Stability indicating RP-HPLC method for development and validation of
Teneligliptin Hydrobromide Hydrate in pure and tablet dosage forms
Bhimireddy Rama Chandra 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
A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC
method was developed and validated for the simultaneous determination of Teneligliptin
hydrobromide hydrate in commercial tablets. The method has shown adequate separation for
Teneligliptin hydrobromide from its associated main impurities and their degradation products.
Separation was achieved on a Chromocil C18 column, 5μm, 250mm × 4.6 mm i.e. column using
a mobile phase consisting of Methanol: Acetonitrile in 75:25(v/v) with pH 5.3 at a flow rate of
0.9ml/min and UV detection at 212nm. The drugs are subjected to oxidation, hydrolysis,
photolysis and heat to apply stress conditions. The linearity of the proposed method was
investigated in the range of 40-140μg/ml (r = 0.998). The limit of detection was 1μg/ml and the
limit of quantification was 3.5μg/ml. Degradation products produced as a result of stress studies
did not interfere with the detection of AT and AM and the assay can thus be considered stabilityindicating.
Key words: Teneligliptin hydrobromide hydrate Hydrochlorothiazide,HPLC, Validation,
Stability, Degradation.
1. Introduction
Teneligliptin hydrobromide hydrate
is a dipeptidyl peptidase 4 (DPP4)
inhibitor is highly effective in lowering
blood glucose levels.
Figure 1: Molecular structure of Teniligliptin hydrobromide hydrate
Teneligliptin hydrobromi hydrate is a
highly potent, competitive, and long-lasting
DPP-4 inhibitor that improves postprandial
2. Experimental
2.1. Materials:
hyperglycemia and dyslipidemia(1,2,3,4). This
Teneligliptin hydrobromide hydrate
dipeptidyl
was obtained as gift sample from Micro
peptidase-4 which degrades incretin, a
Labs Ltd, Bangalore, India. Methanol,
hormone adjusting blood glucose level.
Acetonitrile and Water (HPLC grade) were
Consequently, it enhances insulin secretion
purchased from Merck Mumbai Ltd., India.
depending on blood glucose level, and
All other chemicals and reagents employed
improves blood glucose control. It is
were of analytical grade and were purchased
effectively used to treat type 2 diabetes
from S.D Fine chemicals, India.
drug
inhibits
the
enzyme
mellitus.
The
2.2
most
commonly
reported
adverse reactions include hypoglycemia,
constipation, feeling of enlarged abdomen ,
abdominal discomfort, nausea, abdominal
pain, meteorism, stomatitis, eczema, rash,
pruritus, dermatitis and malaise. Literature
survey reveals that very few HPLC methods
have been reported for the estimation of
Teneligliptin hydrobromide hydrate in pure
and tablet dosage forms.
Instrumentation
and
Chromatographic method:
The analysis of the drug was carried
out on a Peak HPLC system equipped with a
reverse phase Kromosil C18 column, Peak
pump with 20 µl injection loop and a
detector
running
on
Peak
Empower
Software. The mobile phase consists of
Methanol: Acetonitrile (75:25 v/v) and the
flow rate was maintained at 0.9ml/min. The
mobile phase was freshly prepared and
passed through nylon membrane filter of
pore size of 0.45µm and it was degassed by
hydrobromide hydrate at a concentration of
sonicating for 5 min before it was used. The
40-100µg/ml. Each of these drug solution
elution was monitored at wavelength of 212
(2µl) was injected into the column and the
nm with UV detector, and the injection
peak area and retention time were recorded.
volume was 20µl.
2.3.
2.4. Assay of Teniligliptin Hydrobromide
Determination
of
maximum
hydrate tablets:
absorbance:
The
Ten
standard
of
Teniligliptin
of
hydrobromide hydrate were weighed and
Teneligliptin hydrobromide hydrate were
average weight of a single tablet was
scanned in the range of 200-400 nm against
calculated. Tablets were crushed and mixed
mobile
using a mortar and pestle. Then drug sample
phase
as
solutions
tablets
blank.
Teneligliptin
hydrobromide hydrate showed maximum
equivalent
absorbance at 212nm. Thus the wave length
hydrobromide hydrate is accurately weighed
selected
of
and transferred into a 100ml volumetric
is
flask and mixed with known amount of
for
Teneligliptin
the
determination
hydrobromide
hydrate
212nm.
to
100mg
of
Teniligliptin
methanol and the active pharmaceutical
2.4 Preparation of stock and standard
solutions:
ingredients were extracted into the methanol
by
vortex
mixing
followed
by
ultrasonication and then filtered through a
Accurately
Teneligliptin
weighed
100mg
of
nylon membrane of pore size 0.45µm. The
hydrobromide
hydrate
is
drug sample was diluted by adding methanol
dissolved in 100 ml volumetric flask
to obtain a stock solution of 100µg/ml.
containing 70 ml of Methanol which is
considered as stock solution. Working
standard
solution
of
3. Method development
Teneligliptin
The HPLC method developed in this
hydrobromide hydrate was prepared by
study
making various dilutions of the drug
chromatographic system capable of eluting
solution from the stock solution. Six sets of
and resolving Teniligliptin hydrobromide
the drug solution were prepared in the
hydrate and its degradation products with
mobile
satisfying system suitability conditions. To
phase
containing
Teneligliptin
was
aimed
at
finding
the
develop the conditions various parameters
linearity, precision, recovery, ruggedness,
such as mobile phase, pH, flow rate and
robustness,
solvent ratio were changed and suitable
Chromatographic condition was established
chromatographic
been
such that it could be suitable for separation
developed for routine analysis of drug
of drug and it’s degradation products
samples. Initial trails were carried out by
separating impurities during elution from the
using Zodiac column taking Methanol and
chromatographic column. The proposed
Acetonitrile in various proportions with flow
method is simple, rapid and statistically
rate
of
condition
1.2ml/min.
and
LOQ.
column
was
validated for its accuracy. No interfering
phase.
The
peaks were found in the chromatograms
chromatograms obtained after injecting drug
indicating that the tablet excipients did not
samples and maintained with run time of
interfere in the analysis of drugs.
maintained
with
The
has
LOD
gradient
10min reported the poor resolution in
separation and peaks were observed broad
with thick peak heads and high retention
time. Further trails were carried out varying
the flow rate, changing the chromatographic
column, pH conditions and mobile phase
composition.
The
best
resolution
was
reported during a trail when Mobile phase
3.1. Method validation
The Proposed method was validated
according to ICH guidelines
[5]
parameters
linearity,
assessed
were
. The
precision, accuracy, stability, LOD and
LOQ.
3.2. Linearity:
was taken as Methanol and Acetonitrile in
Linearity of the proposed method
the ratio (75:25 v/v); flow rate of 0.9ml/min,
was evaluated according to the ICH
acidic pH of 5.3 in the solvent system and
guidelines by the analysis of working
sharp peak was depicted at retention time of
solutions of Teneligliptin hydrobromide
6.12min, peak was narrow, sharp and with
hydrate at different concentrations ranging
high resolution compared to other peaks
from 40-140 µg/ml. The linearity of an
obtained in different trails. Thus, these
analytical procedure is the ability to obtain
chromatographic conditions were used for
test results that are directly proportional to
studying the different properties of drug
the concentration (amount) of an analyte in
such as degradedness and also used to
the sample within a given range. Linearity
validate various method parameters like
was evaluated by linear-regression analysis.
Corresponding peak area values of different
of test results to the precision of assay is the
concentrations were determined and graph
direct measure of ruggedness of the method.
was plotted between concentration on x-axis
3.5. Robustness:
and peak area values on y-axis.
Robustness
3.2. Precision:
expresses
agreement
the
between
measurements
the
measure
of
closeness
a
obtained
series
from
small, deliberate variations in the method
of
parameters. It provides its reliability during
of
normal usage.
multiple
3.6. Limit of detection:
sampling of the same homogeneous sample
under
the
analytical method to remain unaffected by
The precision of the analytical
method
is
prescribed
conditions.
The
Limit of detection of the individual
analytical
method
is
the
lowest
precision is usually expressed in variance,
concentration of analyte in the sample that
standard deviation or coefficient of variance
the method can detect but not necessarily
of a series of measurements.
quantify
3.3. Recovery:
conditions. LOD not only depend on
The
accuracy
of
method
was
under
stated
experimental
procedure of analysis but also on type of
determined by recovery, by spiking of
instrument.
standard drug solution to pre analyzed
LOD was calculated using the formulae,
sample at three different levels i.e., at 50,
LOD=S/N where Average Baseline Noise
100, and 150%.
The resultant solutions
obtained from Blank was named as (S),
were then re-analyzed by the developed
Signal Obtained from LOD solution (0.25%
method. At each concentration, sample was
of target assay concentration) was named
injected thrice to check repeatability and
(N).
from the data it was analyzed that the
3.7. Limit of quantification:
method was accurate.
Limit
degree
quantification
of
the
individual analytical method is the lowest
3.4. Ruggedness:
The
of
of
reproducibility
concentration of analyte in the sample,
obtained by analyzing the same sample
which can be quantitatively determined with
under variety of normal test conditions such
suitable precision and accuracy under stated
as different analyst, instrument, day, reagent
experimental conditions. The quantification
columns Etc. Comparison of reproducibility
limit
is
used
particularly
for
the
determination of impurities and degraded
exposing the powdered drug form in Petri
products.
the
dish to lab light and UV light. After 48
formulae, LOQ= S/N, where S was Average
hours, the powdered drug forms were added
Baseline Noise obtained from Blank, N was
with diluents and solutions were injected
Signal Obtained from LOD solution (0.75%
into
of target assay concentration).
Chromatograms
4. Stress degradation studies
degradants and compared with initial values.
LOQ
is
calculated
by
Stress degradation was carried out on the
the
chromatographic
were
column.
evaluated
for
4.1 .Thermal degradation
drug in order to check the stability of the
In general, rate of a reaction increase
drug by providing various stress conditions
with increase in temperature. Hence, the
like light, heat, acid, base and oxidation
drugs are susceptible to degradation at
compared against blank solution stored
higher
under normal conditions. The purpose of
pharmaceutical ingredients were sensitive to
stability indicating assay method is to
heat or tropical temperatures. Thermal
provide evidence that the analytical method
degradation involves different reactions like
is
pyrolysis,
efficient
in
determination of drug
temperature.
Many
hydrolysis,
Active
decarboxylation,
substances in commercial drug product in
isomerization,
the presence of its degradation products.
polymerization. Thermal degradation study
4.1. Photolytic degradation
is carried out at 40˚C to 80˚C. The most
Photolytic degradation is carried out by
rearrangement
widely accepted temperature is 70˚C for
exposing the drug substance (in solid as well
thermal
degradation.
as in the solution form) or drug product to a
(>80˚C)
may
combination of visible and UV light. The
degradation pathway [8, 9].
most commonly accepted wavelength of
light is in the range of 300-800 nm to cause
the photolytic degradation
[6, 7]
. To study the
and
In
not
order
to
High
temperature
produce
predictive
study the
thermal
degradation, drug samples were kept in petri
dish and exposed to high temperature around
photolytic degradation of the drug sample,
40°C to 80°C in hot air oven.
Teneligliptin hydrobromide hydrate was
exposing the samples to high temperature
exposed to UV light and normal light. The
for 48 hours, solutions were prepared and
drug was dissolved in diluent at zero hour
injected into the chromatographic column.
and photolyzed sample was prepared by
After
studying
the
After
chromatograms
degradedness was evaluated and compared
sample to 20ml of 0.1 N Sodium Hydroxide
with initial values.
solution. After 48 hours, 5ml of base
4.2 .Acid hydrolysis:
Hydrolytic study under acidic and basic
condition involves catalyzation of ionisable
functional groups present in the molecule.
HCl and NaOH are employed for generating
acidic and basic stress samples, respectively
[10]
.
Acid induced degradation was carried on
drug sample by studying the chromatograms
of solutions prepared in zero hour and 48
hours. Drug sample at zero hour was
hydrolyzed sample was neutralized with 5ml
of 0.1 N Hydrochloric acid in 25ml
volumetric flask and made up to the mark
with diluents. The drug solutions prepared
earlier were injected into the HPLC column
after system suitability solution has been
injected
and
degradants
chromatograms
were
in
the
evaluated
and
compared with initial values without base
hydrolysis.
4.4. Oxidative degradation
prepared by dissolving the drug in diluent
Many
drug
substances
undergo
and for the preparation of acid hydrolyzed
autoxidation i.e., oxidation under normal
sample 300mg of drug sample was dissolved
storage condition and involving ground state
in 0.1 N Hydrochloric Acid. After 48 hours,
elemental oxygen. Therefore it is an
5ml of acid hydrolyzed sample solution was
important degradation pathway of many
transferred into 25ml volumetric flask and
drugs. Autoxidation is a free radical reaction
5ml of 0.1 N sodium hydroxide solution was
that requires free radical initiator to begin
added and made up with diluent. The zero
the chain reaction. Hydrogen peroxide,
hour and 48 hour drug samples were
metal ions, or trace level of impurities in a
injected after the system suitability solution
drug
has been injected and degradedness in the
autoxidation [11]
substance
act
as
initiators
for
chromatogram was evaluated by comparing
with initial values.
4.3. Base hydrolysis:
In
order
to
study
oxidative
degradation under hydrogen peroxide drug
To study the base hydrolysis, the
samples at zero and 48hours were prepared.
drug samples were prepared at zero and 48
The oxidized drug sample solution was
hours. The preparation of base hydrolyzed
prepared by accurately weighing 300mg of
sample involves adding 300mg of drug
drug sample and mixing it with 3% of 20ml
of hydrogen peroxide. After 48 hours, 5ml
5. Results and discussions:
of oxidized drug sample was transferred to
25ml volumetric flask and made up to the
5.1. Linearity
The calibration curve showed(Fig.1)
mark with diluents. The prepared drug
samples
were
chromatographic
injected
column
suitability solution
chromatograms
degradants
in
compared
with
after
has
been
were
the
into
the
system
injected;
evaluated
chromatograms
initial
for
and
values (without
good linearity in the range of 40-140μg/ml,
for Teneligliptin
hydrobromide hydrate
(API) with correlation coefficient (r2) of
0.998. A typical calibration curve has the
regression equation of y = 5255 + 16344.
Results are given in Table 1.
oxidation).
S.NO
Concentration in µg/ml
Peak Area
1
40
232121
2
60
320581
3
80
440369
4
100
546959
5
120
642503
6
140
753391
7
40
232121
Slope
5256
Intercept
16344
Cc
0.998
Table 1: Table showing values of concentration vs. area
800000
y = 5255.3x + 16344
R² = 0.9989
700000
600000
500000
400000
300000
200000
100000
0
0
20
40
60
80
100
120
140
160
Figure 1: Linearity results of Teniligliptin hydrobromide hydrate
on consecutive days. The % RSD was found
5.2. Precision:
to be 0.53 for intraday precision and 0.65 for
Intraday precision was carried out
using test samples prepared and analyzed on
the same day. Interday precision was
interday precision. The low % RSD values
below 2 indicate that the method is precise.
The results are given in table 2&3.
assessed by analysis of the same solutions
Intraday Precision:
S.NO
Concentration in µg/ml
Peak Area
1
80
448228
2
80
448877
3
80
444376
4
80
445691
5
80
442470
6
80
445263
RSD
0.53
Table 2: Table showing results of intraday precision
Interday Precision:
S.NO
Concentration in µg/ml
Peak Area
1
80
441290
2
80
445559
3
80
446747
4
80
446168
5
80
442985
6
80
449641
RSD
0.65
Table 3: Table showing results of interday precision
the method was accurate as % recovery
5.3. Recovery:
At each concentration, sample was
injected thrice to check repeatability and
from the RSD values it was analyzed that
values found to be in the range of 100.34 %
to 101.05 % at three different concentrations
60, 80 and 100µg/ml.
Recovery
% of
Target Conc.,
Spiked conc.,
Final Conc.,
Conc.,
Recovery
(µg/ml)
(µg/ml)
(µg/ml)
Obtained
40
20
60
60.63
101.05
40
20
60
60.12
100.20
40
20
60
60.17
100.29
40
40
80
80.03
100.04
40
40
80
81.06
101.33
40
40
80
80.02
100.03
50%
100%
% of Assay
150%
40
60
100
99.90
99.90
40
60
100
99.92
99.92
40
60
100
100.34
100.34
Table 4: Table showing results of recovery
robustness of the method. The results were
5.4. Robustness:
Small
in
in favor of (% RSD < 2%) the developed
chromatographic conditions such as change
RP-HPLC method for the analysis of
in mobile phase ratio (+ 10ml), change in
Teniligliptin hydrobromide hydrate. The
pH (±1) and detection wavelength of (±
results are given in table 5.
2nm)
were
deliberate
studied
to
changes
determine
the
Condition
Mean area
% difference
Standard
440369
…….
Mp Changes 80:20
440904
0.12
Mp Changes 85:15
441360
0.22
WL Changes 214nm
443932
0.80
WL Changes 210nm
447505
1.62
PH changes 5.1
446937
1.49
PH changes 5.5
445637
1.19
Table 5: Table showing results of Robustness
5.5. Ruggedness:
values were reported in repeated manner
The RSD value 0.71 illustrates that method
thus showing the efficiency of the method
was suitable to analyze different drugs as
and can be used to estimate various other
values were in the order of repeatability
drug samples using this method.
depicting the precision of the method.
Inspite of changing the analyst the peak area
S.NO
Concentration in µg/ml
Peak Area
1
80
444167
2
80
444577
3
80
441121
4
80
442825
5
80
447561
6
80
449863
RSD
0.71
Table 6: Table showing results of Ruggedness
5.4. Limit of Detection (LOD) and Limit
of Quantification (LOQ):
5.5. Stability indicating studies:
The LOD of was found to be ----
RP-HPLC study of samples obtained
µg/ml and the LOQ ---µg/ml estimated by
on
stress
testing
of
using the standard formulas. The low values
hydrobromide
of LOD and LOQ illustrate that the
conditions using mixture of methanol and
developed method was sensitive, accurate
acetonitrile in the ratio 75:25 (v/v) as a
and precise as it can detected and quantify
mobile
with very low concentration.
following degradation behavior.
hydrate
solvent
Condition
Report
Base
Main peak split into 2 peaks
Light
Main peak split into 4 peaks
Peroxide
Main peak split into 6 peaks
Thermal
Main peak split into 2 peaks
Acidic
Main peak split into 3 peaks
system
Teniligliptin
under
different
suggested
The chromatograms obtained on stress degradation, like photolytic degradation and similarly
other conditions were shown in figure 2, 3, 4, 5.
the
Figure 2: Base hydrolysis
Figure 4: Oxidation
Figure 3: Photolysis
Figure 4: Thermal degradation
Figure 5: Acid hydrolysis
Figure 6: Standard chromatogram of Teneligliptin hydrobromide hydrate
can be used for the routine analysis of the
6. Conclusion:
drug in pure and tablet dosage forms.
The study shows that the developed
HPLC Method is simple, precise, specific,
accurate
and
stability
indicating.
The
stability-indicating method resolved the drug
peak and also the peaks of degradation
products formed under variety of conditions.
After
exposure
of
Teneligliptin
hydrobromide hydrate to stress conditions, it
was concluded that the drug is susceptible to
photolysis,
acid
and
base
7. References:
hydrolysis,
oxidation and thermal degradation, but
peaks were unaffected inspite of degraded
products. Therefore this method can be
employed for monitoring the stability of
Teneligliptin hydrobromide hydrate and
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