Simultaneous HPLC Method Development and
Validation of Bilastine and Montelukast in Bulk and Formulation
Ankita Shinde*, G.B. Gajeli, Sneha Ubale, Vinod Matole
D.S.T.S Mandal’s College of Pharmacy,
Solapur - 413004,
Maharashtra, India.
*Corresponding Author E-mail: ankitashinde3005@gmail.com
ABSTRACT:
A new, simple, rapid, selective, precise
and accurate reverse phase high performance liquid chromatography assay has
been developed for simultaneous estimation of Bilastine, and Montelukast in
tablet formulations. The separation was achieved by using Phenomenax Kinetex XB
C-18 column (150 x 4.6mm, 5
.) using mobile phase Methanol: 0.1% TFA water (80:20).
Injection volume was 10µl. The flow rate was 1.0mL.min-1 and the
separated drugs were detected using UV detector at the wavelength of 270nm. The
retention time of Bilastine and Montelukast was noted to be 1.27, and 4.86 respectively, indicative of rather
shorter analysis time. The method was validated as per ICH guidelines. The
proposed method was found to be accurate, reproducible, and consistent. It was
successfully applied for the analysis of these drugs in marketed formulations
and could be effectively used for the routine analysis of formulations
containing any one of the above drugs, or a combination, without any alteration
in the chromatographic conditions.
KEYWORDS: Liquid
Chromatography; Bilastine, Montelukast;
Combined dosage forms; Simultaneous
estimation.
INTRODUCTION:
Bilastine or 2-[4-[2-[4-[1-(2-ethoxyethyl)
benzimidazol-2-yl]piperidin-1-yl]ethyl]phenyl]-2-methylpropionic
acid, is selective Histamine
H1 receptor antagonist,
leading to decreased nasal congestionand urticaria. It reduces the development
of allergic symptoms by binding to and preventing activation
of the H1 receptor. It is used for management of seasonal
rhinitis andspontaneous urticaria.1-6
Montelukast sodium
[R-(E)}-1-[[[1-[3-[2-(7-Chloro-2 quinolinyl) ethynl] phenyl]-3-[2-(1-hydroxy-1-methylethyl)
phenyl] propyl]thio]methyl] cyclopropaneactic acid, monosodium salt is a Cysteinylleukotriene 1( CysLT1)
receptor antagonist. It is
used for management of asthma, exercise induced
bronchoconstrucionand allergic rhinitis. It
works by blocking the action of leukotrine D4 in the lungs resulting in
decreased inflammation and relaxation of smooth muscle.7-10
The structures of these two drugs are
shown in Fig. 1.
Fig.1.The structures of Bilastine and
Montelukast
Reverse phase high performance liquid
chromatography (RP-HPLC) method for single drug Bilastine and Montelukast sodium are reported1,7-10.
However no HPLC method for simultaneous estimation of these two drugs has been
reported in combine dosage form till date11-12. In the present
study, an attempt has been made to develop a method for the simultaneous
estimation of this drugs- Bilastine (I), and
Montelukast (II). It can also be applied for routine
analysis of either one or of any combinations of in these drugs dosage forms.
EXPERIMENTAL:
Chemicals and Reagents:
Pharmaceutical grade (>99%) drugs (I),
(II) were obtained from Aadhar Life Sciences Pvt. Ltd., (Solapur, Maharashtra State, India). Water and
Acetonitrile (HPLC grade) were obtained from Merck Specialties Pvt. Ltd.
Mumbai, India. All other chemical of analytical grade were procured from local
sources unless specified. All dilutions were performed in standard volumetric
glassware.
Instrumentation and Chromatographic Conditions:
The instrument used was a Agilent 1260
Infinity II, equipped with Control Panel Open Lab Version 4.8 Software, G4212B
UV-Visible detector, and (G7129A) auto injector. The samples were filtered
through Axiva Sichem Biotech RC membrane 0.45
and injected using autoinjector.
Chromatographic separation was performed on Phenomenax Kinetex XB C-18 column(
150 x 4.6mm, 5.). The mobile phase was composed ofMethanol:0.1% TFA
Water (80:20 v/v). The flow rate was maintained at 1.0mL.min-1. The
column effluent was monitored on UV detector set at 270nm.
Prepration of Standard Stock Solution:
Standard stock solution of Bilastine – Add 10mg of drug in 10ml of Volumetric
flask and add 5ml diluent,mix for 2 minutes and make the the volume to 10 ml
with diluent. (Conc. Of Bilastine - 1000µg.mL-1
).
Standard stock solution of Montelukast:
Add 5mg of drug in 10ml of volumetric
flask and add diluent, mix for 2 minutes and make the volume to 10ml with
diluent. (concentration of Montelukast - 500µg. mL-1).
Then add 1.0ml of standard stock solution
of Bilastine and 1ml of standard stock solution of Montelukast in 10 ml
volumetric flask and add 5ml diluent and vortex. Make up the volume with
diluent.
Preparation of Sample Solution:
Mixture of sample solutions was prepared
from formulation containing:
Bilastine (20mg) and Montelukast (10mg)
Tablet stock solution:
Ten tablets of the formulation (Billargic
M; CIPLA LTD) were weighed and crushed to a fine homogenous powder. Quantity
equivalent to 10mg Bilastine and 5mg Montelukast was accurately weighed into a
10mL volumetric flask add add 5ml of diluent, sonicate for 10 minutes. The
powdered mixtures were dissolved in the mobile phase and volume was made up. (Conc. Of Bilastine - 1000µg.mL-1,
Montelukast - 500µg.mL-1).
Tablet sample solution:
1ml of filtred tablet stock solution was
transferred to 10 ml voliumetric flask and 5ml diluent was added, sonicate for
10 minutes and volume was made up to 10 ml with diluent. (Conc. Of Bilastine -
100µg.mL-1, Montelukast - 50µg.mL-1).
Method Validation6-14:
The method was validated in terms of stability,
linearity, specificity, accuracy, precision, limit of detection (LOD) and limit
of quantization (LOQ).
RESULTS AND DISCUSSION:
Optimization of Chromatographic
Conditions:
Spectroscopic analysis of compounds showed
that (I),and (II),) have maximum UV absorbance (λmax) at
281.60nm, and 286.5nm respectively. Therefore, the chromatographic detection
was performed at 270nm using a UV-Visible detector. It was observed that when a
combination of drugs was injected, Bilastine and Montelukast together gave a
single peak. Chromatographic conditions were optimized by changing the mobile
phase composition and their ratios. Different experiments were performed to
optimize the mobile phase but adequate separation of drugs could not be
achieved. By altering the ratio of mobile phase a good separation was achieved.
0.1% TFA water was used as a modifier for better peak shape. The optimized
mobile phase was determined as a mixture of Methanol: 0.1% TFA Water (80:20) at
a flow rate of 1.0mL.min-1. Under these conditions (I), and (II) were eluted at 1.27and 4.86 minutes
respectively with a run time of 10 min.
A typical chromatogram for simultaneous
estimation of Bilastine and Montelukast obtained by using the above mentioned
mobile phase from 100 µL of the assay preparation is illustrated in Fig. 2.
Fig.2. HPLC chromatogram obtained during
simultaneous determination of Bilastine and Montelukast.
Method Validation:
Linearity and Calibration standards:
0.50, 0.75, 1, 1.25 and 1.50ml of Std Bilastine and Montelukast
diluted to get 50, 75, 100, 125 and 150 µg.mL-1 of Bilastine and 25, 37.4, 50, 62.5 and
75 µg.mL-1 of Montelukast. The response was measured as peak area. The calibration curve obtained by plotting peak area
against concentration showed linearity in the concentration range of 50 to 150µg.mL-1
for Bilastine and 25 to 75µg.mL-1 for Montelukast. The best fit for
the calibration curve could be achieved by a linear regression equation of Bilastine
and Montelukast were found to be y =20580x + 19350, and y = 26793x +30637,
respectively and the regression coefficient values (r2) were found
to be 1 and 0.999, respectively indicating a high degree of linearity for both
drugs.
Table1. Linearity of Bilastine.
|
Bilastine
|
|
% Level
|
Conc (ug/ml)
|
Area
|
|
50
|
50
|
1045884
|
|
75
|
75
|
1563318
|
|
100
|
100
|
2079008
|
|
125
|
125
|
2597319
|
|
150
|
150
|
3101438
|
Fig3: (Calibration curve of Bilastine)
Specificity:
Individual samples of Bilastine and
Montelukast were prepared of 100µg.mL-1 and 50 µg.mL-1
respectively and peaks were identified. Blank was injected to ensure there is
no blank peak interfering with the main analyte peaks. The specificity studies
revealed the absence of any other excipent interference, since none of the peaks
appeared at the same retention time of Bilastine and Montelukast, shown in
Fig. 2. The interaction study of all
four drugs in standard solution was also carried out by comparing peak of each
drug, individually Vs peaks of drug mixture. Interaction studies indicated that
the analyses did not interact with each other and were well within the
acceptance level of ± 2.0%.
Table No.2 Linearity of Montelukast
|
Montelukast
|
|
% Level
|
Conc.(ug/ml)
|
Area
|
|
50
|
25
|
693459
|
|
75
|
37.5
|
1037286
|
|
100
|
50
|
1380566
|
|
125
|
62.5
|
1707100
|
|
150
|
75
|
2033145
|
Fig4: Calibration Curve of Montelukast
Accuracy:
Samples were prepared of 75%,100%, and
125% concentration by spiking the same amount of concentration given below in
table for both Bilastine and Montelukast. Samples were injected in duplicate to
calculate % RSD. %Recovery also calculated. The results are shown in Table 3.
Table3. Accuracy Result of Bilastine.
|
Bilastine
|
|
|
|
|
|
|
Std Wt. (mg)
|
Purity (%)
|
Potency (ug/ml)
|
|
|
|
|
|
|
10
|
99.58
|
995.8
|
|
|
|
|
|
|
Std Area
|
2081297
|
|
|
|
|
|
|
|
Sample ID
|
Conc (ug/ml)
|
Area
|
Amount Recovered (ug/ml)
|
% Recovery
|
Average
|
STDEV
|
RSD
|
|
75% Rep 1
|
74.69
|
1563318
|
74.80
|
100.15
|
100.22
|
0.10
|
0.10
|
|
75% Rep 2
|
74.69
|
1565434
|
74.90
|
100.29
|
|
|
|
|
100% Rep 1
|
99.58
|
2079008
|
99.47
|
99.89
|
99.95
|
0.09
|
0.09
|
|
100% Rep 2
|
99.58
|
2081616
|
99.60
|
100.02
|
|
|
|
|
125% Rep 1
|
124.48
|
2597319
|
124.27
|
99.83
|
99.81
|
0.04
|
0.04
|
|
125% Rep 2
|
124.48
|
2595977
|
124.20
|
99.78
|
|
|
|
Table 4: Accuracy Result of Montelukast
|
Montelukast
|
|
|
|
|
|
|
Std Wt. (mg)
|
Purity (%)
|
Potency (ug/ml)
|
|
|
|
|
|
|
5
|
99.75
|
498.75
|
|
|
|
|
|
|
Sample ID
|
Conc (ug/ml)
|
Area
|
Amount Recovered (ug/ml)
|
% Recovery
|
Average
|
STDEV
|
RSD
|
|
75% Rep 1
|
37.41
|
1037286
|
37.69
|
100.77
|
100.84
|
0.10
|
0.10
|
|
75% Rep 2
|
37.41
|
1038735
|
37.75
|
100.91
|
|
|
|
|
100% Rep 1
|
49.88
|
1380566
|
50.17
|
100.59
|
100.31
|
0.39
|
0.39
|
|
100% Rep 2
|
49.88
|
1372999
|
49.89
|
100.04
|
|
|
|
|
125% Rep 1
|
62.34
|
1707100
|
62.03
|
99.50
|
99.33
|
0.24
|
0.24
|
|
125% Rep 2
|
62.34
|
1701359
|
61.82
|
99.17
|
125% Rep 2
|
|
|
Instrument Precision:
A single sample was prepared of 100µg.mL-1
as described and 5 injections were made from same sample. Results are as shown
in Table 5.
Table 5. Instrument Precision of Bilastine and
Montelukast
|
Sample ID
|
Area of Bilastine
|
Area of Montelukast
|
|
Rep 1
|
2079008
|
1380566
|
|
Rep 2
|
2081616
|
1372999
|
|
Rep 3
|
2080004
|
1370787
|
|
Rep 4
|
2083151
|
1371702
|
|
Rep 5
|
2082708
|
1366516
|
|
Average
|
2081297
|
1372514
|
|
STDEV
|
1763.616
|
5115.853
|
|
RSD
|
0.08
|
0.37
|
LOD and
LOQ:
For determining the limit of detection
(LOD) and limit of quantitation (LOQ), the method based on the standard
deviation and slope was adopted The limit of detection (LOQ) for Bilastine and
Montelukast was 0.99 µg.mL-1,1.38 µg.mL-1respectively,
and the limit of quantitation (LOQ) Was 2.99 µg.mL-1, 4.19 µg.mL-1
Analysis of Tablets:
The values of analysis of tablets obtained
by the proposed method were between 99.05% and 100.39% (Table 4), which showed that
the estimation of dosage forms were accurate within the acceptance level of 95%
to 105%.
Table 6. Assay of Bilastine and Montelukast
|
|
Bilastine Area
|
Montelukast Area
|
Bilastine Assay
|
Montelukast Assay
|
|
WS
|
2081297
|
1372514
|
-
|
-
|
|
DP
|
2061621
|
1377905
|
99.05
|
100.39
|
System suitability parameters:
For system suitability parameters, three
replicate injections of mixed standard solution were injected and parameters
such as the Resolution, Capacity factor, Tailing factor, Theoretical plate,
Retention volume and Asymmetry factor of the peaks were calculated. The results
are shown in Table 7.
Table7. System suitability data of the method
|
BILASTINE
|
MONTELUKAST
|
|
Sample ID
|
RT
|
Asymmetry
|
TP
|
Resolution
|
RT
|
Asymmetry
|
TP
|
Resolution
|
|
Rep1
|
1.27
|
1.14
|
4149
|
0.00
|
4.87
|
0.95
|
10254
|
26.51
|
|
Rep2
|
1.27
|
1.10
|
4216
|
0.00
|
4.89
|
0.94
|
10119
|
26.49
|
|
Rep3
|
1.28
|
1.02
|
4203
|
0.00
|
4.87
|
0.95
|
10185
|
26.38
|
|
Rep4
|
1.27
|
1.10
|
4230
|
0.00
|
4.87
|
0.96
|
10167
|
26.48
|
|
Rep5
|
1.28
|
1.03
|
4217
|
0.00
|
4.87
|
0.96
|
10150
|
26.39
|
CONCLUSION:
An RP-HPLC method for simultaneous estimation
of Bilastine and Montelukast was developed and validated. Results of analysis
of the formulations are tabulated in Table 1. The amounts obtained by the
proposed method are between 98.35% and 101.62%, within the acceptance level of
95% to 105%. The results obtained indicate that the proposed method is rapid,
accurate, selective, and reproducible. Linearity was observed over a concentration
range of 50 to 150µg.mL-1 for Bilastine and 25 to 75µg.mL-1
drugs. The method has been successfully applied for the analysis of marketed
tablets. It can be used for the routine analysis of formulations containing any
one of the above drugs or their combinations without any alteration in the
assay. The main advantage of the method is the common chromatographic conditions
adopted for all formulations. Therefore, the proposed method reduces the time
required for switch over of chromatographic conditions, equilibration of column
and post column flushing that are typically associated when different
formulations are analyzed.
ACKNOWLEDGEMENTS:
The authors express their gratitude to Aadhar
Life Sciences Pvt. Ltd. Solapur, (India) for gift samples of drugs and for
proiding facility to perform the project work.
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