ISSN No: 2321 8630, V 1, I 1, 2014

Journal Club for Pharmaceutical Sciences (JCPS)

Manuscript No: JCPS/RES/2014/17, Received On: 03/08/2014, Revised on: 09/08/2014, Accepted On: 13/08/2014

RESEARCH ARTICLE
Stability Indicating HPLC Method Development for Estimation of Montelukast
Sodium and Acebrophylline in Combined Dosage Form
Thesia DU1, Patel BP1
1
S. J. Thakkar College of Pharmacy, Avadh Road, Kalawad Road,
Rajkot, Gujarat
ABSTRACT
Analysis of pharmaceutical product is very important as it concerned with life. Combination
of Montelukast sodium and Acebrophylline is used in bronchial asthama and allergic
rhinitis. In this Research work, Montelukast sodium and Acebrophylline stock solution was
subjected to acid and alkali hydrolysis, oxidation, thermal photolytic and thermal
degradation. In this Stability-Indicating method sample was analyzed by reverse phase C18
column (Hibar Lichrospher 100, RP-18e 5 m, 250 mm L 4.6 mm diameter in size) as
stationary phase and Acetonitrile:Methanol (60:40 %v/v, pH 3.2 adjusted with O-phosphoric
acid) as a mobile phase at a flow rate of 0.8ml/min. Quantification was achieved at 260 nm
with PDA detector. Method was validated according to ICH Q2 R1 guideline. The retention
time for Montelukast sodium and Acebrophylline was found to be 15.49 minute and 3.45
minute, respectively. The linearity for Montelukast sodium and Acebrophylline was
obtained in the concentration range of 5-25 g/ml and 100-500 g/ml with mean accuracies
of 99.49-100.81% and 99.45-100.51% respectively. Values of %RSD for Precision Study
and Robustness was found < 2%. % label claim was found to be 99.23% for MTKT and
100.83% for ACBR. The developed method meets all the acceptance criteria for the
validation of analytical method as per the ICH Q2 R1 guideline. The degraded product peaks
were well resolved from the pure drug peak with significant difference in their retentiontime values. A simple, precise and accurate stability indicating RP-HPLC method was
developed for estimation of Montelukast sodium and Acebrophylline in combined Dosage
form.
KEYWORDS
HPLC, Montelukast sodium, Montelukast, Acebrophylline, Stability Indicating HPLC,
Degradation study, ACBR, MTKT
INTRODUCTION

[3-[(1E)-2-(7-Chloro-2-quinolinyl)

Montelukast sodium (MTKT) 1-[[[(1R)-1-

ethenyl]

*Address
for
Correspondence:
Purav Talaviya,
S. J. Thakkar College of Pharmacy,
Avadh Road, Rajkot, Gujarat.
Email ID: pvtalaviya@gmail.com

methylethyl) phenyl] propyl] thio] methyl]

phenyl]-3-[2-(1

hydroxy-1-

cyclopropaneacetic acid (Trade name:

Singulair, Montair, Emlucast, Montek,
Montelast, Monti) is a selective and orally
active leukotriene receptor antagonist that

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99

inhibits the Cysteinyl leukotriene type-1

Though

individual

estimation

of

receptor (CysLT1) and blocks the action

Montelukast sodium was done by HPLC,

of Leukotriene D4 (and secondary ligands

Voltammetric,

LTC4 and LTE4) on the Cysteinyl

methods5-19

leukotriene receptor CysLT1 in the lungs

estimation was done by HPLC, HPTLC

and bronchial tubes by binding to it. It is

spectrophotometric

used for the treatment of asthma and to

method is available for the estimation of

relieve symptoms of seasonal allergies.1-3

Montelukast sodium and Acebrophylline

Spectrophotometric
and

Acebrophylline
methods.20-24

No

in their combined dosage forms. So it is

Acebrophylline

1,2,3,6-

thought of interest to develop and validate

1,3-dimethyl-2,6-dioxo-7H-

chromatographic method for estimation of

Purine-7-aceticacid compound with trans-

these drugs in presence of degradation

4-[[(2-amino-3,5 dibromophenyl) methyl]

products in combined dosage form.

tetrahydro-

amino]

(ACBR)

cyclohexanol

(Trade

name:

cebofyl, AB phylline) is used in bronchial

asthma

and

pulmonary

diseases.

It

contains Ambroxol and Theophylline-7acetic acid, the former facilitates the

biosynthesis

of

while

raises

later

ambroxol,

by

pulmonary
blood

stimulating

surfactant
levels

of

surfactant

production. By deviating phosphatidyl

choline

towards

surfactant

In proposed method, both the drugs are

applied for forced degradation in acid,
alkaline, oxidative, photolytic and thermal
environment and these solutions were
analysed by RP-HPLC method. Stress
study was performed according to ICH
guidelines for stability testing. Method
was developed and validated according to
ICH guidelines.

synthesis,

making it no longer available for the

After systematic and detailed study, the

synthesis of inflammatory mediators such

following procedures are recommended

as the leukotrienes, acebrophylline also

for the determination of Montelukast

exerts

sodium

an

inflammatory

effect.

Acebrophylline is obtained by targeted

and

Acebrophylline

in

pharmaceutical formulations.

salification of the Ambroxol base and

Theophylline-7-acetic acid.4
This combination is launched by Alkem
Healthcare Ltd. in market as brand name
Abrofyl-M.

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MATERIALS & METHODS

Montelukast sodium working standard
grade was supplied by Torrent Research
Centre,

Ahmedabad.

Acebrophylline

100

working standard grade was supplied by

a flow rate of 0.8 mL/min was carried out.

Ami Life science, Baroda. Acetonitrile,

The detection was monitored at 260 nm

methanol, water for Chromatography -

and injection volume was 20 L. The peak

Lichrosolv (Merck Specialities Pvt. Ltd.,

purity was checked with the photodiode

Mumbai), Ortho phosphoric acid 88% GR

array detector (PDA).

(Merck Specialities Pvt. Ltd., Mumbai),

Preparation of standard solutions and

Hydrogen peroxide (30%), LR grade

calibrations

(Merck, India), Sodium hydroxide AR

Standard stock solution of ACBR (2000

grade

ltd,

g/mL) and MTKT (100 g/mL) were

Mumbai), Hydrochloric acid 35% pure

prepared in methanol. To study the

AR

Ltd.,

linearity range, serial dilutions of ACBR

Sartorius Filter Paper 0.45

and MTKT were prepared from 100-500

micron (Sartorius, Germany) and Abrofyl-

g/mL and 5-25 g/mL in methanol and

M tablet formulation-Each tablet contains

injected

10 mg Montelukast sodium and 200 mg

construction of calibration curves, five

Acebrophylline manufactured by Alkem

standard solutions in concentration range

Laboratories was purchased from local

mentioned above were prepared and

market. Identification of both the drugs

injected on to column. Calibration curves

were done by interpreting the IR spectra

were prepared as concentration of drugs

of pure API of drug sample.

versus peak area response. The system

(Merck

(Merck

Mumbai),

specialties

specialities

Pvt

Pvt.

HPLC Instrumentation and Conditions

The HPLC system consisted YL-Clarity
9100

HPLC

System

(YOUNG-LIN

INSTRUMENT, The Republic of Korea),

on

to

column.

For

the

suitability test was carried out from six

replicates of standard solution of both the
drug containing 100 g/mL of ACBR and
5 g/mL MTKT.

column heater and PDA detector (Waters

System

2998). Data collection and analysis was

Analysis of Formulations

performed using YL- Clarity software.

The SST ensures the validating power of

Separation

Hibar

the analytical method as well as confirms

Lichrospher 100, RP-18e (5 m), (250

the resolution between different peaks of

was

achieved

on
o

Suitability

Test

(SST)

and

4.6mm) columns maintained at 25 C using

interest. All critical parameters tested met

column oven. Isocratic elution with

the acceptance criteria on all days.

Methanol:Acetonitrile (40:60 %v/v), pH

Adequate resolution of ACBR and MTKT

adjusted to 3.2 with O-phosphoric acid at

peaks ensured the specificity of the

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101

method. The system suitability assessment

containing 100, 300 and 500 g/mL of

for

ACBR and 5, 15 and 25 g/mL of MTKT

the

analytical

established

HPLC

instrument

method

performance

were

used

for

precision

study.

parameters such as peak area, %RSD,

Repeatability study was determined by

Theoretical Plates (N) and Tailing factor

taking six replicates of ACBR (300

(Tf) for both the analytes. For analysis of

g/mL) and MTKT (15 g/mL). Assay

marketed formulations quantity of powder

method was evaluated with the recovery

from 20 tablets equivalent to 300 mg of

of the standards from excipients. Three

ACBR or 15 mg MTKT were weighed

different quantities (80%, 100% and

and transferred to a 100 mL volumetric

120%) of the standards were added to pre

flask containing about 20 mL of methanol,

analyzed formulation and were analyzed

ultrasonicated for 5 min and solution was

using the developed HPLC method.

filtered through Sartorius Filter paper No.

Values of Limit of Detection (LOD) and

45 into a 100 mL volumetric flask. Filter

Limit

paper was washed with the solvent and

calculated by using (standard deviation

volume was made up to mark. The

of response) and s (slope of the calibration

solution

with

curve) and by using equations, LOD =

methanol to get a concentration of 300

(3.3 x )/s and LOQ = (10 x )/s. To

g/mL of ACBR and 15g/mL of MTKT.

determine the robustness of the method,

The sample solution was then filtered and

the final experimental conditions were

20 L of the test solution was injected and

intentionally altered and the results were

chromatogram was recorded for the same

examined by changing one factor at the

and the amounts of the drugs were

time. The parameters considered (

calculated.

values) for the robustness study, flow rate

was

suitably

diluted

of

Quantitation

(LOQ)

were

( 0.1 mL/min.) and pH ( 1) were

Analytical Method Validation

studied.

The stability indicating RP-HPLC method

was validated in terms of precision,
accuracy,

specificity,

sensitivity,

robustness and linearity according to ICH

guidelines. Method precision (inter-day
and intraday) was determined using three
concentrations and three replicates of each
concentration.

Standard

solutions

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Degradation study of API and Tablet

formulations
ACBR and MTKT were subjected to
various forced degradation conditions
individually to effect partial degradation
of the drug preferably in 10-20% range.
The

forced

degradation

study

was

performed for the drug product ACBR and

102

MTKT bulk to determine whether any

exposed to same stress conditions and

observed degradation occurred because of

stress degraded samples were analysed by

drug properties or was due to drug

following above procedure described for

excipients

bulk analytes.

interactions.

Forced

degradation of the drug product was

RESULTS AND DISCUSSION

carried out under thermal, photolytic,

conditions. For photolytic stress, drug

Optimization of Chromatographic
condition
A well-defined symmetrical peak was

product in the solid state was exposed

obtained upon measuring the response of

with UV radiation. Minimum desired

eluent under the optimized conditions

exposure (200 Wh/m2) was observed after

after thorough experimental trials that can

irradiation for 24 h. Sample solution

be summarized. The mobile phase was

containing 2000 g/mL of ACBR and 100

selected on the basis of best separation,

g/mL were subjected to selected stressed

peak symmetry, theoretical plate etc. A

conditions. Samples except for photo

number of trials were taken for the

oxidation were protected from light. For

selection of mobile phase as mentioned

Acid and base degradation, solutions

here. Initially Methanol and Water were

containing ACBR and MTKT of the drug

tried in different ratios but result achieved

were prepared in 0.1N HCl and 1N NaOH

was not satisfactory. After that Methanol

analysed after 2 h exposure. Oxidative

and Acetoitrile as well as Acetonitrile and

degradation solution was prepared in

water were tried in different ratio and

water containing 3% v/v of H2O2 and

again result was found non-satisfactory.

analysed after 4 h. Sample for photolytic

Finally

studies were exposed UV radiations for 24

Acetonitrile and Methanol in the ratio of

h and were used. During dry heat study

60:40% v/v gave good separation and

samples were exposed to 800C for 24 h in

resolution. The effect of pH (adjusted with

oven and analysed. After exposure to

O-phosphoric acid) was studied initially at

desired

condition,

higher pH values and it shows baseline

samples were diluted with methanol to

disturbance and peak tailing. pH 3.2 was

achieve the nominal concentration of 300

found optimum after few of trial and

g/mL of ACBR and 15 g/mL of MTKT

errors. The effect of various flow rates on

which were based on their label strength

the formation and separation of peaks of

in

the analytes was studied and a flow rate,

acid/alkaline

and

stress

standard

containing

oxidative

degradation

solution.

ACBR and

stress

Formulation
MTKT

were

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mobile

phase

consisting

of

0.8 ml/min was optimum with reasonable

103

time of analysis. UV detector response of

The linearity range for MTKT and ACBR

ACB was studied and the best wavelength

was found to be in the range of 5-25 g/ml

was found to be 260 nm showing highest

and

sensitivity. Development studies revealed

Calibration data for MTKT and ACBR is

that methanol: acetonitrile (40:60 %v/v)

presented in table 1 and 2. Overlay

pH adjusted to 3.2 with O-phosphoric acid

Chromatogram of Std. API mixture of

at a flow rate of 0.8 ml/min was suitable

both

conditions

1.Calibration curve of both the drugs are

for

stability

indicating

100-500

the

drugs

g/ml

is

respectively.

shown

in

fig.

method. ACBR was having retention time

shown in fig. 2 and 3.

3.45 min and MTKT was having retention

System Suitability Test (SST)

time 15.49 min. degraded products of

SST results are presented in Table 3.

ACBR and MTKT were well separated.

Chromatogram for system suitability is

Analytical Method Validation

shown in fig. 4.

The method was validated according to

ICH guidelines. The following validation
characteristics were addressed: linearity,
range, accuracy, precision, sensitivity
(LOQ

and

LOD)

and

robustness.

Specificity of the method was determined

by analyzing solutions containing drug
product, excipients and stress degraded
samples.

All

chromatograms

were

examined to determine if ACBR and

Accuracy
The data for accuracy for MTKT and
ACBR are presented in table 4 and 5
respectively. The recovery range for
MTKT and ACBR were found to be
99.49-100.81%

and

99.45-100.51%

respectively.
Precision
Repeatability

MTKT and its stress degraded product

The data for repeatability of MTKT and

coeluted with each other or with any

ACBR is depicted in table 6. The %RSD

excipient peak. Peak purity of stressed

was found to be 0.511% and 0.863% for

samples of ACBR and MTKT were

MTKT and ACBR respectively.

checked by using PDA detector. The

purity angle within the purity threshold
limit obtained in all stressed samples
demonstrated

the

analyte

peak

homogeneity.
Linearity, Range, LOD and LOQ

Intraday precision
The data for intraday precision of MTKT
and ACBR are presented in table 7. Range
of %RSD was found to be 0.179-0.701%
for MTKT and 0.142-0.586% for ACBR.
Interday precision

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104

The data for interday precision of MTKT

the labelled amount of each component of

and ACBR are summarized in table 8.

tablet.

Range of %RSD was found to be 0.0770.391% for MTKT and 0.088-0.383% for

Stability Indicating Study

Analytes and its stress

ACBR.

product were well separated. Although the

Robustness

conditions used for forced degradation

The data for robustness for MTKT and

were attenuated to achieve degradation in

ACBR

the

are

presented

in

table

9.

range

1030%.

degradation

The

drug

was

Robustness of the method was evaluated

extensively degraded by acid hydrolysis,

by i) change in flow rate ii) change in pH.

alkaline hydrolysis, photolytic, Thermal

Assay of formulation
Formulation was procured commercially
from

the

market.

Formulation

was

and oxidative condition. Chromatograms

of acid, alkali, oxidative, Photolytic and
Thermal

degradation of ACBR

and

analyzed for simultaneous estimation of

MTKT in combined formulation are

MTKT and ACBR by the RP-HPLC

shown in Fig. 5 to 9, respectively. Stress

method. The assay values for MTKT and

conditions used and are presented in Table

ACBR are presented in Table 10. The

11 and 12. Chromatographic peak purity

result

data was obtained from the spectral

of

dosage

form

analysis

by

developed method was compatible with

analysis report

Figures and Tables

Fig. 1 : Overlay chromatogram of Std. API mixture of MTKT and
ACBR

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105

Fig. 2 : Calibration curve of

MTKT

Fig. 3: Calibration curve of

ACBR

Fig. 4: Chromatogram of Standard MTKT (5 g/ml) and ACBR (100 g/ml)

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106

Fig. 5 : Degradation peak of Standard API mixture of ACBR and MTKT in 0.1 M HCl
after 2 hrs.

Fig. 6 : Degradation peak of Standard API mixture of ACBR and MTKT in 1M NaOH
after 2 hrs

Fig. 7 : Degradation Peak of std. API mixture of ACBR and MTKT in 3%v/v H2O2
after 4 hrs.

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107

Fig. 8 : Degradation peak of std API mixture of ACBR and MTKT after 24 hrs of UV
exposure

Fig 9 : Degradation peak of std API mixture of ACBR and MTKT after 24 hrs. in Hot
air oven for 80oC

Table 1: Data of Linearity

Area SD

Conc. in g/ml
Sr. No.

MTKT

ACBR

MTKT

ACBR

100

1109.784 7.3970

1143.628 3.6021

10

200

2331.780 6.1427

2541.217 5.0723

15

300

3445.345 4.0549

3462.522 5.8670

20

400

4715.748 5.7981

4593.376 6.9692

25

500

6016.050 8.5052

5837.654 6.4161

Correlation co-efficient

0.999

0.999

Slope

243.9

11.64

Intercept

135.2

16.66

Regression equation

243.9x - 135.2

11.64x - 16.66

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108

Table 2: Data of LOD and LOQ

MTKT(g/ml )

ACBR(g/ml )

LOD

0.4807

1.412

LOQ

1.4570

4.281

Table 3 : Data of System suitability parameters

Sr. No.

1
2
3
4
5
Average
SD
%RSD
Retention time
Theoretical plates
Tailing Factor
Resolution

Standard Response (mV*S)

MTKT
1109.784
1118.458
1114.298
1102.259
1121.655
1113.262
1.8017
0.4871
16.49
5056
1.359

Std. Value

ACBR
1143.628
1151.912
1131.324
1147.584
1160.235
1146.935
1.1800
0.0590
3.450
10303
1.568
2.180

2%
> 2000
Not more than 2
>2

Table 4 : Accuracy data of MTKT

Conc.
Conc.
Spiked
in
added
Total
level sample
in
conc.
(%)
(g/ml) (g/ml) (g/ml)

80%

10
10
10

8
8
8

18
18
18

100%

10
10
10

10
10
10

20
20
20

120%

10
10
10

12
12
12

22
22
22

Conc.
Recovered

%
Recovery

18.17
18.12
18.09
Avg.
20.16
20.19
20.14
Avg.
21.86
21.89
21.92
Avg.

100.94
100.67
100.50
100.70
100.80
100.95
100.70
100.81
99.35
99.50
99.64
99.49

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SD

%RSD

0.2218

0.2210

0.1258

0.1248

0.1450

0.1457

109

Table 5 : Accuracy data of ACBR

Conc.
Conc.
Spiked
in
added
Total
level sample
in
conc.
(%)
(g/ml (g/ml) (g/ml)

80%

100%

120%

Conc.
Recovered

%
Recovery

200

160

360

361.89

100.52

200

160

360

361.94

100.54

200

160

360

361.77
Avg.

100.49
100.51

200

200

400

397.51

99.38

200

200

400

398.13

99.53

200

200

400

397.85
Avg.

99.46
99.45

200

240

440

442.32

100.53

200

240

440

441.98

100.45

200

240

440

441.86
Avg.

100.42
100.46

SD

%RSD

0.0251

0.0250

0.0750

0.0755

0.0568

0.0566

Table 6 : Data of Repeatability study

Peak area at 260 nm (mV*s)
Sr No.

MTKT

ACBR

3445.345

3462.522

3421.257

3445.147

3455.236

3489.371

3425.347

3411.482

3467.589

3465.548

3440.951

3476.193

Mean

3442.620

3458.377

S.D.

17.59291

27.27936

% RSD

0.511

0.863

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110

Table 7 : Data of Intraday Precision

Area
Mean % SD
(mV*s)

Conc. (g/ml)

MTKT ACBR

%RSD

Area
Mean % SD
(mV*s)

MTKT

%RSD

ACBR

100

1110.835 7.7917

0.7015

1147.62 6.7337

0.5863

15

300

3495.249 6.2657

0.1795

3505.40 5.0075

0.1426

25

500

5962.317 11.0949

0.1859

5803.62 8.9770

0.1547

Avg.

0.3014

Avg.

0.2945

Table 8 : Data of Interday Precision

Conc. (g/ml)

Area
Mean % SD
(mV*s)

MTKT ACBR

%RSD

Area
Mean % SD
(mV*s)

MTKT

%RSD

ACBR

100

1112.546 4.3711

0.3912

1145.32 4.4734

0.3891

15

300

3497.478 3.0412

0.08702

3508.12 7.0500

0.2009

25

500

5959.987 4.6282

0.07761

5801.99 5.1117

0.0880

Avg.

0.1852

Avg.

0.226

Table 9: Data for Robustness study

Robustness
parameter

Drug

Rt(min.) SD

%RSD

MTKT

15.60 0.0551

0.5314

ACBR

3.41 0.0300

0.2327

MTKT

15.59 0.0252

0.2561

ACBR

3.44 0.0550

0.3097

MTKT

15.66 0.0351

0.3349

ACBR

3.39 0.0569

0.4397

MTKT

15.64 0.0473

0.4835

ACBR

3.47 0.0153

0.1276

0.9 0.1ml/min.

Change in flow rate

1.0 0.1ml/min.

3.5

Change in pH
4

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111

Table 10 : Data for Analysis of marketed formulation

Amount taken
(mg)

Average amount
found (mg) SD

% Label claim SD

MTKT

15

14.88

99.23 1.1229

ACBR

300

302.5

100.83 0.2544

Table 11 : Data of Degradation study of MTKT

Degradation
condition

Time
(hrs)

Area

Acidic/ 0.1 M
HCl /2 hr./
Solution
Alkaline/1.0 M
NaOH/RT/
2 hr/ Solution
Peroxide/ 3%
H2O2/4 hr/
Solution
Photo/under UV
ligtht /
24 hr/Solid

3449.22

14.92

99.46

2
0

415.94
3441.88

1.79
14.90

87.40
99.33

825.884

3.57

75.75

3446.75

14.93

99.53

654.74

2.83

80.72

3439.32

14.86

99.06

24

343.89

1.49

89.39

Thermal / 80C/
24 hr/ Solid

Conc.
%
% Assay
(g/ml)
Degradation

Time
(hrs)

Area

Acidic/ 0.1 M HCl

/2 hr./ Solution

3468.54

301.23

100.41

471.64

39.16

86.35

Alkaline/1.0 M
NaOH/RT/
2 hr/ Solution
Peroxide/ 3%
H2O2/4 hr/
Solution
Photo/under UV
ligtht /
24 hr/Solid

3465.23

300.12

100.04

658.35

57.02

81.11

3470.15

300.21

100.07

728.70

63.04

78.34

3462.65

299.72

99.90

24

3482.12

29.97

88.35

3466.25

299.23

99.74

24

242.62

20.94

93.05

Thermal / 80C/
24 hr/ Solid

12.60

2.91

24.45

6.22

19.28

7.30

10.61

2.89

0
3440.02
14.88
99.20
3.60
24
123.84
0.54
96.40
Table 12 : Data of Degradation study of ACBR

Degradation
condition

Conc.
% Assay
(g/ml)

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Retention
Time

6.78

%
Degradation

Retention
Time

13.65

14.98

18.89

13.95

21.66

14.09

11.65

6.96

6.95

13.92

112

CONCLUSION
Stability-indicating RP-HPLC method for
estimation of Montelukast sodium and
Acebrophylline in their solid dosage form
was established and validated as per the
ICH guidelines. Different degradation
products were found for drug substance
and drug product in acidic, alkaline,
oxidative,

thermal

and

photolytic

degradation. Peak of degraded products

were not interfering with that of main
drug. The developed method is simple,
precise, accurate, specific, and robust.
Hence it can be used for the routine
analysis of Montelukast sodium and
Acebrophylline.

Further

method

be

could

this

helpful

present
for

the

identification of impurities through LCMS, degradation kinetic studies and

establishment of probable degradation
pathway.
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HOW TO CITE THIS ARTICLE

Thesia, D. U., Patel, B. P. (2014). Stability Indicating HPLC Method Development for Estimation
of Montelukast Sodium and Acebrophylline in Combined Dosage Form Journal Club for
Pharmaceutical Sciences (JCPS), 1(I), 99-114
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114