Development and Validation of RP-HPLC
Method for the Estimation of Montelukast Sodium in
Bulk and In Tablet Dosage Form
Nishant Sarode1*, G.
S. Chhabra2, Shailesh Luhar1 and Anil Jadhav1
1Smt. BNB Swaminarayan College of Pharmacy, Salvav,
Vapi. Gujarat 396191
2SPTM, NMIMS, Babulde, Bank of Tapi River, Mumbai – Agra Road, Shirpur,
Dist. Dhule, Maharashtra - 425405
ABSTRACT:
A simple,
selective and rapid reverse phase high performance liquid chromatographic
(RP-HPLC) method for the analysis of Montelukast
Sodium in bulk and in tablet dosage form has been developed and validated.
Sample was resolved on a Luna Phenomenax, C18 (150mm
X 4.6 mm i.d., particle size 5μ) column. The
mobile phase consisted of Acetonitrile l : 20 mM phosphate buffer ( 80:20 v/v pH adjusted to 6.0 by using
KOH ) was delivered at a flow rate of 1.0 ml/min at ambient temperature and the
retention time was about 9.92 minutes. Studies were performed on an HPLC system
equipped with a UV/Visible detector at 213nm. The method is specific to Montelukast sodium and able to resolve the drug peak from
formulation excipients. The calibration curve was
linear over the concentration range of 25-150 μg/ml
(R=0.9991).The results of analysis of formulation was found to be 99.80 ± 0.3268. The proposed method is applicable to routine analysis
of Montelukast sodium in bulk and in tablet dosage
form.
KEYWORDS: Montelukast Sodium, RP-HPLC,
validation.
1. INTRODUCTION:
Montelukast is an
oral selective leukotriene receptor antagonist that
inhibits the cysteinyl leukotrienes
cysLT1 and has been shown to be effective in the treatment of chronic asthma
and chemically is [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)
ethenyl] phenyl]-3-[2-(1-hydroxy-1 methylethyl) phenyl] propyl]thio] methyl] cyclopropaneacetic
acid, monosodium salt.1-3 Literature survey revealed that
only the HPLC methods with fluorescence detector 4,5, stereoselective high performance liquid chromatography with
column–switching6 and Montelukast
in human plasma by LC-MS methods was reported.7,8 Therefore, this
study focused on the development of simple and rapid isocratic RP-HPLC method
which can be employed for the routine analysis of Montelukast
sodium in bulk and in tablet dosage form. Fig 1 shows structure of Montelukast sodium
2. EXPERIMENTAL:
2.1.
Materials:
Montelukast
sodium was received as a gift sample from Micro Labs Ltd. (Bangalore, India)
and was used as-received. HPLC grade acetonitrile and
methanol was purchased from Merck (Darmstadt, Germany). HPLC grade water was
obtained from Milli-Q water purification system
(Millipore, Milford, USA). All material used for study is of analytical grade.
MONTAIR (Cipla) tablets containing 10 mg of Montelukast
sodium was purchased from local market.
Fig 1 Structure of Montelukast
sodium
2.2.
Preparation of stock solution:
Montelulast
sodium stock solution was prepared by accurately weighing 52.0 mg of Montelulast sodium to a 100 ml volumetric flask and
dissolving this quantity in HPLC grade methanol. The solution was sonicated for 3 min and volume was made up to 100 mL with methanol. All the solutions were prepared in
triplicate. Before being subjected to analysis, all the working standard
solutions were filtered through a 25 mm nylon membrane syringe filter (pore
size 0.45 lm). Before injecting solutions, the column was equilibrated for at
least 30 min with the mobile phase flowing through the system.
2.3. Instrumentation and analytical condition:
The
LC system used for the analysis was Perkin Elmer 200B/250 with a variable
wavelength UV Visible Detector and injector fitted with a 20 μl loop. The output signal was monitored and processed
using Total Chrome navigation software (Perkin Elmer, Waldbronn, Germany). The chromatographic separation
was carried out under isocratic reversed phase conditions on a Phenomenex (C18, 250mm × 4.60mm, 5 μm). The mobile phase used was Acetonitrile
: 20mM Potassium di hydrogen orthophosphate buffer,
pH adjusted to 6.0 with KOH (80:20 % v/v) with detection wavelength 213nm (λmax obtained from the scan spectra in the range of
200 to 400nm. All analyses were done under isocratic conditions at a flow-rate
of 1.0 mL/min, run time of 15 min and at room
temperature.
2.4.
Validation of the developed method:
The
developed chromatographic method was validated for selectivity, linearity,
precision, accuracy, and robustness. The method was also applied for the drug
content analysis from commercial tablet. The method was validated according to
the ICH guidelines Q2 (R1) 9 for validation of analytical methods.
2.4.1.
Linearity:
Linearity
test solutions for the assay method were prepared from stock solutions at six
concentration levels from 25.0 to 150.0 μg/ml in
the mobile phase. The linearity was evaluated by the least square regression
method with triplicate determinations at each concentration level.
2.4.2.
Precision:
The
precision of the assay was studied with respect to both repeatability and
intermediate precision. The system precision of the assay was investigated by
performing six replicate analyses of three standard samples of Montelukast sodium (50 μg/ml,
n = 6 ) on the same day and on eighth day and evaluated by relative standard
deviation (RSD) of the peak area of the analyte. The
method precision of the developed LC method was determined by preparing the
tablet samples of the same batch in six replicate determinations. The RSD value
of the assay results, expressed as a percentage of the label claim, was used to
evaluate the method precision.
2.4.3.
Accuracy:
The accuracy
was determined by standard addition method. Known amounts of Montelukast sodium were added to the samples and analysed by the proposed methods. Method accuracy was
tested (% recovery and % RSD of individual measurements) by analyzing samples
of Montelukast sodium at three different levels in
pure solutions using three preparations for each level. In this study,
different concentrations of pure drug were added to a known analysed
formulation sample and the total concentration was determined using the
proposed methods (n = 3). The percent recovery of the added pure drug was
calculated as, % recovery = [(Cv-Cu)/Ca] * 100, where
Cv is the total drug concentration measured after
standard addition; Cu, drug concentration in the formulation; Ca, drug
concentration added to formulation.
2.4.5.
Robustness:
Robustness
study was conducted by making small but deliberate changes to the optimized
method parameters. The design of the experimentation technique was used to
identify critical chromatographic factors and their effect on method
performance. The mobile phase ratio by ± 2 ml (in organic phase) the detector
wavelength by ±2 nm and flow rate by ± 0.1ml and the effects were monitored.
2.4.6
.Specificity:
Specificity
is the ability of the test method to measure an analyte
without interference from other samples and the matrix components. In
quantitative analysis, a method is called completely selective when it produces
correct analytical signal. A method is called completely selective when it
produces correct analytical results for mixture without any mutual interaction
of the components. Blank (methanol), Standard and Sample solution was injected
and interference was observed.
3.
RESULTS AND DISCUSSION:
3.1.
Validation of the method:
3.1.1.
Linearity:
The response
for the drug was found to be linear in the investigated concentration range.
Table 1 shows linearity of response
|
Concentration (mcg/ml)
|
Average Area (n=3)
|
|
25
|
2897263.66
|
|
50
|
5794527.32
|
|
75
|
8980647.89
|
|
100
|
12508946.16
|
|
125
|
15901172.58
|
|
150
|
18887735.19
|
|
Slope
|
130057.8191
|
|
Intercept
|
- 551677
|
|
Correlation Coefficient
|
0.9991
|
Fig 2 Calibration curve
for Montelukast sodium
3.1.2.
Precision:
Data obtained
from precision experiments are given in Table 2, for repeatability and
intermediate precision studies. The RSD values, 0.3275 % for repeatability
study and from 0.3275 to 0.5570 % for intermediate precision study,
respectively, confirm that the method was precise.
Table 2 shows
observation of precision study
|
Sample ID
|
Day I –Analyst 1
results
|
Day VIII
–Analyst 2 results
|
|
% Label claim
|
% Label claim
|
|
1
|
99.63
|
99.58
|
|
2
|
99.80
|
99.61
|
|
3
|
99.86
|
99.21
|
|
4
|
99.90
|
100.23
|
|
5
|
100.30
|
99.82
|
|
6
|
99.30
|
100.78
|
|
Mean
|
99.80
|
99.87
|
|
SD
|
0.3268
|
0.5563
|
|
% RSD
|
0.3275
|
0.5570
|
3.1.3.
Accuracy:
As shown
from the data in Table 3, recoveries of the drug in the range from 99.70 to 100.41 % were made at
various added concentrations.
3.1.4.
Robustness
Robustness
can be described as the ability to reproduce the method in different
laboratories or under different circumstances without the occurrence of
unexpected differences in the obtained results. Robustness of the proposed
method was assessed with respect to small alterations in the acetonitrile concentration (80 ± 2 ml), the detector
wavelength, pH of buffer and flow rate. With these modifications, the retention
time of Montelukast sodium showed small changes, but
the symmetry of the peak was conserved, indicating there was no negative effect
on the analysis.
3.3.5.
Specificity
Blank
(methanol), Standard and Sample solution was injected and interference was
observed. Peak of analyte was not interfering with
placebo and blank, hence method was specific and selective. Overlain chromatogram shows in Fig.
3
Fig 3 Chromatogram of Montelukast
sodium tablet in marketed formulation
3.2.
Assay of Tablet
The
validated method was applied for the assay of commercial tablets containing 10
mg of Montelukast sodium.
Preparation of sample solution: Accurately weigh not less than 20 tablets and determine the average
weight and powder all tablets. Transfer 5 intact tablets (equivalent to 50 mg
of montelukast) into a 100.0 ml of volumetric flask
and dissolved in sufficient quantity of HPLC grade methanol and volume was made
up to the mark with methanol, sonicated for 5 min.
From that pipette out 5.0 ml in 50.0 ml volumetric flask and volume was made up
to the mark with methanol. (Concentration: 50 µg/ml Montelukast).
The solution was filtered through 0.2 µm membrane filter.
Fig 4 Overlain chromatogram for specificity study.
The
resulting chromatogram is shown in the Fig. 4. Each sample was analyzed in
triplicate after extracting the drug as mentioned in assay sample preparation
of the experimental section and injections were carried out in triplicate. Fig
4 shows an LC chromatogram of Montelukast sodium in
tablet. None of the tablet ingredients interfered with the analyte
peak. The results presented in Table 4 are in good agreement with the labelled content. Assay results, expressed as the
percentage of the label claim, were found to be in 99.30 to 100.30 % for Montelukast sodium. The above results demonstrated that the
developed LC method achieved accurate determination of Montelukast
sodium and could be used for the determination of Montelukast
sodium in drug substance and tablets.
4.
CONCLUSIONS:
A validated
isocratic RP-HPLC method has been developed for the determination of Montelukast sodium in tablets. The proposed method is
simple, accurate, precise, linear and
specific. Therefore, it is suitable for the routine analysis of Montelukast sodium in pharmaceutical dosage forms. The
simplicity of the method allows for application in laboratories that lack
sophisticated analytical instruments such as LC–MS or GC–MS that are
complicated, costly and time consuming rather than a simple LC–UV method.
5. REFERENCES:
1)
Martinedale – The Complete
Drug Reference; 35th ed., Vol 1;
Pharmaceutical Press, 2007; 1010-1011.
2)
http://www.rxlist.com/singulair-drug.htm (Accessed on 20th Dec, 2010).
3)
http://www.drugbank.ca/drugs/DB00471 (Accessed on 20th Dec, 2010).
4)
Ochiai H, Uchiyama
N, Takano T, Kamei T. Determination of montelukast
sodium in human plasma by column switching high-performance liquid
chromatography with fluorescence detection
. J. Chromatogr. B
1998; 713: 409-414.
5)
Alsarra I A. Development of a
stability-indicating HPLC method for the determination of montelukast
in tablets and human plasma and its applications to pharmacokinetic and
stability studies. Saudi Pharm. J. 12: 2004 : 136-143.
6)
Liu L, Cheng H, Zhao, Jamie J, Rogers JD. Determination of montelukast (MK-0476) and its S-enantiomer
in human plasma by stereoselective high performance
liquid chromatography with column–switching. J. Pharm. Biomed. Anal. 1997; 15: 631-638.
7)
Papp R, Luk P, Mullett WM, Kwong EA. Rapid and sensitive method for the quantitation of montelukast in
sheep plasma using liquid chromatography/ tandem mass spectrometry. J. Chromatogr. B 2007; 858: 282-286.
8)
Bharathi DV, Hotha KK, Jagadeesh
B, Mullangia R, Naidu A. Quantification of montelukast, a selective cysteinyl
leukotriene receptor (CysLT1) antagonist in human
plasma by liquid chromatography mass spectrometry: validation and its
application to a human pharmacokinetic study. Biomed. Chromatogr. 2009; 23: 804-810.
9) ICH, Harmonized Tripartite Guidelines (1996)
Validation of Analytical Procedure; Text and Methodology Q2 (R1).