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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
RESEARCH ARTICLE
International Journal of Pharma and Bio Sciences



A VALIDATED STABILITY INDICATING UPLC METHOD FOR MONTELUKAST
IMPURITIES IN MONTELUKAST SODIUM ORAL GRANULES

HANIMI REDDY BAPATU
A*
,MARAM RAVI KUMAR
B
, LOVLEEN KUMAR GARG
C
, DAMA VENUGOPAL
C

AND A. MALLESWARA REDDY
C

a
Department of Chemistry, J.N.T.University, Kukatpally, Hyderabad-500072, A.P, India.
b
Dr.Reddy's Laboratories Ltd. CPS, Bachupally, Hyderabad-500072, A.P, India.
c
Dr.Reddy's Laboratories Ltd. IPDO, Bachupally, Hyderabad-500072, A.P, India.


*Corresponding author





















ABSTRACT

Objectives: Montelukast sodium is a selective and orally active leukotriene receptor
antagonist. The main objective of this research is to develop a RP-UPLC method for
the determination of impurities in Montelukast sodium Oral Granules.
Methods: Chromatographic separation was achieved by using 100 x 2.1 mm, 1.7m
Acquity C18 column, 0.03 M phosphate buffer and 1% of Sodium perchlorate, pH
adjusted to 4.9 was used as buffer, mobile phase containing a gradient mixture of
solvent-A: (Buffer and Acetonitrile in 70:30 v/v ratio) and Sol-B: (Buffer and
Acetonitrile in 30:70 v/v ratio). Gradient program was 0-4min, sol-B: 40-50; 4-11min-
sol-B: 50-50; 11-22min- sol-B: 50-70; 22-25min- sol-B: 70-80; 25-28min- sol-B: 80-90,
28-38min- sol-B: 90-40 and 38-45min- sol-B: 40-40. Column temperature was
maintained at 30C, 4L injection volume and run time was 45min. Analytes
absorbance was measured at 225 nm.
Results: The developed method was validated as per ICH guidelines with respect to
specificity, limit of detection, limit of quantification, precision, linearity, accuracy,
robustness and system suitability. Validation results were found to be satisfactory and
the method applicable for bulk and formulation analysis.

HANIMI REDDY BAPATU
Department of chemistry, J.N.T.University, Kukatpally, Hyderabad-500072, A.P,
India.
ANALYTICAL CHEMISTRY


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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
KEYWORDS

Montelukast sodium, Impurities, RP-UPLC, leukotriene receptor antagonist.

INTRODUCTION

Montelukast sodium
(1-4)
is a selective
and orally active leukotriene receptor antagonist
(LTRAs). It works by blocking the action of
substances in the body that cause the
symptoms of asthma and allergic rhinitis. It is
used to prevent breathing problem, chest
tightness, wheezing and coughing caused by
asthma. Montelukast safety results reveal that
intravenous doses of montelukast sodium from
3 to 18 mg and a 10-mg oral dose are well
tolerated
(5)
. Montelukast treatment has given
best results for significant asthma control
(chronic asthma and seasonal aeroallergen
sensitivity) during the allergy season compared
with placebo
(6)
. Figure-1 represents the
chemical structure of montelukast and its
impurities.



Figure-1
Chemical structure of Montelukast and its related compounds.


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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
Montelukast have chemical and
instrumental methods.
(7-10)
But no method was
reported by using ultra peroformance liquid
chromatography (UPLC). The present research
is to develop a simple, short runtime and
stability indicating UPLC method for the
determination of montelukast and its six related
compounds. The method was developed with
low particle size (1.7 micron) and short 100 mm
column. Hence a rapid simple reproducible
stability indicating UPLC method was developed
for the quantitative determination of six
Montelukast impurities in pharmaceutical oral
dosage forms. Out of all six impurities two are
degradents MOK-3 Sulphoxide and Styrene
impurity. MOK-3 Sulphoxide is a metabolite
impurity. MOK-3 Sulphoxide is having an
isomer; so it gives two peaks in chromatogram.

MATERIALS AND METHODS

Chemicals and reagents
Pure standards were used for this study
(Montelukast and its six impurities namely
MOK-3 Sulphoxide, QUID-8, Saturated
analogue, MOK-3 Keto, MOK-1 Nitrile and
Styrene). HPLC grade acetonitrile and analytical
grade NaH
2
PO
4
, Sodium perchlorate and ortho
phosphoric acid were purchased from Merck,
Darmstadt, Germany. High purity water was
prepared by using Millipore MilliQ Plus water
purification system.

Equipment
Waters make UPLC system was used
(equiped with binary solvent manager, a sample
manager and a UV detector, Empower
software). Julabo, Seelbach, Germany make
Water bath equipped with MV controller, Sanyo,
Leicestershire, UK photo stability chamber.
MACK Pharmatech, Hyderabad, India make dry
air oven were used. Waters make Acquity C18
100 x 2.1 mm, 1.7m UPLC column was used
for this study.

Chromatographic Conditions
Column: Acquity C18 100 x 2.1 mm, 1.7m
Buffer: 0.03M phosphate buffer and 1% of
Sodium perchlorate and pH was adjusted to 4.9
with ortho phosphoric acid
Mobile phase: Sol-A: Buffer and
acetonitrile in 70:30 v/v
Sol-B: Buffer and Acetonitrile in 30:70 v/v
Gradient prog: 0-4min, sol-B: 40-50; 4-
11min- sol-B: 50-50; 11-22min- sol-B: 50-70;
22-25min- sol-B: 70-80; 25-28min- sol-B: 80-90,
28-38min- sol-B: 90-40 and 38-45min- sol-B:
40-40.
Flow rate: 0.5 mL/min
Wavelength: 225 nm.
Injection volume: 4 l.
Column temp: 30 C
Diluent : Milli-Q water : Acetonitrile 40 : 60 v/v

Standard Solutions: A stock solution of
Montelukast (1.0 mg/mL) was prepared by
dissolving appropriate amount of drug in the
diluent. Working solutions of 50.0 and 2.5
g/mL were prepared from the above stock
solution for the related substance determination.
A stock solution of impurity (mixture of MOK-3
Keto and styrene impurity) at 0.16 and 0.14
mg/mL was also prepared in the diluent. System
suitability solution Montelukast (0.5 mg/mL),
MOK-3 Keto (3.2 g/mL) and styrene impurity
(2.8 g/mL) were prepared by using the above
impurity stock.

Sample Solution: Equivalent to 24 mg
Montelukast was then transferred to a 50 mL
volumetric flask, 35 mL diluent was added,
sonicated for 20 min and diluted to volume to
give a solution containing 480g/mL. This
solution was centrifuged at 4,000 rpm for
10 min.

CALCULATIONS
100(1/F) CS / CT) (ri / rS)
Here, F is the relative response factor of the
impurity (Table-2); CS is the concentration of


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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
standard preparation; CT is the concentration of
Test preparation; ri is the peak area for any
impurity in the Test solution and rS is the peak
area for Standard solution.

RESULTS AND DISCUSSIONS

Development trials were performed with
phosphate buffer and acetonitrile as mobile
phase, C8 50mm columns were used but the
montelukast and keto-impurity were eluted at
same retention time. Finally the separation of all
impurities and montelukast was achieved with
optimized method. System suitability was
evaluated with the replicate injections of the
standard solution. Diluent and diluted standard
solution chromatograms were represented in
figure-2 and 3. Interference of the placebo was
studied and it was found that there is no
interference with all the impurities and
montelukast. Figure-4 represents the placebo
chromatogram. All known impurities were
spiked to test sample and the recovery results
of the impurities were calculated. Figure-5
represents the un-spiked test sample
chromatogram and figure-6 represents the
impurities spiked sample. Table-2 represents
the retention time and relative retention time of
montelukast and impurities.

Table-2
Retention time of Montelukast and its impurities.

COMPOUND RT RRT
a

MOK-3 Sulphoxide 5.47 & 6.13 0.26 & 0.29
QUID-8 12.01 0.56
Saturated Analogue 16.77 0.79
Montelukast 21.29 1.00
MOK-3 Keto 22.25 1.05
MOK-1 Nitrile 28.27 1.33
Styrene impurity 33.76 1.59

a
Relative retention times (RRT) were calculated against the retention time (RT) of Montelukast


Figure-2
Typical chromatogram of Blank



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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012

Figure-3
Typical chromatogram of Montelukast Standard


Figure-4
Typical chromatogram of Montelukast System suitability solution


Figure-5
Typical chromatogram of placebo


Figure-6
Typical chromatogram of test preparation spiked with impurities


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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
METHOD VALIDATION
Specificity: Specificity is the ability of the
method to measure the analyte response in the
presence of its potential impurities. Stress
studies were performed for Montelukast sodium
oral granules to provide an indication of the
stability indicating property and specificity of the
proposed method. Intentional degradation was
attempted to stress condition of UV light (200
watt hours / square meter), sun light (1.2 Million
Lux hours ), heat (70C), acid (0.1N HCl), base
(0.1N NaOH) and oxidation (0.1% H
2
O
2
) to
evaluate the ability of the proposed method to
separate Montelukast from its degradation
product. Peak purity test was carried out for the
Montelukast peak by using PDA detector in
stress samples. Degradation has been
summarized in table -3 and stress study
chromatograms are represented in figure-7 to
11.



Figure-7
Chromatogram of Acid stressed Montelukast Sodium Oral Granules 4 mg Test



Figure-8
Chromatogram of Base stressed Montelukast Sodium Oral Granules 4 mg Test



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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012

Figure-9
Chromatogram of Oxidation stressed Montelukast Sodium Oral Granules 4 mg Test


Figure-10
Chromatogram of Sun light stressed Montelukast Sodium Oral Granules 4 mg Test


Figure-11
Chromatogram of UV Light stressed Montelukast Sodium Oral Granules 4 mg Test



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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
Table-3
Stress study results.
Stress Condition
Drug product % Impurities
M
O
K
-
3

S
u
l
p
h
o
x
i
d
e

Q
U
I
D
-
8

S
a
t
u
r
a
t
e
d

a
n
a
l
o
g
u
e

M
O
K
-
3

K
e
t
o

M
O
K
-
1

N
i
t
r
i
l
e

S
t
y
r
e
n
e

i
m
p
u
r
i
t
y

Maximum
Unknown
Impurity
R
R
T

%

I
m
p
u
r
i
t
y

0.1N HCl solon for ~ 1 hour at 70C 0.32 0.02 0.00 0.00 0.0 2.88 0.35 0.54
0.1N NaOH solution for ~ 4 hours at 70C 3.38 0.02 0.00 0.00 0.0 0.06 0.12 0.05
Bench top with 0.1% H
2
O
2
for ~ 15 minutes 8.14 0.02 0.00 0.00 0.0 0.06 0.12 0.04
Water bath with purified water for ~ 10hours
/70C.
0.37 0.02 0.00 0.00 0.0 0.06 0.12 0.04
Sunlight about 1.2 Million Lux hours. 1.07 0.03 0.00 0.01 0.02 0.06 1.73 1.23
UV light both at shorter and longer
wavelengths for ~ 200 watt hours / square
meter.
1.34 0.07 0.00 0.02 0.03 0.05 0.70 1.41
Dry heating done at 70 C for ~ 12 hrs. 1.39 0.03 0.00 0.00 0.0 0.06 0.70 0.16
Humidity at 25C, 90% RH ~7 days 3.54 0.02 0.00 0.00 0.0 0.06 0.12 0.04

Precision: The % R.S.D. for impurity of MOK-3
Sulphoxide, QUID-8, Saturated analogue, MOK-
3 Keto, MOK-1 Nitrile and Styrene in related
substance method precision study was within
2%. The % R.S.D. for impurity MOK-3
Sulphoxide, QUID-8, Saturated analogue, MOK-
3 Keto, MOK-1 Nitrile and Styrene were well
within 2 %, conforming good precision of the
method. %RSD values are presented in table-4.
The precision of the related substance
method was checked by injecting six individual
preparations of Montelukast (0.48 mg/mL) spiked
with 0.50% of MOK-3 Sulphoxide, QUID-8,
Saturated analogue, MOK-3 Keto, MOK-1 Nitrile
and Styrene with respect to Montelukast analyte
concentration. %R.S.D. of % of impurity for each
MOK-3 Sulphoxide, QUID-8, Saturated
analogue, MOK-3 Keto, MOK-1 Nitrile and
Styrene was calculated. The intermediate
precision of the method was also evaluated
using different analyst and different instrument in
the same laboratory.

Table-4
Regression and Precision Data.

PARAMETER
Mok-3
Sulphoxide
QUID-8
Saturated
analogue
MOK-3 Keto
MOK-1
Nitrile
Styrene
LOD (g/ml) 0.004 0.009 0.008 0.013 0.009 0.011
LOQ (g/ml) 0.016 0.026 0.022 0.036 0.029 0.036
Slope (b) 28715.8874
30753.403
4
35476.764
8
19272.2868 19272.6941
26337.999
2
Intercept (a) 349.0255 745.8856 161.3521 540.1722 225.5690 202.5683
Correlation coefficient 0.999 0.999 0.999 0.999 0.999 0.999
Precision (%RSD) 0.3 0.7 0.5 0.5 0.4 0.4
Inter. precision(%RSD) 0.8 0.8 0.5 0.6 0.4 0.8
Precision at LOQ 6.1 3.0 3.3 2.1 3.2 2.2


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ISSN 0975-6299 Vol 3/Issue 1/Jan Mar 2012
Limits of Detection (LOD) and Quantification
(LOQ): The LOD and LOQ for MOK-3
Sulphoxide, QUID-8, Saturated analogue, MOK-
3 Keto, MOK-1 Nitrile and Styrene were
determined at a signal-to-noise ratio of 3:1 and
10:1, respectively, by injecting a series of dilute
solutions with known concentrations. Precision
study was also carried out at the LOQ level by
injecting six individual preparations of MOK-3
Sulphoxide, QUID-8, Saturated analogue, MOK-
3 Keto, MOK-1 Nitrile and Styrene and
calculating the % R.S.D. of the % of impurity.
The determination limit of detection, limit of
quantification of all the impurities namely
Montelukast, MOK-3 Sulphoxide, QUID-8,
Saturated analogue, MOK-3 Keto, MOK-1 Nitrile
and Styrene are reported in table-4. The
precision at the LOQ concentrations for
Montelukast, MOK-3 Sulphoxide, QUID-8,
Saturated analogue, MOK-3 Keto, MOK-1 Nitrile
and Styrene were below 7.5 %.
Linearity: The result shows that an excellent
correlation existed between the peak area and
concentration of the analyte. Linear calibration
plot for the related substance method was
obtained over the calibration ranges tested, i.e.
LOQ to 200 % for MOK-3 Sulphoxide, QUID-8,
Saturated analogue, MOK-3 Keto, MOK-1 Nitrile
and Styrene. The correlation coefficient obtained
was 0.999 greater than 0.997 (table 4). The
above results show that an excellent correlation
existed between the peak area and the
concentration of MOK-3 Sulphoxide, QUID-8,
Saturated analogue, MOK-3 Keto, MOK-1 Nitrile
and Styrene.
Linearity test solutions for the related
substance method were prepared by diluting
stock solutions to the required concentrations.
The solutions were prepared at six concentration
levels from LOQ to 200% of the specification
level. Correlation coefficient value for the slope
and Y-intercept of the calibration curve was
calculated.
Accuracy: The accuracy study of impurities was
carried out in triplicate at LOQ, 50%, 100%,
150%, and 200% of the target concentration
level, 0.5 % of Montelukast analyte concentration
(480g/mL). The percentages of recoveries for
impurities were calculated. The percentage
recovery of impurities in Montelukast samples
varied from 90 to 110 % at LOQ, 50%, 100%,
150%, and 200% levels of target 0.5 % level.
The LC chromatogram of spiked sample at 0.5%
level of all six impurities in Montelukast oral
solution is shown in figure 2. % Recovery values
for impurities are presented in table-5.
Table-5
Evaluation of Accuracy

% Accuracy

M
O
K
-
3

S
u
l
p
h
o
x
i
d
e

Q
U
I
D
-
8

S
a
t
u
r
a
t
e
d

a
n
a
l
o
g
u
e

M
o
k
-
3

K
e
t
o

M
o
k
-
1

N
i
t
r
i
l
e

S
t
y
r
e
n
e


LOQ
% Recovery 102.2 107.5 105.7 102.8 103.0 99.8
% RSD 3.50 1.93 5.19 2.72 3.59 4.18
50 %
% Recovery 92.0 105.9 100.7 97.8 102.4 101.8
% RSD 4.85 1.80 0.65 1.39 0.34 1.92
100 %
% Recovery 100.9 103.5 97.4 95.8 98.0 101.4
% RSD 1.18 0.97 0.93 0.71 1.33 1.25
150 %
% Recovery 105.4 103.3 100.7 101.0 98.2 100.7
% RSD 1.90 0.44 0.35 0.23 0.56 0.73
200 %
% Recovery 101.9 104.6 96.6 102.3 99.7 101.5
% RSD 0.88 0.78 0.54 0.31 1.14 1.29
% RSD values calculated with three sample recovery at each level.


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Robustness: In all the deliberate varied
chromatographic conditions (flow rate, column
temperature and composition of organic
solvent), the resolution between critical pairs,
i.e. Montelukast and MOK-3 Keto was greater
than 1.5, illustrating the robustness of the
method.
The robustness was evaluated,
experimental conditions were deliberately
altered and the resolution between Montelukast,
MOK-3 Sulphoxide, QUID-8, saturated
analogue, MOK-3 Keto, MOK-1 Nitrile and
Styrene was recorded. The flow rate of the
mobile phase was 0.5 mL/min. To study the
effect of flow rate on the resolution, flow was
changed by 0.1 units from 0.4 to 0.6 mL/min.
The effect of the column temperature on
resolution was studied at 25 and 35 C instead
of 30 C. The effect of the percent organic
strength on resolution was studied by varying
acetonitrile by 10% +10%; while other mobile
phase components was held constant as stated
in Section 2.3.

Solution Stability and Mobile Phase
Stability: The solution stability of Montelukast
and its impurities in the related substance
method was carried out by leaving spiked
sample solutions in tightly capped amber
coloured volumetric flasks at room temperature
for 48 hours. Content of MOK-3 Sulphoxide,
QUID-8, Saturated analogue, MOK-3 Keto,
MOK-1 Nitrile and Styrene were determined for
every 24 hours interval upto the study period.
The mobile phase stability was also carried out
for 48 hours by injecting the freshly prepared
sample solutions for every 24 hours interval.
Content of MOK-3 Sulphoxide, QUID-8,
Saturated analogue, MOK-3 Keto, MOK-1 Nitrile
and Styrene were checked in the test.
No significant changes were observed in
the content of impurities namely MOK-3
Sulphoxide, QUID-8, Saturated analogue, MOK-
3 Keto, MOK-1 Nitrile and Styrene during
solution stability and mobile phase stability
experiments when performed using the related
substance method. The Standard solution
stability is stable for 5 days on bench top and
Test solution is stable on bench top for 1 day.
Mobile phases used during the related
substance determination were stable for 5 days.

CONCLUSIONS

The gradient UPLC method developed
for Montelukast and related substances in
pharmaceutical dosage forms is precise,
accurate, linear, robust, rugged and specific.
Satisfactory results were obtained from
validation of the method. The method is
stability-indicating and can be used for routine
analysis of production samples and to check the
stability of samples of Montelukast oral
granules.

ACKNOWLEDGEMENT

We wish to express our sincere thanks to the
Managements of Dr. Reddys Laboratories,
Hyderabad, India for their support and
encouragement.
Cooperation from colleagues and of Research &
Development and Analytical Research &
Development of Dr.Reddys Laboratories Ltd. is
appreciated.







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