Determination
of Montelukast by New Spectrophotometric Method using Bromocresol Green
Nazira Sarkis1, Saad Antakli2, Zakaa Alhamada1
1Department
of Analytical and Food Chemistry, Faculty of Pharmacy, Aleppo University,
Syria.
2Department
of Chemistry, Faculty of Science, Aleppo University, Syria.
*Corresponding Author E-mail:
nazirasarkis@gmail.com
ABSTRACT:
Simple,
sensitive and accurate spectrophotometric method has been developed for the
determination of Montelukast Sodium in raw and tablets formulation. The method is based
on the formation of yellow ion-pair complex between Montelukast Sodium
and Bromocresol green in Dichloromethane medium. The absorption maximum of the complex was
found to be at 405 nm. Different parameters affecting the reaction such as:
effect of solvents, reagent concentration, correlation
ratio, etc. were carefully studied and optimized. The formed complex was
quantified spectrophotometrically at absorption maximum. Linearity range was 2
– 19 µg/mL, regression analysis showed a good correlation coefficient R2
= 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) were
to be 0.50 µg/mL and 1.51 µg/mL. The developed methods could be successfully
applied to pharmaceutical formulations. The proposed method is simple, direct,
sensitive and do not require any extraction process. Thus, this method could be
readily applicable for the quality control and routine analysis.
KEYWORDS: Montelukast
Sodium, Bromocresol Green, Spectrophotometric method.
INTRODUCTION:
Montelukast Sodium (MTK) Fig. (1, a) is chemically a
Sodium[1-[[[(1R)-1-[3-[(E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]sulfanyl]methyl]
cyclopropyl]acetate1. Which is a leukotriene receptor antagonist
used as an alternative to anti-inflammatory medications in the management and
chronic treatment of asthma and exercise-induced bronchospasm (EIB)2.
It is usually administered orally. Montelukast
is a CysLT1 antagonist; it blocks the action of leukotriene D4 (and secondary
ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT1 in the
lungs and bronchial tubes by binding to it. This reduces the bronchoconstriction otherwise caused by the
leukotriene and results in less inflammation3.
Several
methods have been reported in the literature for the analysis of Montelukast
Sodium such as Liquid chromatographic (LC)4-5, High performance
liquid chromatographic (HPLC) and high-performance thin layer chromatographic
(HPTLC)6-7, Voltammetric method8-9, Spectrophotometric
method10-11, Reverse phase high performance liquid chromatographic
(RP-HPLC) method12-13, Ultra Performance Liquid Chromatography
(UPLC) method14.
Bromocresol Green (BCG) as shown in Fig. (1, b) is a
sulphonphthalein dye commonly used as indicator and spectrophotometric reagent.
Fig. (1,
a): Structural formula of Montelukast Sodium.
Fig. (1, b):
Structural formula of Bromocresol Green.
MATERIALS
AND METHODS:
Apparatus:
All
spectral measurements were carried out using a Spector Scan T80+, UV/Vis
spectrophotometer instrument Ltd (UK), connected to computer, quartz cells 1
cm. Ultrasonic processor
(Hwashin Technology, Power sonic 405, KOREA),
and analytical balance (Sartorius, model 2474, GERMANY).
Chemical
regents:
Dichloromethane from Riedel-de Haën (GERMANY) was used to
prepare the solutions. Pharmaceutically
pure samples were Montelukast Sodium, purity 99.6 %, was obtained from (JAYCO CHEMICAL,
INDIA). Bromocresol Green from (BDH, ENGLAND).
Pharmaceutical
formulations:
The
listed commercial products were subjected to analytical procedure:
· Asmalair® tablets, UNIPHARMA pharmaceutical
company, Damascus – Syria, labeled 4,5,10 mg/tab of Montelukast.
· Lukast® tablets, ALFARES pharmaceutical
company, Damascus – Syria, labeled 4,5,10 mg/tab of Montelukast.
· Monkast® Tablets, PHARMASYR pharmaceutical
company, Damascus – Syria, labeled 4,5,10 mg/tab of Montelukast.
Standard
preparation:
Montelukast
Sodium stock solutions:
Stock
solutions 1.64×10-3 M of Montelukast
Sodium (MW = 608.169g/mol) was
prepared by dissolving 10mg of pharmaceutically pure raw material
sample equivalent to 10.04mg (by taken the purity in consideration) in
volumetric flask 10mL of Dichloromethane, then 1mL of the solution was taken to
volumetric flask 10mL and diluted
with Dichloromethane to give concentration 1.64×10-4
M equivalent to 100𝜇g/mL. The
working standard solutions of Montelukast
Sodium samples were prepared
by appropriate dilutions among (100 - 950)𝜇L of 100𝜇g/mL in
volumetric flask 5mL and added 1mL of
Bromocresol Green 2.4×10-3 M then completed to volume with Dichloromethane to give concentrations between (2 - 19)𝜇g/mL of Montelukast
Sodium.
Reagent stock solution:
Bromocresol Green
2.4×10-3 M was prepared by dissolving 42.73mg of pure Bromocresol
Green (MW = 698.014g/mol) in volumetric flask 25mL
and completing to volume with Dichloromethane.
Calibration
Curve:
To construct the calibration
curve, five standard solutions for each concentration were prepared and the absorbance was measured of each
solution five times.
Sample preparation:
Ten tablets weighed and finely powdered
and an accurate weight equivalent to labeled content (4, 5 or 10 mg/tab) was weighed accurately, dissolved in volumetric flask 25ml of Dichloromethane and
sonicated for 15 minutes. Then sample was filtered by using BÜCHNER funnel.
After that, further dilutions were made to obtain an appropriate concentration
(among the range of linearity) and then the general procedure was conducted.
RESULTS AND
DISCUSSION:
Montelukast Sodium forms with Bromocresol Green at 25±5şC yellow ion-pair complex and Stability of the complex was 24 hours. The result solution was scanned in the range of wavelengths 300 - 550nm against a blank of BCG prepared in Dichloromethane, and then measured the absorbance at maximum wavelength 405 nm. We studied all the parameters of the colored result solutions to obtain the optimal conditions. Fig. 2 shows the spectrum of complex between Montelukast Sodium and Bromocresol Green in Dichloromethane medium.
Fig. 2: a- Spectrum of complex MTK-BCG in Dichloromethane medium for MTK (2×10-5 M).
b- Spectrum of BCG (1×10-4 M) in Dichloromethane medium.
Effect the kind of solvent:
In order to select a suitable solvent for preparation of the reagent solutions used in the study, the reagents were prepared separately in different solvents such as aceton, chloroform, dichlorometane and ethyl acetate, and the reaction of MTK and BCG was followed. Dichlorometane was preferred as the most suitable solvent because in this medium, the reagent blank gave negligible blank absorbance and the formed ion-pair complex was found to exhibit higher sensitivity and stability. In other solvents, the reagent blank yielded high absorbance values.
Effect of reagent concentration:
To study the effect of reagent concentration on the
colored complex solution, we made a series of 10mL of separated volumetric
flasks, by adding 1mL of Montelukast Sodium 2×10-4
M equivalent to 20µM and added between (0.025 – 2.5mL) of (BCG) 1×10-3
M, equivalent to (5 - 500µM), and completed to 10mL by
Dichloromethane. The absorbance at 405nm for every added
(BCG) reagent was measured against the blank of Dichloromethane.
It was found that the completed colored complex formation was 1.5mL of (BCG)
solution. The best concentration addition of (BCG) was 300µM with fifteen times
of Montelukast concentration.
Stoichiometric Relationship:
The stoichiometric ratio between drug and dye in the complex MTK-BCG was determined by Molar ratio method and Job's method of the continuous variation method as following:
1. Molar ratio method:
We have prepared a series of complex solutions MTK-BCG in the medium of the Dichloromethane. The concentration of Montelukast Sodium changes within the ratio (7.83×10-6 – 7.83×10-5) M while the concentration of the reagent was constant in each solution and equal to 3.132×10-5 M. We measured the absorbance values of these solutions at the wavelength of the maximum absorbance 405nm against the blank of BCG in Dichloromethane. The absorption changes of the molecular ratio of the Montelukast to the reagent permitted us to measure correlation ratio, we obtained the curve A = f([MTK]/[BCG]) shown in Fig. (3, a) where the correlation ratio is (1:1).
2. Job's method of the continuous variation:
We have prepared a series of complex solutions MTK-BCG in the medium of the Dichloromethane. The concentration of the reagent and the concentration of Montelukast Sodium changes in solutions between (0 - 4×10-5 M) where the sum of both concentrations remains constant and equal to 4×10-5 M. We plotted the spectral curves of each solution at the wavelength 405 nm and plotted the absorption changes of the solutions of the formed complex in terms of molecular fraction of Montelukast Sodium. We obtained the curve A = f( [BCG] /{[BCG] + [MTK]}) shown in fig. (3, b) where the correlation ratio is (1:1).
Fig. 3, a: molar ratio of MTK-BCG complex (1:1).
Fig. 3, b: Job's method of the continuous variation of MTK-BCG complex
Method validation:
The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose. The objective of the analytical procedure should be clearly understood since this will govern the validation characteristics which need to be evaluated. Typical validation characteristics which should be considered are accuracy, precision, detection limit, quantitation limit, linearity and range.
Range and linearity of Montelukast Sodium:
We studied the linearity of Montelukast Sodium
concentrations at the optimal conditions where we made a series of 5mL of
separated volumetric flasks, each one contains the equivalent volume of BCG
2.4×10-3 M, and Montelukast Sodium stock solution 1.6×10-4
M, and completed to 5mL with Dichloromethane, finally we measured the absorbance at 405nm for each
concentration against the blank of BCG in Dichloromethane.
Fig. 4 presents the Montelukast Sodium spectra. The range of linearity was
obeyed to Beer’s law in concentration (2 - 19) μg/mL and the
linearity curve is presented in Fig. 5.
Accuracy
and precision:
To determine the precision and accuracy of the
proposed method, five replicate determinations were carried out on three
different concentrations of standards (MTK). The validation results are shown
in table 1.
|
Fig. 4: spectra of
(MTK-BCG):
C1: 2
𝜇g/mL, C2: 4 𝜇g/mL, C3: 6 𝜇g/mL
C4:8 𝜇g/mL, C5:10 𝜇g/mL, C6: 12 𝜇g/ mL
C7:14
𝜇g/mL, C8: 16 𝜇g/ mL, C9: 18 𝜇g/ mL
C10: 19 𝜇g/ mL.
|
Fig. 5:
Calibration curve for (MTK-BCG):
C1: 2 𝜇g/mL, C2: 4 𝜇g/mL, C3:
8 𝜇g/mL
C4:12
𝜇g/mL, C5:16 𝜇g/mL.
n = 5 for each concentration.
|
Table
1: Precision and accuracy for determination of Montelukast Sodium.
|
inter-day n=5
|
intra-day n=5
|
Theoretical concentration
(μg/mL)
|
|
Accuracy (%)
|
Precision
(RSD %)
|
SD
µg/mL
|
 found concentration (μg/mL)
|
Accuracy (%)
|
Precision
(RSD %)
|
SD
µg/mL
|
 found concentration (μg/mL)
|
|
97.57
|
2.39
|
0.14
|
5.85
|
97.66
|
1.36
|
0.08
|
5.86
|
6
|
|
100.44
|
0.44
|
0.06
|
13.56
|
100
|
1.55
|
0.21
|
13.50
|
13.5
|
|
101.06
|
0.64
|
0.11
|
17.18
|
100.41
|
2.10
|
0.36
|
17.07
|
17
|
: mean of five replicated determinations, Accuracy (%)
= (observed concentration/theoretical concentration) ˟
100,
Precision (RSD %) = (standard deviation/mean concentration) ˟100.
Detection limit,
Quantitation limit and Sensitivity Sandell’s:
The
mean molar absorptivity ε, Sandell’s
sensitivity, limit of detection (LOD) and limit of quantification (LOQ)
were calculated from the standard deviation of the absorbance measurements
obtained from Beer’s law and are presented in table 2.
Table2:
Analytical parameters of Montelukast Sodium determination
|
Parameter
|
Value
|
|
λ max (nm)
|
405
|
|
Beer’s law limits
(μg/mL)
|
2 – 19
|
|
Stability of the complex
|
24 hours
|
|
Temperature
of solution
|
25 ± 5
oC
|
|
Solvent
|
Dichloromethane
|
|
CBCG:
CMTK , M
|
≥15
|
|
Molar absorptivity, ε
(L/mol.cm)
|
65550
|
|
Regression equation
|
Y = 0.1019X + 0.0421
|
|
Slope (b)
|
0.0421
|
|
Intercept (C)
|
0.1019
|
|
R2
|
0.9998
|
|
LOD (µg/mL)
|
0.50
|
|
LOQ (µg/mL)
|
1.51
|
|
Sandell’s sensitivity SS
(µg/cm2)
|
0.0092
|
RECOVERY:
The recovery was studied by three addition standards
for every product. Table 3 presents the recoveries result for the two products
(Asmalair UNIPHARMA 4mg and Lukast ALFARES 5mg).
Application
of the method: Estimation of Montelukast Sodium in nine tablets Syrian
products:
The
developed method was applied for quantitative determination and identification
of Montelukast Sodium in nine Syrian pharmaceutical products. The samples were
prepared as described in the section of samples preparation and analyzed.
Quantitative analysis was done by using calibration curve. The obtained results
are summarized in table 4 for all products.
In
general, the concentrations of the detected Montelukast Sodium compounds in the
products were within in the allowed limits under BP legislation) the tablets must contain not
less than 98.00 percent and not more than 102.00 percent of labeled amount(. Therefore, the obtained results are
conformed to BP legislation1.
Table
3: Recoveries for Asmalair UNIPHARMA 4 mg and Lukast ALFARES 5 mg Syrian
products.
|
Recovery
Average %
|
RSD%
|
SD
µg/mL
|
Recovery %
|
Total Found
 µg/mL
|
Added
µg/mL
|
Sample
µg/mL
|
Dosage as montelukast sodium
|
product name
|
|
100.05
|
1.28
|
1.28
|
100.00
|
10.96
|
4.8
|
6.16
|
4.2 mg/tab
|
Asmalair 4
|
|
1.09
|
1.09
|
99.74
|
12.14
|
6
|
|
0.90
|
0.90
|
100.42
|
13.39
|
7.2
|
|
100.75
|
1.82
|
1.84
|
101.30
|
11.07
|
4.8
|
6.212
|
5.2 mg/tab
|
Lukast 5
|
|
1.39
|
1.40
|
100.50
|
12.24
|
6
|
|
1.28
|
1.28
|
100.46
|
13.44
|
7.2
|
Mean for five separate determinations were performed
and calculated the mean.
Table 4: Results of Montelukast Sodium in Syrian pharmaceutical
products.
|
product name
|
Label claim (Montelukast
Sodium)
|
Found Concentration
 mg/tablet
|
SD
mg/tablet
|
RSD %
|
Per %
|
|
Asmalair 4
|
4.2 mg/tab
|
4.18
|
0.016
|
0.39
|
99.52
|
|
Asmalair 5
|
5.2 mg/tab
|
5.22
|
0.085
|
1.63
|
100.38
|
|
Asmalair 10
|
10.4 mg/tab
|
10.49
|
0.186
|
1.77
|
100.86
|
|
Lukast 4
|
4.2 mg/tab
|
4.26
|
0.046
|
1.085
|
101.43
|
|
Lukast 5
|
5.2 mg/tab
|
5.23
|
0.081
|
1.56
|
100.58
|
|
Lukast 10
|
10.4 mg/tab
|
10.49
|
0.110
|
1.05
|
100.86
|
|
Monkast 4
|
4.2 mg/tab
|
4.26
|
0.043
|
1.02
|
101.43
|
|
Monkast 5
|
5.2 mg/tab
|
5.24
|
0.104
|
2.00
|
100.77
|
|
Monkast 10
|
10.4 mg/tab
|
10.61
|
0.103
|
0.975
|
102.02
|
Mean for five replicates.
CONCLUSION:
We
developed a new method was applied for the identification and quantification of
Montelukast Sodium in Syrian tablets formulation. A good percentage of
recovery shows that the method can be successfully used in routine analyses.
The proposed method is simple, sensitive, rapid, a little cost and could be
applied for quality control of Montelukast
Sodium. The levels of Montelukast Sodium compounds were within the permissible limits set by
the BP legislation1.
ACKNOWLEDGEMENT:
The
Ministry of High Education and scientific research in Syria financially and
technically supported this work through department of Chemistry, Faculty of
Pharmacy, University of Aleppo, Syria.
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