A Validated Stability Indicating LC Method for Amlexanox in Bulk Drugs

doi:10.4236/ajac.2011.25063

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American Journal of Anal yt ical Chemistry, 2011, 2, 533-538

doi:10.4236/ajac.2011.25063 Published Online September 2011 (http://www.SciRP.org/journal/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

E-mail: [email protected]

Received March 2, 2011; revised May 10, 2011; accepted July 4, 2011

Abstract

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

Amlexanox:

O N

COOH

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.

534 B. S. SUNDAR ET AL.

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

Samples

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

robustness.

B. S. SUNDAR ET AL.

535

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

experiments.

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).

B. S. SUNDAR ET AL.

536

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

12%.

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

min

USP

Resolution

(Rs)

USP

Tailing facto

(T)

Theoretical

plates

(N)

Closest eluted

degradant 4.9 - 0.99 6363

Amlexanox 5.9 2.9 1.34 4476

B. S. SUNDAR ET AL.

537

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% -

100.2%.

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

2.9

2 Flow rate

(a) At 0.8 mL·min–1

(b) At 1.2 mL·min–1 2.9

2.7

3 pH (a) At 4.7

(b) At 4.9 2.8

2.7

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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