American Journal of Anal yt ical Chemistry, 2011, 2, 533-538
doi:10.4236/ajac.2011.25063 Published Online September 2011 (
Copyright © 2011 SciRes. AJAC
A Validated Stability Indicating LC Method for
Amlexanox in Bulk Drugs
Bethanabhatla Syama Sundar*, Mohammed Nazeerunnisa
1Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, India
Received March 2, 2011; revised May 10, 2011; accepted July 4, 2011
A novel and sensitive stability indicating RP-HPLC method has been developed for the quantitative deter-
mination of amlexanox in bulk drugs. The separation was accomplished on C18 column using 10 mM ammo-
nium dihydrogen orthophosphate (pH adjusted to 4.8 by using ortho phosphoric acid) and methanol (30:70
v/v) as mobile phase in an isocratic elution mode at a flow rate of 1.0 mL min-1. The eluents were monitored
by PDA detector at 245 nm. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis
and thermal degradation. Significant degradation was found under basic, acidic stress and UV light. The
resolution (Rs) between amlexanox and its degradation products was found to be greater than 2.5. Regression
analysis shows correlation coefficient greater than 0.999 for amlexanox. The inter and intraday precision
values for amlexanox were found to be within 1.0% RSD. The method has shown good and consistent re-
coveries for amlexanox in bulk drugs (98.86% - 101.05%). The developed method was validated with re-
spect to linearity, accuracy, precision and robustness.
Keywords: RP-HPLC, Amlexanox, Degradation, Validation, Stability indicating
1. Introduction
Amlexanox is a novel anti-inflammatory and anti-aller-
gic agent that has been evaluated for the treatment of
recurrent aphthous ulcers (RAU) and is currently the
only clinically proven product approved by the US FDA
for the treatment of aphthous ulcers. The chemical name
of amlexanox is 2-acid amino-7-isopropyl-5-oxo-5H-
chromeno [2,3-b] pyridine-3-carboxylic acid (Figure 1).
RAU is the most prevalent oral mucosal disease in hu-
mans. Prior to amlexanox available treatment was largely
symptomatic, with patient management being either en-
tirely empiric or based on clinicians’ perception of the
cause of the ulcers. Amlexanox effectively treats aphth-
ous ulcers by accelerating healing of ulcer and by accel-
erating complete resolution of pain. Amlexanox is com-
mercially available as 5% oral paste and as biodegrad-
able muco-adhesive disc. Amlexanox potently inhibits
the release of histamine and leukotrienes from mast cells,
basophils and neutrophils under invitro settings, possibly
through increasing intracellular cyclic AMP content in
inflammatory cells, a membrane-stabilizing effect or
inhibition of calcium influx [1-5].
A few HPLC methods were reported in the literature
for the analysis of amlexanox which includes like va lida-
tion of HPLC-FL assay for the determination of amlexa-
nox in human serum [6], RP-HPLC method for three
distinct anti-allergic drugs to bind the proteins: Amlexa-
nox, cromolyn and tranilast [7]. Study on pharmacoki-
netics and demonstratio n of clinical safety for amlexanox
5% oral paste [8,9], stable viscous liquid formulations of
amlexanox for the prevention and treatment of mucosal
diseases and disorders [10].
Extensive literature survey reveals that there is no sta-
bility-indicating LC method for determination and for the
quantitative estimation of amlexanox in bulk drugs. An
ideal stability indicating chromatographic method for
estimation of any drug should be able to resolve from
IUPAC Name: 2- acid amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]
pyridine-3-carboxylic acid
Molecular Formula: C16H14N2O4
Molecular weight: 298g/mol
Figure 1. Chemical Structures and labels of Amlexanox.
degradation products. The present drug stability test
guideline Q1A (R2) issued by International Conference
on Harmonization (ICH) suggests that stress studies
should be carried out on a drug to establish its inherent
stability characteristics, leading to the separation of deg-
radation products and hence supporting the suitability of
the proposed analytical procedures. It also requires being
stability indicating besides validated fully. [11-1 3].
Hence, an attempt has been made to develop an accu-
rate, rapid, specific and reproducible method for the de-
termination of amlexanox in bulk drug samples along
with method validation as per ICH norms. The stability
tests were also performed on both drug substances as per
ICH norms.
2. Experimental
2.1. Chemicals
Amlexanox and its standard were obtained from Star Lab
Tech Pharmaceuticals, Hyderabad, India. HPLC grade
methanol, ortho phosphoric acid and analytical reagent
grade ammonium dihydrogen phosphate were of Merck,
Darmstadt, Germany. High purity water was prepared by
using Millipore Milli-Q plus water purification system.
All samples and impurity used in this study were of
greater than 99.6 % purity.
2.2. Equipment
The LC system used for method development, forced
degradation studies and method validation were Waters
2695 binary pump plus auto sampler and a 2996 photo
diode array detector (Waters Corporation, MA, USA).
The output signal was monitored and processed using
Empower software on Pentium computer (Digital equip-
ment Co) and Agilent 1200 series DAD (diode array de-
tector) with Empower soft ware. Photo stability studies
were carried out in a photo stability chamber (MACK
Pharmatech). Thermal stability studies were performed
in a dry air oven (Mack Pharmatech, Hyderabad, India).
2.3. Chromatography
The chromatographic column used was Intersil ODS-4,
(4.6 × 250) mm; 5 µm. The mobile phase consists of a
mixture of buffer and methanol in the ratio of 30:70 v/v.
Buffer consists of 10 mM ammonium dihydrogen ortho-
phosphate, pH adjusted to 4.8 using ortho phosphoric
acid. The column temperature was maintained at 25˚C
and the detection was monitored at a wavelength of 245
nm. The injection volume was 10 µL. Mobile phase be-
ing used as diluent.
2.4. Preparation of Standard Solutions
About 12 mg portion of amlexanox standard was weighed
into standard 100 ml volumetric flask, dissolved in and
diluted to volume with diluent. 5 ml of above solution
was transferred into a 10 ml volumetric flask and diluted
to volume with diluent.
2.5. Preparation of Stress/System Suitability
About 50 mg of amlexanox sample was taken into a 50
mL volumetric flask and made up to the mark with 0.1 N
sodium hydroxide and refluxed at 100˚C for 1 hr. An
aliquot of 0.6 mL was pipette out into a 10 mL volumet-
ric flask, neutralized with 1N HCl and made up to the
volume with mobile phase.
2.6. Specificity/ Application of Stress (Forced
Degradation Study)
Specificity is the ability of the method to measure the
analyte response in the presence of its potential impuri-
ties [14] which can help to iden tify the lik ely d egradatio n
products and establish its pathways and the intrinsic sta-
bility of the molecule an d validate the stability ind icating
power of the analytical procedures used
The specificity of the developed LC method for am-
lexanox was determined in the presence of its degrada-
tion products. Forced degradation studies were also per-
formed on amlexanox to understand th e stability indicat-
ing property an d specificity of the proposed method. The
stress conditions employed for degradation study in-
cludes light (carried out as per ICH Q1B), heat (105˚C
for 48 hrs), acid hydrolysis (1N HCl), base hydrolysis
(0.1N NaOH), water hydrolysis and oxidation (5% H2O2).
Amlexanox is exposed to 200 w/hm2 UV light in solution
and solid states. Amlexanox is exposed to 1.2 million
flux hours fluorescent light in solution and solid state.
Amlexanox solution is exposed to ultrasonic bath for 1 hr
at 25˚C. Peak purity of stressed samples of amlexanox
was checked by using 2996 Photo diode array detector of
Waters (PDA).
Assays were carried out for the stress samples against
a qualified reference standard. The mass balance (% as-
say + % of impurities + % of degradation products) was
calculated for all of the samples.
2.7. Analytical Method Validation
The developed chromatographic method was validated
for linearity, range, precision, accuracy, sensitivity, and
Copyright © 2011 SciRes. AJAC
2.7.1. Precision
The precision of the amlexanox method was checked by
injecting six individual preparations of (60 µg mL–1) in
triplicate (intraday) on the same day. The %RSD area of
amlexanox was calculated. Precision study was also de-
termined by performing the same procedures on three
different days (inter-day precision).
The intermediate precision (ruggedness) of the method
was also evaluated by different experimenter, different
column and different instrument in the same laboratory.
Assay method precision was evaluated by carrying out
six independent determinations of test sample of amlex-
anox against qualified reference standard. The %RSD
values of six determinations obtained were calculated.
The intermediate precision of the assay method was
evaluated by different experimenter and by using differ-
ent instrument from the same laboratory.
2.7.2. Sensitivity
Sensitivity was determined by establishing the Limit of
detection (LOD) and Limit of quantitation (LOQ) for
amlexanox estimated at a sign al-to-noise ratio of 3:1 and
10:1 respectively by injecting a series of dilute solutions
with known concentration. The precision study was also
carried out at the LOQ level by injecting six individual
preparations of amlexanox and the values of %RSD cal-
culated for the areas of the amlexanox.
2.7.3. Linearity and Range
To establish linearity of the assay method, calibration
solutions were prepared from stock solution at five con-
centration levels from 80% to 120% of assay analyte
concentrations (48, 54, 60, 66 and 72 µg mL–1). Linearity
was checked for three consecutive days in the same con-
centration range. Upper and lower levels of range were
also established.
2.7.4. Accu racy
The accuracy of the assay method was evaluated in trip-
licate at three concentration levels, i.e. 48, 60 and 72 µg
mL–1 in bulk drugs. For each concentration, three sets
were prepared and injected in triplicate. The percentage
of recovery was calculated at each level.
2.7.5. Robustness
To determine the robustness of the developed method,
experimental conditions were deliberately changed and
the resolution (Rs) between amlexanox and its degradant
in basic condition was evaluated. The effect of flow rate
on the resolution was studied with 0.8 and 1.2 mL·min–1
while the optimized flow rate of the mobile phase was
1.0 mL·min–1. The effect of column temperature on
resolution was studied at 20˚C and 30˚C instead of 25˚C.
The effect of pH on resolution of impurity was studied
by varying ± 0.1 pH units (i.e. buffer pH altered from 4.8
to 4.7 and 4.9). In the all above varied conditions, the
components of the mobile phase were held constant.
2.7.6. Solution Stability and Mobile Phase Stability
The solution stability of amlexanox in the assay method
was carried out by leaving the test solutions of samples
in tightly capped volumetric flasks at room temperature
for 48 hrs. The same sample solutions were assayed at 0
hrs, 18 hr s, 24 hrs, 42 hrs and 48 hrs against freshly pre-
pared standard solutions. The mobile phase stability was
also carried out by determining the freshly prepared
sample against freshly prepared reference standard solu-
tions at 0 hrs, 18 hrs, 24 hrs, 42 hrs and 48 hrs. The
%RSD of assay of amlexanox was calculated for the
study period during mobile phase and solution stability
3. Results and Discussion
3.1. Method Development and Optimization
The objective of the present work was to develop a sta-
bility-indicating liquid chromatographic analytical method
for the determination of amlexanox in bulk drugs. Am-
lexanox standard was used during the method develop-
ment. To develop a rugged and suitable LC method for
the amlexanox, different mobile phases and stationary
phases were employed. Preliminary trial was carried on
mobile phase containing 10 mM potassium dihydrogen
phosphate monohydrate, pH adjusted to 4.0 with phos-
phoric acid and methanol (50:50, v/v) was chosen on a
C18 stationary phase with a 25 cm length, 4.6 mm ID
and 5 µ particle size and retention time found to be high
and peak is not in good shape. The propo rtion of the mo-
bile phase compon ents was optimized to reduce retention
times and enable good resolution of amlexanox from the
degradation products obtained by base degradation.
When pH increased towards basic side (pH 4.8) the re-
tention time and the resolution between the degradants
and amlexanox was improved. To further reduce the re-
tention time, the methanol proportion was increased and
the observed retention time of Amlexanox was found to
be about 5.9 min.
Under optimized concentration of 10mM Potassium
dihydrogen phosphate monohydrate, pH adjusted to 4.8
with phosphoric acid and methanol (30:70 v/v) as mobile
phase, the typical retention times of degradants in basic
condition an d amlexanox were found to be abou t 4.2, 4.9,
7.2 for degradants and 5.9 for amlexanox respectively
(Figure 2).
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Copyright © 2011 SciRes. AJAC
Figure 2. Typical chromatograms of (a) Amlexanox sample, (b) acid stress, (c) base stress and (d) UV e x pose d str e ss sample s.
Buffer pH and percentage of methanol played a key
role in achieving the good separation between the de-
gradants and amlexanox besides enhancing chroma-
tographic efficiency. The system suitability results were
given in [Table 1].
3.2. Results of Forced Degradation Studies
The drug was exposed to 0.1 N methanolic NaOH re-
fluxed at 100˚C temperature for 1 hr. Amlexanox has
shown significant sensitivity towards the treatment with
0.1 N NaOH leading to observed degradation of about
The drug was exposed to 1N methano lic HCl refluxed
at 60˚C for 3 hrs caused significant degradation (8.3%).
UV Light exposed solution caused significant degrada-
tion (6.58% de graded at UV 200 W/hm2).
No major degradation products were observed when
the sample was stressed in an oxidative condition (5%
methanolic H2O2, heated at 60˚C for 7hrs), neutral, fluo-
rescent and thermal conditions.
From the degradation studies, Peak purity test results
derived from PDA detector, confirmed that the amlexa-
nox peak was homogeneous and pure in all the analyzed
stress samples. The mass balance of stressed samples
was close to 99 .97%. No degradants wer e observed after
45 minutes in the extended runtime of 90 minutes of all
the amlexanox samples. The developed LC method was
found to be specific in the presence of its degradation
products confirm the stability indicating power of the
developed method.
3.3. Method Validation
3.3.1. Precision
The %RSD of amlexanox during precision study and
intermediate precision study was 0.1% and confirming
the good precision of the developed analytical method.
3.3.2. Linearity and Range
Linear calibration plot for assay method was obtained
over the calibration ranges tested, i.e. 48-72 µg mL–1 and
the correlation coefficient obtained was greater than
0.999. The result shows an excellent correlation existed
between the peak area and concentration of the analyte.
Table 1. System suitability data.
Name Retention
time (tr) in
Tailing facto
Closest eluted
degradant 4.9 - 0.99 6363
Amlexanox 5.9 2.9 1.34 4476
The best-fit linear equation obtained was Y = 36422x
+ 338053. At all concentration leve ls, standard deviation
of peak area was significantly low and RSD was below
1.0%. Analysis of residuals indicated that residuals were
scattered within ± 2.0% with respect to 100% concentra-
tion response.
3.3.3. Accu racy
The percentage recovery of amlexanox in bulk drug
samples ranged from 98.86% - 101.05% [Table 2].
3.3.4. Robustness
Close observation of analysis results for deliberately
changed chromatographic conditions (flow rate, pH and
column temperature) revealed that the resolution be-
tween closely eluting degradant in basic condition and
amlexanox was always greater than 2.5, illustrating the
robustness of the method [Table 3].
3.3.5. Solution Stability and Mobile Phase Stability
The %RSD of assay of amlexanox during solution stabil-
ity and mobile phase stability experiments was within
1.0% RSD. No significant changes were observed in the
content of amlexanox during solution stability and mo-
bile phase stability experiments. The solution stability
and mobile phase stability experiments data confirms
that sample solutions and mobile phase used during as-
say and related substance determination were stable up to
the study.
3.3.6. Ass ay Analysis
Analysis was performed for different batches of amlex-
anox in bulk drug samples (n = 3) ranged from 99.5% -
Table 2. Results of Accuracy study for Bulk drugs.
Added (g)
(n = 3) %Recovery for
Bulk drugs %RSD for
Bulk drugs
48 101.05 0.23
60 100.27 0.39
72 98.86 0.04
n =3, Number of determinations
Table 3. Results of robustness study.
S. No Parameter Variation Resolution (Rs)
between base degradant
and Amlexanox
1 Temperature (a) At 20˚C
(b) At 30˚C 2.8
2 Flow rate
(a) At 0.8 mL·min–1
(b) At 1.2 mL·min–1 2.9
3 pH (a) At 4.7
(b) At 4.9 2.8
4. Conclusions
The Stability Indicating RP-LC method developed for
quantitative determinatio n of amlexanox in bu lk drugs is
precise, accurate and specific. The method was com-
pletely validated showing satisfactory data for all the
method validation parameters tested. The developed
method is stability indicating and can be used for the
routine analysis of production samples and also to check
the stability of amlexanox sample.
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