HPLC method
for Alogliptin an Anti-Diabetic drug in its bulk form. Alogliptin was subjected to stress
degradation under different conditions recommended by International Conference on
Harmonization. The sample so generated was used to develop a stability indicating high
performance liquid chromatographic method. The peak Alogliptin was well resolved from the
peaks of degradation products, using a kromasil-C18 (250mm, 4.6mm, 5µm) column and mobile
phase comprising of Methanol: Acetonitrile: Water in the ratio of 80:10:10(v/v) using the
isocratic method at a flow rate of 1ml/min. Detection was carried out using a UV detector at
280nm. The degradation product peak was well resolved from drug peak. The method proved to
be specific to the drug and its degradation products. The developed HPLC method was validated
with respect to linearity, accuracy, precision and robustness. All the results were found to be
within the specification limit.
Key Words: Alogliptin, Alogliptin Benzoate, Stress degradation, Assay Method, Liquid
Chromatography.
1. Introduction:
Alogliptin is an anti-diabetic drug in
[(3R)-3-aminopiperidin-1-yl]-3-methyl-2,4dioxo-3,4-dihydropyrimidin-1(2H)-
the class of DPP-4 inhibitors. It’s a
yl}methyl)benzonitrile benzoate salt. The
prescription medicine used along with diet
molecular
and exercise to improve blood sugar
C18H21N5O2 .C7H6O2The dug is having a
(glucose) control in adults with type 2
molecular weight of 461.513 g/mol
diabetes. IUPAC name of the drug is 2-({6-
formula
of
the
drug
is
[1]
.
Figure 1: Structure of Alogliptin Benzoate
Chemically, alogliptin is available in
Very few analytical methods have
the market as a benzoate salt. It is well-
been
known as Alogliptin benzoate. The drug acts
Alogliptin in bulk and pharmaceutical
as a dipeptidyl peptidase-4 inhibitor (DPP-4)
dosage
which acts against incretin hormones, which
spectrophotometer in
slows down the inactivation of the hormone
Metformine
by inhibition of GLP-1 (glucagon-like
reported method was found to be less
peptide-1)
suitable for the estimation of Alogliptin.
and GIP
(glucose-dependent
[3,4]
insulin tropic peptide)
administered
orally
and
different dosage forms.
. The drug is
available
in
Clinical trials
reported
forms
[9, 10]
for
the
using
estimation
[7,
RP-HPLC
of
8]
,
combination with
and Pioglitazone
[11]
. The
There is a need of developing a new simple
analytical method for the estimation of
Alogliptin.
Hence
a
simple
stability
analysis stated that the drug shows its action,
indicating analytical method is developed
without depending on the dietary habits and
for the estimation of Alogliptin and the
body mass index (BMI)
[5]
. Clinical trials
further confirm that alogliptin is an add-on
agent; it is taken in combination with other
efficient
anti-diabetic
drugs
such
as
metformin and pioglitazone. The alogliptin
when combined with other anti-diabetic
drugs, showed superior impact on diabetes
biomarkers [6].
results are reported here.
2. Materials and Method:
2.1. Instrumentation:
To develop a High Pressure Liquid
Chromatographic method for quantitative
estimation of Alogliptin an isocratic PEAK
HPLC
instrument
with
kromasil
C18
column (250mm x 4.6mm, 5μ) was used.
The instrument is equipped with a LC 20AT
calibration curve were prepared by proper
pump for solvent delivery and variable
dilution from the stock solution.
wavelength programmable LC – 7000 UVdetector. A 20μL Rheodyne inject port was
used for injecting the samples. Data was
analyzed by using PEAK software.
2.4. Preparation of tablet assay solution:
Preparation of tablet assay solution
was carried out by using commercially
available tablets. Average weight of 20
tablets was calculated and 20tablets of
2.2. Chemicals and Solvents:
All the solvents like Methanol,
Alogliptin (NESINA-25mg) were crushed
Acetonitrile and Water was of HPLC grade
and grinded using mortar and pestle.
and purchased from Merck Specialties,
Accurately weighed 10mg of drug sample
Mumbai, India and was used as received.
was introduced into the 10ml volumetric
The pure drug form of Alogliptin was
flask containing the solvent. From this tablet
obtained as gift sample from Chorus Labs
solution, a concentration of 80µg/ml was
Limited,
prepared. The prepared solution was injected
available
Hyderabad.
tablet
Commercially
formulation
(NESINA-
in to HPLC system, the peak area of the
25mg) was purchased from local pharmacy
obtained chromatogram was used for the
and was used for the verification of the
formulation assay estimation.
effectiveness of the developed method.
Sodium hydroxide, hydrochloric acid and
hydrogen peroxide were purchased from
Merck, Mumbai, India.
2.5. Method Development:
The HPLC method developed in this
study
was
aimed
at
finding
the
chromatographic system capable of eluting
2.3. Preparation of Standard solution:
10mg of the standard drug was
and resolving Alogliptin and its degradation
products with satisfying system suitability
weighed accurately and was dissolved in
conditions.
10ml of solvent. The stock solution of
conditions various parameters such as
concentration 1000µg/ml was obtained. The
mobile phase, pH, flow rate and solvent
solution was filtered through 0.45micron
ratio
meter nylon membrane filter paper. The
chromatographic condition was developed
solution was preserved safely and required
for routine analysis of drug samples. Initial
concentrations
trails were carried out by using kromasil
for the
construction of
were
To
develop
changed
the
and
method
suitable
C18 column taking Methanol, Acetonitrile
The accuracy of the assay method
and water in various proportions with flow
was
rate of 1.0ml/min. The chromatograms
concentration levels (50, 100 and 150%),
obtained after injecting drug samples were
and
maintained with run time of 10min, poor
calculated. The study was carried out in
separation and broad peaks were observed
triplicate at 60, 80 and 100µg/mL. The
with thick peak heads and high retention
robustness
time. Further trails were carried out varying
established by introducing small changes in
the flow rate, changing the chromatographic
the
column, pH conditions and mobile phase
wavelength (283 and 277 nm), percentage of
composition.
mobile phase and pH of the mobile phase
The method was validated for the
following parameters: linearity, precision,
accuracy, selectivity, robustness, limit of
(LOD) and system suitability [12].
method were prepared from a stock solution
at different concentration levels and 20mL
of each solution was injected into the HPLC
peak
at
three
recoveries
were
of the assay method
conditions
which
was
included
area
using
six
concentration level
replicates
of
at
a
80 mg/mL of
Alogliptin.
The LOQ and LOD were based on
the standard deviation of the response and
Linearity test solutions for the assay
the
triplicate
percentage
HPLC
studied
quantification (LOQ), limit of detection
and
the
in
(4.8 and 5.0). Robustness of the method was
2.6. Method Validation:
system
evaluated
of
the
chromatogram obtained was noted. The
intra-day precision of the assay method was
evaluated at a concentration of 80µg/mL
against a qualified reference standard. The
inter-day precision study was performed on
the slope of the constructed calibration curve
(n ¼3), as described in International
Conference on Harmonization guidelines Q2
(R1)
[12]
. The solutions extracted from the
marketed formulations were also injected
into the HPLC system and the peak area of
the chromatograms was noted. A calibration
curve was plotted by taking concentration of
the drug solution on the X-axis and the
corresponding peak area on the Y-axis.
three different days i.e. day 1, day 2 and day
The formulation analysis for the
3 at concentration of 80µg/mL. The %RSD
determination of the ability of the method
of the obtained assay values was calculated.
for the routine analysis of bulk and
pharmaceutical formulations was confirmed
thermostat the sample solution was cooled
by prepared 80µg/mL of the solution. The
and diluted accordingly with the mobile
solution was injected in to HPLC system.
phase.
Peak area of the resultant chromatogram was
used for the estimation of formulation assay.
2.7. Forced degradation studies:
For thermal stress testing, the drug
solution (1mg/mL) was heated in thermostat
at 80 oC for 30 min, cooled and used. The
The study was intended to ensure the
drug solution (1mg/mL) for photo stability
effective separation of Alogliptin and its
testing was exposed to UV light for 4h UV
degradation peaks of formulation ingredients
light chamber (254nm) and analyzed.
at the retention time of Alogliptin. Forced
degradation studies were performed to
evaluate the stability indicating properties
and specificity of the method [13].
3.0. Results and discussion:
The study was aimed to develop
a stability indicated RP-HPLC method
All solutions for use in stress studies
for the determination of Alogliptin in bulk
were prepared at an initial concentration of
and pharmaceutical dosage form. Isocratic
1mg/mL of Alogliptin and refluxed for 30
elution was simple, requires only one pump
min at 800C. All samples were then diluted
and flat baseline separation for easy and
in mobile phase to give a final concentration
reproducible results. So, it was preferred for
of 80 mg/mL and filtered before injection.
the current study over gradient elution. In
Acid decomposition was carried out
in 0.1M HCl and alkaline degradation was
conducted using 0.1M NaOH and refluxed
for 30 min at 800C.
After cooling the
solutions were neutralized and diluted with
case of RP-HPLC various columns are
available, but here kromasil C18 column
(250 X 4.6 mm, 5μ) was preferred because
using this column peak shape, separation
and absorbance was good.
mobile phase.
Solutions for oxidative stress studies
were prepared using 3% H2O2 at a
concentration of 1 mg/mL of Alogliptin and
after refluxation for 30 min at 800C on the
Mobile
preparation
of
phase
and
various
diluent
samples
for
were
finalized after studying the solubility of API
in different solvents of disposal (methanol,
acetonitrile and water). The drug was found
to
be
highly
soluble
in
Methanol,
Acetonitrile.
Using these solvents with
Alogliptin elute at a retention time of
appropriate composition newer methods can
5.80min (Figure 2). The results found to be
be developed and validated. Conditions were
specific and it shows there is no interference
optimized with a mobile phase ratio of
and co-elution of any other peaks with the
Methanol:
Water
retention of Alogliptin. The optimized
80:10:10(v/v), flow rate of 1.0ml/min, UV
conditions and system suitable parameters
detection at 280nm with a run time of
were
Acetonitrile:
showed
in
table
1
and
2.
10min. in these conditions, standard drug
Parameter
Condition
Mobile phase
Methanol: Acetonitrile: Water 80:10:10(v/v)
Pump mode
Isocratic
pH
4.9
Column
kromasil C18 column (250 X 4.6 mm, 5μ)
Column Temp
Ambient
Wavelength
280nm
Injection Volume
20μl
Flow rate
1.0ml/min
Run time
10minutes
Pump Pressure
6.0±5MPa
Table 1: Optimized chromatographic conditions for Alogliptin
Parameter
Condition
Retention Time
5.80minits
Theoretical plates
3151
Tailing Factor
0.88
Table 2: System suitable parameters for Alogliptin
The representative chromatogram
concentration range 20–120µg/mL (Table 3)
obtained for Alogliptin is shown in Fig. 1B
and the regression equation was found to be
and those of marketed formulations are
y = 7827.8x + 69895 with correlation
shown in Fig. 1D. The calibration curve
coefficient
(Figure
1.C)
was
linear
over
the
of
0.9994.
Figure 1B: Standard chromatogram of Alogliptin Standard
Concentration of
Level
Alogliptin In µg/ml
Peak area
Level – 1
20
234513
Level – 2
40
380753
Level – 3
60
535769
Level – 4
80
684894
Level – 5
100
855184
Level – 6
120
1015916
Slope:
7827.8
Range:20-120µg/ml
Intercept:
69895
2
r:
0.9994
Table 3: Linearity Graph for Alogliptin
Calibration Curve for Alogliptin
1200000
y = 7827.8x + 69895
R² = 0.9994
Peak Area
1000000
800000
600000
400000
200000
0
0
50
100
Concentration in µg/mL
Figure 1.C: Calibration curve for Alogliptin
150
The RSD in precision studies was
and robustness studies were found to be less
found to be 0.37(Intra-day), 0.48% (Inter-
than 2.0%, indicating that the method is
day) and 0.58 Ruggedness studies (Table 4).
precise, accurate and robust.
The % RSD in accuracy studies (Table 5)
Peak Area at 80µg/ml
Intraday Interday Ruggedness
Precision Precision
1
686994
685352
690456
2
682351
685959
683561
3
680313
689764
695086
4
684571
683151
689663
5
686211
680640
686011
6
682939
681745
687269
%RSD
0.37
0.48
0.58
Table 4: Precision results for Alogliptin
S. No
Recovery
50%
100%
150%
Stress
Concentration in µg/ml
Target Spiked Final Amount found % of Assay
40
20
60
59.6756
99.4593
40
20
60
59.4379
99.0632
40
20
60
59.5783
99.2971
40
40
80
79.1905
98.9882
40
40
80
79.2364
99.0455
40
40
80
79.6448
99.556
40
60
100
100.642
100.642
40
60
100
100.458
100.458
40
60
100
99.9239
99.9239
Table 5: Accuracy Studies results for Alogliptin
degradation
0.20
0.31
0.37
were
products are also observed with different
carried out to determine the behavior of the
retention times in the chromatogram. In
molecule under the stressed conditions. The
peroxide condition, the molecule breaks in
molecule was stable in all the stressed
to more than 3 compounds and they are
conditions under the study. This confirmed
identified with different retention times. The
by the retention time in all the stressed
method can be able to separate the
conditions was equal to the standard
degradation products also. Results of the
retention time of the drug. Few of the
stress degradation studies were shown in
molecules
table 6.
degrade
in
studies
RSD of Recovery
to
different
degradation products and the degradation
S. No
1
2
3
4
5
6
7
Condition
Results
3% Peroxide
Alogliptin degraded in to more compounds
0.1 N Basic
Alogliptin degraded in to two compounds
0.1 N Acidic
Alogliptin degraded in to two compounds
Sun light
Alogliptin degraded in to one compound
UV light
Alogliptin degraded in to three compounds
Aqueous (HPLC)
Alogliptin degraded in to one compound
Thermal (thermal) Alogliptin degraded in to Two compounds
Table 6: Stress degradation studies results for Alogliptin
Formulation Dosage Concentration Amount found
% Assay
NESINA
25mg
80µg/ml
79.59µg/ml
99.48
Table 7: Formulation analysis results for Alogliptin
Figure 1.D: Formulation chromatogram for Alogliptin
The LOQ was found to be 1.5µg/ml
indicating that the developed method was
and the LOD was found to be 0.5µg/ml. The
robust and can be used for the estimation of
proposed method was applied for the
Alogliptin in pharmaceutical formulations.
determination of Alogliptin tablets and the
results of these assays yielded 99.48(Table
7) and formulation chromatogram was
shown in figure 1.D. The theoretical plates
were 3151 (more than 2000) and tailing
factor was 0.88 (less than 2) for the
Alogliptin
peak.
The
value
of
assay
determined under original conditions and
robustness conditions was less than 2.0%,
4.0. Conclusion:
A rapid and robust method for the
analysis of Alogliptin was developed and its
applicability as a method for analyzing
stability was checked. The method was
found to be specific, accurate, precise, and
reproducible. Force degradation studies confirmed its ability to determine stability
because no interference from degradation
peptidase
products was observed. Moreover, no
Chem. 50 (10): 2297–2300
influence
from
excipients
was
found,
allowing it to be used in final drug product
analysis. The method was also validated in
accordance
with
ICH
and
FDA
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Med.
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