ISSN 0974-3618 www.rjptonline.org
RESEARCH ARTICLE
Analytical Method Development and Validation for Simultaneous
Estimation of Montelukast and Ebastine by HPLC
Baokar Shrikrishna1,2*, Ranpise Nisharani3
1Shivnagar
Vidya Prasarak Mandal’s College of Pharmacy, Malegaon (Bk.), Tal- Baramati,
Dist- Pune, Maharashtra, India- 413115.
2Department
of Pharmaceutical Sciences, Pacific Academy of
Higher Education and Research (PAHER) University, Udaipur, Rajasthan, India-
313003
3Department of
Pharmaceutical Sciences, Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune, Off
Sinhgad Road, Pune, Maharashtra, India- 411041.
*Corresponding Author E-mail: krishnabaokar@gmail.com
ABSTRACT:
A RP-HPLC method was developed
and validated for simultaneous estimation of montelukast and ebastine in bulk
as well as in tablet formulation according to ICH guidelines. The chromatographic separations of drugs were
achieved on Younglin (S.K) isocratic system with Lichrocart C18 column
(4.6 mm×250 mm, 5µm). The mobile phase
consisted by methanol and water (80: 20) at pH 3 adjusted by
ortho phosphoric acid. The flow rate was
adjusted to 1 ml/min and UV detection was carried out at 268 nm. The retention
time for montelukast and ebastine were found to be 6.56 and 2.95 min
respectively. The detector was showed
linear responses over the concentration range 5 – 25 µg/mL for both drugs by
showing a good correlation coefficient of 0.999. This proposed method is highly sensitive,
precise and accurate which reduces cost of analysis; hence recommended for
routine quality analysis in laboratories.
KEYWORDS: Montelukast; ebastine;
validation; simultaneous estimation.
INTRODUCTION
Montelukast
(MTS) is 1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl) ethenyl]
phenyl]-3-[2-(1-hyroxy-1-methyl-ethyl) phenyl] propyl] thio] methyl]
cyclopropaneacetic acid.
Fig 1 Montelukast
Sodium1
MTS
is freely soluble in ethanol, methanol, water and practically insoluble in
acetonitrile. MTL is a selective, potent
and orally active antagonist of the cysteinyl, CysTL1, leukotriene receptor
used for the treatment of asthma in children and adults2-4.
Received on 13.09.2014 Modified on 21.10.2014
Accepted on 09.12.2014 © RJPT All right reserved
Research J. Pharm. and Tech. 8(1):
Jan. 2015; Page 01-05
DOI: 10.5958/0974-360X.2015.00001.3
Montelukast blocks the action of leukotriene D4 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
inflammation. Because of its method of
operation, it is not useful for the treatment of acute asthma attacks. Again
because of its very specific locus of operation, it does not interact with
other allergy medications such as theophylline. Literature survey reveals several methods
for MTS such as analytical method development and validation by RP-HPLC5-6, stability indicating HPLC, HPLC coupled with
ESI-MS/MS7, capillary electrophoresis8, derivative
spectroscopy9, HPLC simultaneous estimation with other drugs10-11,
TLC-Densitometry12, HPLC and HPTLC13-14, liquid-liquid
extraction method by using HPLC with fluorescence detector15, derivative
spectrophotometry16. Ebastine
(EBS) is 1-[4-(1,1-Dimethylethyl) phenyl]-4-[4-(diphenylmethoxy) piperidin-1-yl]
butan-1-one.
Fig. 2 Ebastine
EBS
is very soluble in methylene chloride and sparingly soluble in methanol. It is
used in antihistaminic treatment17. Ebastine,
a piperidine derivative, is a long-acting, non-sedating, second-generation
histamine receptor antagonist that binds preferentially to peripheral H1
receptors. It has antihistaminic, antiallergic activity and prevents
histamine-induced broncho-constriction.
It does not have significant sedative or antimuscarinic actions. Literature
survey for EBS revealed several methods such as, individual determination of
EBS by HPLC18-19, stability indicating LC Method20, HPTLC21, spectrofluorimetry22,
HPLC simultaneous estimation with other drugs23, simultaneous
spectrophotometric estimation24, spectrophotometric absorption ratio
method25, HPLC-DAD26. Literature survey reveals several
methods for simultaneous determination of MTS and EBS by HPLC27-30
but this present work describes
new and kinetic validated RP- HPLC method with different retention time.
EXPERIMENTATION:
Instrumentation:
The analysis was performed by using
Younglin (S.K) isocratic system consisting UV detection at 344 nm for MTS and
252 nm for EBS. The separation was achieved on a C18 Lichrocart
column (4.6mm × 250mm, 5µm).
MATERIALS:
Reference
standards for MTS and EBS were gifted by Micro Labs Pvt. Limited,
Pondicherry. Pharmaceutical preparation
for combination of MTS and EBS (Ebast-M) was obtained from local
market. Methanol of analytical reagent
grade and HPLC grade, ortho phosphoric acid, water of HPLC grade were purchased
from Merck Ltd., India.
Preparation of mobile phase:
The mobile phase consisting
methanol: water (80:20; v/v) and pH adjusted to 3 by ortho phosphoric
acid. The flow rate was adjusted to 1 ml/min.
Preparation of standard stock
solutions:
Weighed accurately 10 mg of MTS
and EBS and transferred to separate volumetric flask of 10 ml. Sufficient
amount of mobile phase was added and drugs were dissolved to give a stock
solution of 1000 µg/mL each.
Preparation of test solution:
Weighed accurately powdered tablet
equivalent to 10 mg of MTS and EBS, transferred to volumetric flask, mixed it
well with 100 ml of mobile phase to prepare 100µg/mL. From above solution 1.5 ml was taken and
diluted to 10 ml with mobile phase to get a solution containing 15µg/mL of MTS
and EBS each. The amounts of MTS and EBS
per tablet were calculated by extrapolating the value of area from the
calibration curve by using UV detection at isoabsorptive point of 268 nm (fig.
3). Procedure was repeated six times with the same tablet formulation. And
obtained results are tabulated in Table 1.
Fig.3 Overlaying spectra of MTS and EBS
Table 1 Assay of MTS and EBS
Sr.
No. |
Amount
present (mg) |
Amount
found in mg |
%
Label claim |
|||
MTS |
EBS |
MTS |
EBS |
MTS |
EBS |
|
1 |
10 |
10 |
9.906 |
9.902 |
99.10 |
99.03
|
SD |
|
1.235 |
0.894 |
|||
% RSD |
|
1.246
|
0.903 |
*All the results were average of
6 readings, (n=6)
Calibration:
MTS
and EBS were showed linear responses in the range 5-25 µg/mL at their respective maxima, which
were validated by least square method. For simultaneous estimation of MTS and
EBS, a series of standard solution were prepared by diluting appropriate
volume. The scanning of MTS and EBS were carried out in the range of 200 – 400
nm against selected mobile phase. MTS and EBS showed maximum absorbance at 344
nm and 252 nm respectively.
System
Suitability:
According
to United States Pharmacopoeia, system suitability tests were integral part of
LC method in the course of optimizing the conditions of proposed method. The system suitability test solutions were
injected and chromatographic parameters for MTS and EBS were evaluated for proving
the system suitability. (Table 2)
Table 2 System Suitability study
System Suitability Parameters |
MTS |
EBS |
Retention Time (Min) |
6.58 |
2.98 |
Theoretical Plates |
5302.5 |
3545.4 |
Area Under Curve |
659.13 |
690.34 |
Tailing Factor |
0.96 |
1.16 |
Resolution
of MTS and EBS:
Standard
solution of MTS and EBS were injected to get a chromatogram. The retention time of MTS and EBS were found
to be 6.56 and 2.95 min respectively.
Chromatogram of MTS and EBS is shown in Fig 4.
Fig 4 HPLC
chromatogram of MTS and EBS
RESULTS:
VALIDATION31-37:
Validation of analytical method is a
process to establish performance characteristics of developed method which
meets the requirement with intended analytical applications. This method was
validated according to ICH guideline for linearity, precision, accuracy,
robustness, LOD and LOQ.
Linearity:
The linearity of an analytical method is
the ability to obtain test results which are directly proportional to
concentration in sample.This was studied by analyzing five different concentrations
in selected range of 5 – 25 µg/mL for MTS and EBS. The analytical data for
Linearity is tabulated in Table 3.
Precision:
The precision of an analytical method
expresses the closeness of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the same homogeneous sample
under the prescribed conditions.
Precision was determined by repeatability, inter-day and intra-day
experiments. Standard solution
containing MTS and EBS were injected six times. The mean amount and standard
deviation (SD) value of each constitute were calculated and mentioned in Table
3.
Accuracy:
The accuracy of an analytical method is the
closeness of the test result obtained by that method to true value. The
accuracy was calculated from the test results as the percentage of analyte
recovered by the assay. These studies were performed by standard addition
method at 80 %, 100 % and 120 % levels as stated in ICH Guideline. Results are
tabulated in Table 4.
Robustness:
The
robustness is the capacity of method to remain unaffected by small but
deliberate changes in chromatographic conditions. Robustness was studied by
testing the influence of small changes in column temperature (±5°C), change in
flow rate (±10 %) and changes in mobile phase composition (75:25). The obtained
results are mentioned in Table 5.
LOD and LOQ:
LOD and LOQ for the optimized method were
performed as per ICH guidelines. LOD was expressed by establishing the minimum
level at which the analyte can be reliably detected. LOQ was considered as the
lowest concentration of analytes in standards that can be reproducibly measured
with acceptable accuracy and precision. LOD and LOQ) were separately determined
at a signal to noise ratio (S: N) of 3: 10 and which was based on calibration
curve. The standard deviation of y intercept and slope of the regression line
were used. The LOD and LOQ were calculated by using following formulas and
results are mentioned in Table 6.
LOD = 3.3 × D / S
LOQ = 10 × D / S
Where,
S = Slope of regression line, D = Standard deviation of y-
intercept on the regression line.
Table 3 Validated analytical parameters
Parameters |
MTS |
EBS |
Linearity Range |
5 – 25 µg/mL |
5 – 25 µg/mL |
Intercept |
-0.30 |
-1.53 |
Slope |
0.025 |
0.024 |
Corre. Coef. |
0.999 |
0.999 |
Inter day Preicision (%RSD, n=3) |
0.250 |
0.403 |
Intraday Preicision (%RSD, n=3) |
0.522 |
0.479 |
Table 4 Recovery Studies of MTS and EBS
Level
of Recovery |
Drug |
%
Recovery |
Standard Deviation* |
% RSD |
80 % |
MTS |
99.25 |
0.395 |
0.397 |
EBS |
100.33 |
0.497 |
0.495 |
|
100 % |
MTS |
99.9 |
0.507 |
0.508 |
EBS |
99.76 |
0.475 |
0.476 |
|
120 % |
MTS |
99.91 |
0.409 |
0.409 |
EBS |
100.14 |
0.395 |
0.394 |
*Denotes average of three determinations.
Table 5 Robustness Studies of MTS and
EBS
Factor |
Retention Time |
Factor |
Retention Time |
||
Flow
Rate (mL/min) |
MTS |
EBS |
Mobile Phase (v/v) |
MTS |
EBS |
0.9 |
6.63 |
2.96 |
79:21 |
6.66 |
2.96 |
1.0 |
6.70 |
2.95 |
80:20 |
6.60 |
2.95 |
1.1 |
6.63 |
2.96 |
81:29 |
6.56 |
2.91 |
Mean |
6.65 |
2.95 |
Mean |
6.60 |
2.94 |
S.D. |
0.0404 |
0.0057 |
S.D. |
0.0503 |
0.0264 |
%RSD |
0.6074 |
0.1952 |
%RSD |
0.7618 |
0.8999 |
Table No. 6
Results of LOD and LOQ
Sample |
LOD
(µg/mL) |
LOQ
(µg/mL) |
MTS |
1.05 |
3.20 |
EBS |
1.13 |
3.44 |
DISCUSSION:
In literature various methods are discussed
for MTS and EBS with each other or with another drug combination separately.
Sony LK. et.al. (2012)26 reported simultaneous estimation of ebastin
with phenylephrine hydrochloride by UV absorption method whereas RIM and Tarek
(2012)27 in his study reported HPLC and UV method for MTS and EBS
but this showed more retention time. Anand J et.al (2013)28, Jangid
RK et.al. (2013)29, Savsani JJ et.al (2012)30 and
Ambadkar SG et.al. (2013)31 were reported separately HPLC methods
for simultaneous estimation of MTS and EBS showing linearity range at 5-35
µg/mL, 1.2 ml/min flow rate and etc. this indicating scope to minimize total
cost of analysis.
CONCLUSION:
Proposed study describes HPLC method for
the simultaneous estimation of MTS and EBS in bulk as well as in tablet
formulation. This method showed good separation of two compounds with less
retention time than any method listed in reference. This method is useful to
minimize total cost of analysis. The method is validated and found to be
simple, sensitive, accurate, precise and robust. Hence proposed method is suggested for
routine quality analysis of MTS and EBS in laboratories.
ACKNOWLEDGEMENT:
The
authors are wish to thank Principal and Management of Shivnagar Vidya Prasarak
Mandal’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune, Department of Pharmaceutical Sciences, Pacific Academy of Higher
Education and Research (PAHER) University, Udaipur, Rajasthan and Scan
Research Bioanalytical Laboratories, Bhopal, for providing us required lab
facilities with enthusiastic environment.
REFERENCES:
1.
The Merck Index. An encyclopedia of chemicals, drugs and
biological, Merck and Co., Inc. USA; 2006. 14th ed, Monograph 6258, p1080.
2.
Riccioni G, Vecchia
RD, D'Orazio N, Sensi S, Guagnano MT. Comparison of montelukast
and budesonide on bronchial reactivity in subjects with mild-moderate
persistent asthma. Pulm Pharmacol Ther. 2003;
16(2):111-4.
3.
Simons FE. Montelukast added to budesonide
in children with persistent asthma: a randomized, double blind, crossover
study. Journal of pediatrics.
2001; 138(5): 694.
4.
Claesson HE, Dahlen SE. Asthma and leukotrienes: antileukotrienes
as novel anti-asthmatic drugs. J
Internal Med. 1999; 245: 205-227.
5.
Ibrahim AA. 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. 2004;
12(4): 136-143.
6.
Prashant Kumar K, Akiful H. Stability indicating RP-HPLC method
for the estimation of montelukast in pharmaceutical dosage form. J of Pharm Bio Sci. 2012; 1(6):
31-36.
7.
Singh RM. Development and validation of a RP-HPLC method for estimation
of montelukast sodium in bulk and in tablet dosage form. Ind J Pharm Sci. 2010; 72(2): 235–237.
8.
Balasekhara RC. Method development and validation of montelukast
in human plasma by HPLC coupled with ESI-MS/MS application to a bioequivalence
study. Scientia Pharmaceutica.
2010; 78: 411–422.
9.
Yuliya S. Determination of montelukast sodium by capillary
electrophoresis, J of Separation Sci,
2008; 31(6-7): 1137–1143.
10. Patil SS, Mandrupkar SN. Development and statistical
validation of spectrophotometry method for estimation of montelukast in bulk
and tablet dosage form. J Pharm Res, 2009, 2(4), 714-716.
11.
Kalyankar TM, Development and validation of RP-HPLC method for
estimation of montelukast sodium and fexofenadine hydrochloride in
pharmaceutical preparations. Chem Sci
Tran. 2013; 2(3): 889-899.
12.
Patil S, Pore YV, Kuchekar BS. Determination
of montelukast sodium and bambuterol hydrochloride in tablets using RP HPLC. Ind J Pharm Sci. 2009; 71(1): 58–61.
13.
Sharma S, Sharma MC, Kohil DV. Development
and validation of TLC-densitometry method for simultaneous quantification of
montelukast sodium and levocetirizine dihydrochloride pharmaceutical solid
dosage form. Der Pharmacia Lettre.
2010; 2 (1): 489-494.
14.
Rathore AS, Sathiyanarayanan L, Mahadik KR. Development of validated HPLC and HPTLC methods for simultaneous
determination of levocetirizine, dihydrochloride and montelukast sodium in bulk
drug and pharmaceutical dosage form. Pharmaceutica
Analytica Acta. 2010; 1(1): 1-6.
15.
Revathi R. High performance liquid
chromatographic method development for simultaneous analysis of doxofylline and
montelukast sodium in a combined form. J
Pharm Res. 2011; 2 (4): 223-228.
16.
British Pharmacopoeia; London: The Stationery
Office, 2009. vol
1, p. 735.
17.
Chauhan B, Nivsarkar M. New liquid extraction method for
determination for montelukast in small volume human plasma samples using HPLC
with fluorescence detector. Ind J Pharm
Sci. 2009; 71(1): 58–61.
18.
Radhakrishna T, Satyanarayana A. Simultaneous
determination of montelukast and loratadine by HPLC and derivative
spectrophotometric methods. J Pharm
Biomed Anal. 2003; 31(2): 359-368.
19.
Prabu SL, Dinesh KC. Determination of ebastine in
pharmaceutical formulations by HPLC. Ind
J Pharm Sci. 2008; 70(3): 406-407.
20.
Nelofer SM, Janardhan M. Analytical method development
and validation for the assay of ebastine in ebastine mouth dissolving tablets. Int J Pharm Clin Res. 2012; 4(4):
56-60.
21.
Marcela Z, Simone G. Development and validation of a stability-
indicating lc method for determination of ebastine in tablet and syrup. Chromatographia journal. 2009; 69(2):
195-199.
22.
Ashok P, Meyyanathan SN, Suresh B. Analysis of ebastine in
pharmaceutical preparations by high-performance thin-layer chromatography. Journal
of planar chromatography. 2003; 16 (2): 167-169.
23.
Ibrahim F, Eid M. Spectrofluorimetric determination of some H1
receptor antagonist drugs in pharmaceutical formulations and biological fluids, Int J Pharm Sci Res. 2011, 2(8):
2056-2072.
24.
Wagh R, Hajare R. Method development and validation for
simultaneous determination of ebastine and phenylephrine hydrochloride in
tablet formulation by RP-HPLC. Int J
Pharm Res Dev. 2011; 3(7): 214-220.
25.
Soni LK, Saxena C. Development and validation of UV spectro-
photometric assay protocol for simultaneous estimation of ebastine and
phenylephrine hydrochloride in tablet dosage form using simultaneous equation
method. Int J of ChemTech Res.
2011; 3(4): 1918-1925.
26.
Soni LK, Narsinghani T. UV spectrophotometric estimation
of ebastine and phenylephrine hydrochloride in tablet dosage form using
absorption ratio method. Der Pharmacia
Sinica. 2011; 2 (6): 11-16.
27.
Rim SH, Tarek SB. Gradient HPLC–DAD determination
of two pharmaceutical mixtures containing the antihistaminic drug ebastine. J Chrom Sci. 2012; 50(10): 862-868.
28.
Anand J, Mohan S. Development and
validation of RP-HPLC method for simultaneous estimation of ebastine and
montelukast sodium in combined dosage form. Am J PharmTech Res. 2013; 3(3): 669-777.
29.
Jangid RK, Magdum CS. Development and validation of UV
spectrophotometric method for simultaneous estimation of ebastin and
montelukast sodium in bulk and marketed formulaion. Int J Pharm Res Dev. 2013;
5(3): 51-56.
30.
Savsani JJ, Goti PP. Development and
validation of simultaneous equation method for estimation of ebastine and
montelukast sodium in combined tablet dosage form. Der
Pharmacia Sinica. 2012; 3 (6): 690-698
31.
Ambadkar SG, Chandewar AV, Bombaywala MA. An isocratic HPLC method for
simultaneous determination of montelukast and ebastinee in pharmaceutical
formulations. Int J Drug Form Res.
2013; 4(3): 127-140.
32.
Baokar SB, Ranpise NS. Development and validation of RP-HPLC
method for simultaneous estimation of vildagliptin and metformin. Res J Pharma Dosage Forms and Tech. 2013; 5(2): 95-98.
33.
Mangolkar K. Simultaneous estimation of
lamivudine and tenofovir disproxil fumarate in tablets by isocratic reverse
phase high performance liquid chromatography method. Indian Drugs. 2008; 45(2): 119-122.
34.
Mohammad AM, Marwa AF. Development and validation of a reversed‐phase column liquid chromatographic method for simultaneous
determination of two novel gliptins in their binary mixtures with Metformin. Euro J Chem. 2012; 3(2): 152‐155.
35.
Shaikh KA, Patil SD, Devkhile AB. Development
and validation of a reversed-phase HPLC method for simultaneous estimation of
ambroxol hydrochloride and azithromycin in tablet dosage form. J Pharm Biomed Anal. 2008; 48(5):
1481–1484.
36.
Santhosha B, Sundari CH. Validated method for the simultaneous
estimation of metformin hydrochloride and vildagliptine by RP-HPLC in bulk and the
pharmaceutical dosage form. Int Res J Pharm Appl Sci. 2012; 2(3): 22-28.
37.
De AK, Bera AK, Pal B. Development and validation of same RP-HPLC method for separate
estimation of theophylline and doxofylline in tablet dosage forms. J Curr Pharm Res. 2012; 9 (1): 55-58.