ACADEMIA ROMÂNĂ Rev. Roum. Chim.

,
Revue Roumaine de Chimie 2017, 62(2), 173-179

http://web.icf.ro/rrch/

COMPARATIVE STUDY FOR DIRECT EVALUATION

OF MONTELUKAST SODIUM IN TABLET DOSAGE
FORM BY MULTIPLE ANALYTICAL METHODOLOGIES

Wafa BADULLAa,b,* and Göksel ARLIa

a
Department of Analytical Chemistry, Faculty of Pharmacy, Anadolu University, 26470, Eskisehir, Turkey
b
Department of Analytical Chemistry, Faculty of Pharmacy, Aden University, Aden, Yemen

Received August 1, 2016

Different non-sophisticated analytical methods consisting of Ultraviolet

Spectrometry (UV), Osteryoung Square Wave Voltammetry (OSWV) and
flow injection analysis (FIA) were developed, validated according to ICH
Q2 (R) 1guideline and applied for the determination of Montelukast
Sodium (MLS) in bulk and tablet formulation. In the UV absorbance
spectrum, MLS exhibits more absorption maxims, the main ones being
found at the wavelengths of 211.4 and 344.4 nm, respectively. The
determination was performed at 344.4 nm. In OSWV determinations rely
on the reduction of MLS at the hanging mercury electrode (HMDE). This
reduction is mainly diffusion-controlled in Britton–Robinson buffer, a
cathodic peak appearing at pH 4.0 in 0.15 M LiCl solutions containing also
40% methanol. In FIA determinations, the carrier solvent was 50%
methanol at 1mL /min flow rate. The FIA connected to UV detector. The
calibration graphs obtained for the three analytical methods applied in
determinations were rectilinear over the ranges of 0.9989, 09976 and 0.9997 for UV, OSWV and FIA respectively. The statistical
comparison of the obtained results demonstrates that the three developed methods are very similar in respect of both accuracy and
precision.

INTRODUCTION* high levels after exposure to oxygen. It deteriorates

chemically if the solution remains in contact with
MLS is selective and orally efficacious atmosphere for a long time. Stability studies of MLS
competitor of the cysteinyl, CysTL1, leukotriene have been reported by capillary electrophoresis and
receptor. It is recommended for the treatment of HPLC.6, 7
asthma in kids and grownups.1, 2 It is the main OSWV is considered as a very sensitive and direct
leukotriene modifier accepted by the US Food and analytical technique, which has been used for the
Drug Administration (FDA) in 2008 for use by rapid and sensitive determination of a wide variety of
youngsters from 2 to 12 years old and adults.3 organic molecules.8,9 The merits of OSWV over
The MLS empirical formula is C35H35ClNNaO3S, differential pulse voltammetry are the speed of
and its molecular weight is 608.18 g/mol. It is freely analysis, elevation of the current–response,10 minor
soluble in ethanol, methanol, and water and utilization of electroactive compound result in
practically insoluble in acetonitrile.4 It is photosensi- minimizing the chance of electrode obstruction and
tive, it becomes unstable and cis-isomer is formed as high sensitivity.11
a photo-degradation product when a solution of MLS FIA is a modern technique characterized by its
is exposed to light.5 Sulphoxide impurities found at utility, simplicity of mechanization, large sampling
*
Corresonding author:
174 Wafa Badula and Göksel Arli

capacity and low sample processing before quality acquired from Merck & Co, USA. High purity water
injection into the system. In recent times, there is a was obtained by using a Waters Milli-Q plus distillation
system.
direction to get economical, rapid and green
analysis method; FIA techniques fulfill this need in 2. Instruments
comparison with manual not automatic techniques.
Spectrophotometric estimation was operated on double
Furthermore, in recent years FIA has widespread beam UV-VIS Spectrophotometer Shimadzu 240 version 2.21
utilization as they can also be optimized rapidly for utilizing 10 mm quartz cells with 2 nm slit-width.
the compound analysis in comparison with the Polarographic system was Polaropulse Model BAS100B
conventional chromatographic methods.12,13 with controlled growth mercury electrode as the working
electrode and platinum wire was used as auxiliary electrode
Spectrophotometric techniques were developed
and saturated Ag/AgCl was used as a reference electrode. All
for the determination of MLS in dosage forms14,15 quantification was done at room temperature 25 ± 2 °C. A
and in combination with other drugs such as pure nitrogen gas 99.999% was used for deoxygenating.
Desloratadine16. Several other analytical techniques The FIA study was carried out by a Shimadzu (Kyoto,
such as spectrofluorimetric,17 high performance Japan) HPLC system constructed from the LC-20AT pump
and a model SPD-20A UV-VIS detector with manual injection
liquid chromatographic (HPLC) methods18 were also of standard solutions and samples. Various instrumental and
reported for its determination in human plasma. analytical parameters were examined for each methodology.
HPLC and derivative spectrophotometric method for
determination of MLS and loratadine in combined 3. Standard and sample preparation
tablet was found19. Assay of MLS in human serum The preparation of a stock solution of MLS 163.59 g/mL
specimen and MLS tablets on nickel hydroxide was done by dissolving the standard in 50% methanol then the
nanopetals modified carbon electrode,20 adsorptive working solutions were prepared by further dilution with the
stripping voltammetry of MLS in trade tablets and same solvent. To protect the solution form degradation by
light they were kept in an amber volumetric flask and all
biological specimen (urine and plasma) using experiments were carried out in darkness. The chemical
HMDE21 as well as direct current (DCt), differential stability of MLS was studied by a voltammetric method for a
pulse polarography (DPP) and alternating current period of 5 days; the reference solution was stored at 2–8°C in
(ACt) polarography for determination of MLS in a refrigerator. The voltammetric examination showed a peak
signal on the first day only which indicate that the solution is
dosage form and plasma22 were developed. The stable for 1 day for this reason working solution were prepared
superiority of the current developed OSWV method freshly every day.
over the already found electroanalytical methods are The method was applied to an MLS tablet according to the
no need for special electrode, necessity to accumulate United States pharmacopeia rules.23 For the analysis, 10
the drug on electrode surface which requires time tablets were weighed and pulverized. An amount of the
powder proportional to 10.0 mg of MLS was weighed
before detection, highly qualified analyst. In addition, precisely and transferred into a 100 mL volumetric flask. After
the detection limit of the OSWV developed method addition of a portion of 50% methanol the mixture sonicated
is considered satisfactory for the determination of for 30 min, then the volume of the solution was completed to
MLS in dosage forms. the 100 mL with the same solvent. For UV and FIA methods
the aliquots containing MLS were transferred into centrifuge
Overview of the literature showed that there is no tubes, the solution was centrifuged for 10 min at 20 °C at the
article for evaluation of MLS in tablet dosage form speed of 4000 rpm and then filter of 0.45 µm was used for
neither by FIA nor by OSWV is present till now. The filtration of working solutions. In case of OSWV analysis the
main aim of this study is to cope with the demands of solution examined without centrifuge or filtration. The
pharmaceutical analysis field for reasonable, high- regression equation calculated from the calibration graph of
the standard solution was used to calculate the nominal
throughput, immediate and sensitive determination of content of the tablets.
MLS in tablet dosage. The UV method was also
developed and recommended for the comparison of
OSWV and FIA. These methods would allow RESULTS AND DISCUSSION
determination MLS for routine analysis and quality
control in different laboratories equipped with
different analytical instruments. 1. Ultraviolet Spectrometry

To determine the maximum absorbance of MLS,

EXPERIMENTAL the standard solution of 12.16 µg/mL was scanned in
the range of 200 – 400 nm by 0.1 nm intervals. MLS
1. Materials and Reagents has many absorption maxims. The complex
MLS was supplied from Unimark Remedies Ltd. Batch
absorption spectrum of MLS can be explained by the
No.MNT-0330811-B-XJ; other chemicals were of analytical effect of structure and presence of a certain chemical
 Multiple analytical methodologies 175

functional group on UV absorption. Commonly, a solution pH is appropriate to be optimized at 4 in

molecule has a complex spectrum as it contains more the consequent analytical evaluation.
than one chromophore. MLS has conjugated systems
that connected with each other. Chloroquinolin 2.2. Effect of Solvents
connected to the phenyl by the ethenyl group and the and Supporting Electrolytes
phenyl connected to 2-hydroxypropan-2-yl-phenyl by
propyl group and to the cyclopropyl acetic acid by Supporting electrolyte and solvents are the main
thiomethanyl. The presence of saturated and component of the examined medium where the
electrochemical reaction occurs; they should be inert
unsaturated functional group between the conjugated
over the working potentials. For this purpose, the
systems affects the overall absorption of the
scanning of methanol 20-80%, which is used as a
conjugated systems. Also, MLS contains C-S-C solvent, was carried out, and there was no peak at
linkage which gives absorbance at 215 nm. A 20% methanol. The current was at the maximum
maximum absorbance at 211.4 can be related to the level at 40% methanol, and the peak morphology was
presence of C-S-C linkage and the shift in absorption also good at this percentage. The reason of high
is due to the effect of other functional groups of current and good morphology of the peak at 40%
MLS. A maximum absorbance at 344.4 can be methanol is facilitation of proton transfer between the
related to the presence of the conjugated systems.24 electrode surface and the solution. Supporting
The peak morphology was good at 344.4 nm. Hence electrolytes lower the resistance of the solution,
344.4 nm was observed to be appropriate and reduce electro migration effects, and assure a stable
efficient for the UV determination further study was ionic strength. The molarity of LiCl 0.05-0.3 M
done at this wavelength. which used as supporting electrolyte was examined,
and LiCl concentration of 0.15 M was selected
because the current was stable between 0.1 and 0.2 M
2. Voltammetry and in maximum level at 0.15 M of LiCl
concentration. Thus, the subsequent analytical
2.1. Effect of pH determination of the drug was done at these
The voltammteric study extremely influenced conditions.
by the pH of the electrolysis medium because it
affects the peak morphology, therefore the effect of 2.3. Effect of Instrumental Parameters
pH on the peak potential was fully examined. The Different interrelated instrumental parameters
voltammogram at the HMDE showed a well- may affect the peak current acquired in OSWV
shaped reduction peaks over the pH range of 2.0- such as amplitude and frequency, but in the current
12.0. An ill-shaped peak with a negative shift in study the instrumental parameters have limited
the potential was observed by increasing the pH effect on the peak potential. The optimum working
especially after pH 8. The negative shift indicates conditions such as square wave amplitude 1-200
the protonation of the reactive part of the MLS. mV, frequency 1-150 mV, drop size 2-12 and
The reduction occurs at ethenyl group bonded with initial potential 0-500 mV was examined. Thus, the
the quinoline nucleus and the phenyl group as instrumental parameters were identified and the
represented in Scheme 1. An ill shape and subsequent analytical determination of the drug
reduction in the current may be due to adsorption was studied in the following conditions: Square
of the MLS according to reference 22. The plot of wave amplitude is 25 mV, the frequency is15Hz,
pH versus the cathodic peak current is stable at pH drop size 4 and initial potential is 200 mV.
values from 3.5 to 4.5, which support the reduction
process of MLS at the electrode surface. Thus, the

Scheme 1 – Mechanism of electrochemical reduction process for MLS.

176 Wafa Badula and Göksel Arli

only a small peak of the trans-isomer and the peak

3. Flow Injection Analysis
for reduction of cis-isomer is vanished, which may
The optimum conditions of FIA were be due to further degradation of the compound.
determined for a standard solution of MLS at the The absence of peak for the degraded compound
concentration of 12.16 µg/mL. MLS is soluble in could be explained that they are not faradic. The
methanol and insoluble in acetonitrile for this stability study voltammogram is shown in Fig. 1.
reason different percentage of Methanol-water 10-
100%, v/v was studied as mobile phase. The peak 5. Analytical performance
shape was perfect at 50%, v/v concentration of
methanol the following determination was ICH Q2 (R) 1Guidelines for validation of
performed at this percentage. Different flow rates analytical procedures was applied to evaluate the
over the range 0.2-3.0 mL /min was investigated. developed methods.26 For the three methods, a
The flow rate of 1 mL/min was optimally in series of working solution in the range of 3-16.5
accordance to the peak morphology. µg/mL was prepared by transferring a proper
portion of the standard solution 163.59 g/mL to the
volumetric flask and diluting with the 50%
4. Stability study
methanol. The calibration graphs for the three
In the stability study of MLS by OSWV the methods are illustrated in Figs. 2, 3, 4.
compound showed a reduction peak at - 0.7 V on The investigation of method linearity was
the first day due to reduction of the ethenyl group performed with 7 concentrations in the range of
as mentioned before. But in second day the drug (0.5, 1.5, 3.27, 6.5, 9.8, 13 and 16.5µg/mL).
produced small peak at -0.7 V reduction potential Calibration plots were chosen according to the
which can be explained by the conversion of trans- limit of quantification in this range (3.27, 6.5, 9.8,
isomer to the cis form due to exposure to light 13 and 16.5 µg/mL) and 3 set were examined by
since it is photosensitive compound as mentioned the three techniques. The calibration graph was set
before, so solution contain only a small percentage up by applying the measured absorbance, signal
of trans–isomer. area and current versus concentration of UV,
A hump shaped peak at -1.1 V was appeared OSWV, and the FIA. Various statistical parameters
which could be due to reduction of cis-isomer on for linear regression equation like slope, intercept
the mercury electrode at more negative potential. and sum of square of regression have been
The reason for the negative shift could be calculated respectively and the linear regression
explained by considerable variation in equation was calculated. LOD and LOQ were
electrochemical behavior of isomers and geometric calculated as [(standard deviation of regression
orientation of cis-isomer with the electrode.25 In the equation) / (slope of the regression equation)] by
three following days the voltammogram shows multiplying by 3.3 and 10, respectively.

Fig. 1 – OSWV voltammogram of (12.16 µg/mL) for 5 days.

 Multiple analytical methodologies 177

Fig. 2 – Calibration curve of MLS by FIA.

Fig. 3. Calibration curve of MLS by UV.

Blank

Fig. 4 – Calibration curve of MLS by OSWV.

178 Wafa Badula and Göksel Arli

Table 1
Regression data of the calibration curve for quantitative determination of MLS
Analytical Methods
Parameters Voltammetry FIA UV Spectrometry
Measured potential (V)
and Measured wavelength (nm) 0.6-0.8 344.4 344.4
Linearity Range (µg/mL) 3-16.5 3-16.5 3-16.5
Slope 6.79x10-9 34501 0.0429
Intercept 4.75x10-8 15189 0.0167
Regression 0.9976 0.9997 0.9989
SD of slope 1.29x10-10 514.3 0.00099
SD of intercept 1.4x10-9 5587 0.0108
LOD, µg/mL 1.142 0.8881 0.6384
LOQ, µg/mL 3.461 2.691 1.935
Within-day precision (RSD), % 1.788 1.679 1.288
Between-days precision (RSD), % 1.986 1.848 1.467
Max. 97.96% Max.100.08% Max. 101.45%
Accuracy recovery % Med.100.82% Med.100.24% Med. 99.55%
Low. 98.02% Low. 98.86% Low. 101.52%
SD:Standard Deviation, LOD:Limit of Detection, LOQ:Limit of Quantification, RSD: Relative Standard Deviation

MLS solutions of 3.27µg/mL, 9.8µg/mL, and determination can be performed without any
16.5µg/mL were used for accuracy studies. The separation step because the peak obtained from the
examination of intraday and interday accuracy of dosage form was similar to those obtained from the
the three methods was performed for three MLS standard with no interference from tablet
subsequent days.26 The accuracy results are highly excipients. Both OSWV and UV analysis are
acceptable for the determination of MLS since the convenient and accurate to be applied in
recoveries percent were almost close to 100% for laboratories lacking liquid chromatographic
both drug standard and product. instruments. The uniformity of the mobile solvent
To examine the repeatability of the method in the FIA makes the method considerable in
(Intraday) and intermediate precision (Interday), comparison with the customary chromatographic
medium concentration 9.8 µg/mL of the MLS methods. A slight superiority of FIA over the other
standard solution was examined for three days, six two methods was seen since, it is suitable for
times in a day. Good correlations were obtained for processing numerous samples on a daily basis due
both intra and interday’s experiments indicating to short analysis time, large sample capacity and
that the developed methods are highly precise and low solvent consumption especially in quality
analytically acceptable. The calculated relative control laboratories. To prove the developed
standard deviation (RSD) value is lower than 2% methods applicability the replicate analysis of
deviation from the nominal value of precision.27 pharmaceutical dosage form was performed and
The validation data are given in Table 1. the results were evaluated and validated
statistically in the Table 2.
6. Comparison of the Determination Methods Statistical comparison of the developed
methods was performed at the 95 % confidence
On the basis of the results in Table 1, the three level with the assist of Student’s t and F-Tests. The
methods can be accepted as an analytical technique obtained results from the analysis of MLS in tablet
for analysis of MLS in tablet dosage form. By dosage form by OSWV and FIA are in good
comparing the precision and accuracy of three agreement with those obtained by the UV methods
methods, all of the three methods are adequate which is used as a reference method. On the basis
from an analytical perspective. From the point of of results shown in Table 2, there was no
detection limit, UV analysis showed some considerable variation between the performances
superiority so it can be applied for the of the three methods since the corresponding
determination of low concentration samples with theoretical values for Student’s t and F-Test were
no need for derivatization steps. In the OSWV lower than the calculated values.28
 Multiple analytical methodologies 179

Table 2
The results for the determination of MLS of dosage form

Parameter OSWV FIA UV Spectroscopy

Labeled claim, mg 10 10 10

Amount found, mg 10.13 10.09 9.97

RSD % 1.851 1.732 1.303

Bias % -1.3 -0.9 0.3

t-value 2.09 1.63 T-theoretical: 2.57

F-test 2.018 1.76 5.05

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Electrochem., 2003, 33, 951-957.
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Three resourceful and uncomplicated methods
Lett., 1999, 32, 2977-2988.
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The OSWV analysis showed simplicity over the Method J., 2010, 3, 56-73.
other methods due to absence of matrix effect so 12. F. González, P. Tarin, S. Maspoch and M. Blanco., Arch.
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13. G. Altiokka and K. Kircali, Anal. Sci., 2003, 19, 629-631.
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14. K. Singh, P. Bagga, P. Shakya, A. Kumar, M. Khalid, J.
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17. I. Alsarra, N. Y. Khalil, M. Sultan, R. Al-Ashban and F.
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