1. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
ISSN:2249-5347
IJSID
International Journal of Science Innovations and Discoveries An International peer
Review Journal for Science
Research Article Available online through www.ijsidonline.info
A SIMPLE RP-HPLC METHOD FOR SIMULTANEOUS ANALYSIS OF PSEUDOEPHEDRINE, BAMBUTEROL,
LEVOCETIRIZINE AND MONTELUKAST IN PHARMACEUTICAL DOSAGE FORMS
1Department of Chemistry, JNT University, Kukatpally, Hyderabad, AP, India; 2AR&D, Custom Pharmaceutical Services, Dr.
Hanimi Reddy Bapatu*1, Maram Ravi Kumar2, Useni Reddy Mallu3, R.S. Murthy1 and Harikishan Reddy Ganthi3
Reddys Laboratories Ltd, Bachupally, Hyd-72, India; 3Department of Chemistry, Sri Krishnadevaraya University, Anantapur,
AP, India
A simple and economic reverse phase high performance liquid chromatography
ABSTRACT
(RP-HPLC) method has been developed for the estimation of Pseudoephedrine,
Bambuterol, Levocetirizine and Montelukast in bulk and tablet dosage forms. Separation
was achieved on C18 column (150 x 4.6mm i.d., 5μm) using gradient mobile phase Sol-A:
Received: 13.02.2012
Accepted: 19.06.2012 buffer (weighed accurately 1gm of potassium di-hydrogen phosphate in to 1000mL of
HPLC grade water) and Sol-B: acetonitrile gradient program (0-4min, sol-A:98-98; 4-
8min- sol-A: 98-75; 8-12min- sol-A: 75-70; 12-15min- sol-A: 70-45; 15-20min- sol-A: 45-
*Corresponding Author
20; 20-25min- sol-A: 20-98 and 25-30min- sol-A: 98-98), pumped in to the column at
flow rate of 1.0 ml/min and the detection of eluent from the column was carried out
using variable wavelength detector at 210nm. The total run time was 30 min and the
column oven temperature was maintained at 35°C. The retention times of
Pseudoephedrine, Bambuterol, Levocetirizine and Montelukast were 4.1 min, 10.9 min,
15.4 and 21.1 min, respectively. The standard curves were linear over the concentration
range of 10-60 μg/ml. The method was validated as per ICH guidelines. Validation
studies demonstrated that the proposed RP-HPLC method is simple, specific, rapid,
Address:
reliable and reproducible. The suitability of the proposed method for the routine quality
INTRODUCTION
Name:
control analysis in bulk and tablet dosage forms.
Hanimi Reddy Bapatu
Place:
JNT University,
Hyderabad, India
E-mail:
hanimi.b@gmail.com
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377

2. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
INTRODUCTION
Montelukast sodium is a leukotriene receptor antagonist and used in the treatment of asthma. it is a hygroscopic,
optically active, white to off-white powder and freely soluble in ethanol, methanol, and water and practically insoluble in
acetonitrile. Side effects of montelukast are skin rash, bruising, severe tingling, numbness, pain, muscle weakness, mood or
behavior changes, anxiety, depression, tremors or shaking, severe sinus pain, swelling or irritation or worsening asthma.
General dose of montelukast is for adults and adolescents 15 years of age and older: one 10-mg tablet, for pediatric patients 6
to 14 years of age: one 5-mg chewable tablet and for pediatric patients 2 to 5 years of age: one 4-mg chewable tablet or one
packet of 4-mg oral granules. For pediatric patients 12 to 23 months of age: one packet of 4-mg oral granules. Safety and
effectiveness in pediatric patients less than 12 months of age with asthma have not been established. Studies are in progress
on clinical trials in patients with asthma to evaluate the relative efficacy of morning versus evening dosing. The
pharmacokinetics of montelukast are similar whether dosed in the morning or evening. Efficacy has been demonstrated for
asthma when montelukast was administered in the evening without regard to time of food ingestion.
Bambuterol hydrochloride is a direct acting sympathomimetic with predominantly -adrenergic activity (β2-agonist)
and an ester prod rug of β2 adrenergic agonist and it is known to a producing of terbutaline but has better therapeutic to
toxicity ratio and acts as an endogenous reservoir. Bambuterol is a pro-drug of terbutaline that is slowly converted into the
body to the form.
Pseudoephedrine is a decongestant that shrinks blood vessels in the nasal passages. It is used in the treatment of nasal
and sinus congestion, congestion of the tubes that drain fluid from you inner ears. Do not give pseudoephedrine to a child
younger than 4 years old it can give adverse effects of death.
Pseudoephedrine Bambuterol
Figure-1: Chemical structures of active ingredients
Levocetirizine Montelukast
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3. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
Levocetirizine is a third generation non-sedative antihistamine cetirizine. Chemically it is the active enantiomer of
cetirizine. It works by blocking histamine receptors and it may not prevent the actual release of histamine from mast cells, but
prevents in binding to its receptors. This in turn prevents the release of other allergy chemicals and increased blood supply to
the area and provides relief from the typical symptoms of hay fever.
Dosage forms list
Tablets Montelukast 4 mg chewable tablets
Dosage form Composition
Montelukast 5 mg chewable tablets
Montelukast 10 mg tablets
Montelukast oral granule packets -- containing 4 mg per packet
Psedoepidrine HCl-15mg/60mg
pseudoephedrine hydrochloride USP 120mg/tablet
pseudoephedrine hydrochloride USP 240mg/tablet
Bambuterol Tablets 10 mg
Bambuterol Tablets 20 mg
Levocetirizine dihydrochloride 5mg tablet.
Syrup Bambuterol hydrochloride 5mg/5ml syrup
pseudoephedrine hydrochloride 30mg/5mL
Literature survey reveals that numerous methods (1-27) have been reported for the analysis of Montelukast Sodium,
bambuterol, Pseudoephedrine and Bambuterol for individual and combination drug products but not method available for the
determination of four drugs in single time. The present research work is to develop a single and simple method for the
determination of these four drugs by HPLC method.
MATERIALS AND METHODS
A waters HPLC system consisting of alliance 2695, agilent 1200 series HPLC instrument with UV-Visible detector, two
Instruments:
systems were operated by Empower software. Waters make X-Terra C18 1504.6mm, 5µm column, Mettler Toledo made
analytical balance were used for this study
Pure (not less than 98.5%) standards of all active ingredients, HPLC grade acetonitrile and water; AR grade of
Materials:
Potassium Dr-Hydrogen orthophosphate were used.
Mobile phase:
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4. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
Sol-A: weighed accurately 1g of potassium hydrogen phosphate, transferred in to 1000ml of HPLC water and mixed.
Filtered the final solution through a 0.4μm membrane filter; Sol-B: HPLC grade acetonitrile.
Mixed the HPLC water and acetonitrile in the ratio of 1:1 (v/v).
Diluent:
Transferred the 40mg of Levocetirizine standard in to 100mL volumetric flask and added 10ml of methanol and shake
Standard solution:
well then added 40mg of each Montelukast sodium, Bambuterol and Pseudoephedrine standards in to the same volumetric
flask, 50ml of diluent added and sonicated to dissolve and made up to volume with diluent. Further diluted the 5ml of resulting
solution into 50ml with diluent (each standard 40ppm).
Prepared the all dosage forms to get each active ingredient equal to 40microgram per mL with diluent and analyzed.
Test solution:
Chromatograph : Waters/ Agilent HPLC system with Empower software.
Chromatographic conditions
Mobile phase : Solution-A and solution-B with gradient elution.
Gradient program : (0-4min, sol-A: 98-98; 4-8min-sol-A: 98-75; 8-12min-sol-A: 75-70; 12-15min-sol-A: 70-45; 15-
20min-sol-A: 45-20; 20-25min-sol-A: 20-98 and 25-30min-sol-A: 98-98;)
Column : Waters make X-Terra C18 1504.6mm, 5µm.
Flow rate : 1.0 mL per min
Detection : 210nm
Injection volume : 10 μL
Retention time : Pseudoephedrine-4.1, Bambuterol-10.9, Levocetirizine-15.4 and Montelukast-21.1.
Run time : 30 min.
Column oven temp : 35°C
: All active ingredients were quantified with the following calculation.
Sample area x standard concentration x Potency of standard
Calculation
-------------------------------------------------------------------------- X 100
Standard area x sample concentration x 100
RESULTS AND DISCUSSION
The mobile phase was chosen after several trials with methanol, acetonitrile, water and buffer solutions in various
Method Development
proportions and at deferent pH values. Flow rates between 0.5 and 1.5/min were studied. A flow rate of 1.0 ml/min gave an
optimal signal to noise ratio with a reasonable separation time. Using a reversed-phase C18 column, the retention times of
Pseudoephedrine, Bambuterol, Levocetirizine and Montelukast were 4.1 min, 10.9 min, 15.4 and 21.1 min respectively. Total
time of analysis was less than 30 min. The maximum absorption of Pseudoephedrine, Bambuterol, Levocetirizine and
Montelukast together as detected at 210nm and this wavelength was chosen for the analysis. The chromatogram at 210nm
showed a complete resolution of all peaks (Figure-3).
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5. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
System suitability parameters were established by injecting the freshly prepared standard solution (each active
System suitability
40microgram per mL/five replicate injections) in to the chromatographic system. The percent relative standard deviation for
peak area and retention time results found to be satisfactory. System suitability chromatograms were represented in figure-4
and tabulated the results in table-1 and 2.
The precision of the method was demonstrated by inter day and intraday variation studies. In the intraday studies, six
Precision:
repeated injections of standard and sample solutions were made and the area of drug peaks and percentage RSD were
calculated. In the inter day variation studies, six repeated injections of standard and sample solutions were made for three
consecutive days and area of drug peaks and percentage RSD were calculated. From the data obtained, the developed HPLC
method was found to be precise.
Linearity is determined by calculating the regression line using a mathematical treatment of the linearity results vs
Linearity:
analyte concentration (10microgram per mL to 60microgram per mL for each ingredient) of the standard solution. Linearity
graph was plotted against peak area and concentration of solution. The correlation coefficient value found to be within the
limit 0.999. The linearity chromatograms shown in figure-5 and linearity results tabulated in table-4 and linearity plots were
represented in graph-1.
Each factor selected (except columns from different manufacturers) was changed at three levels (−0.1, 0 and 0.1). One
Robustness and Ruggedness:
factor at the time was changed to estimate the effect. Thus, replicate injections (n = 6) of mixed standard solution at three
concentration levels were performed under small changes of three chromatographic parameters (factors). Insignificant
differences in peak areas and less variability in retention time were observed
Figure-2: Diluent chromatogram
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6. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
Table-1: System suitability (Area %RSD)
Active Ingredient Standard solution Area
Pseudoephedrine
Name Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD
Bambuterol
16524569 16522905 16477184 16701716 16634850 16572245 0.56
Levocetirizine
1868913 1789289 1849078 1841880 1852652 1840362 1.64
Montelukast
2043705 2038930 2033310 2027799 2036356 2036020 0.29
2900859 2902302 2901514 2899794 2916066 2904107 0.23
Figure-3: Standard chromatogram
Table-2: System suitability (Retention time %RSD)
Standard solution Retention time (min)
Pseudoephedrine
Active Ingredient Name
Inj-1 Inj-2 Inj-3 Inj-4 Inj-5 Average %RSD
Bambuterol
4.11 4.12 4.12 4.12 4.12 4.118 0.11
Levocetirizine
10.89 10.9 10.9 10.91 10.91 10.902 0.08
Montelukast
15.38 15.39 15.39 15.39 15.4 15.39 0.05
21.09 21.1 21.11 21.1 21.11 21.102 0.04
Table-3: System suitability (USP Tailing factor)
Standard solution Tailing factor
Pseudoephedrine
Active Ingredient Name
Inj-1 Inj-2 Inj-3 Inj-4 Inj-5
Bambuterol
1.0 1.0 1.0 1.1 1.1
Levocetirizine
1.2 1.1 1.1 1.1 1.1
Montelukast
0.9 0.9 0.9 0.9 0.9
0.8 0.8 0.8 0.8 0.8
Table-4: System suitability (USP Resolution)
Standard solution Resolution
Resolution between Pseudoephedrine and Bambuterol
Active Ingredient Name
Inj-1 Inj-2 Inj-3 Inj-4 Inj-5
Resolution between Bambuterol and Levocetirizine
11.18 11.54 11.69 11.91 12.11
Resolution between Levocetirizine and Montelukast
21.69 2186 21.6 21.48 21.74
26.37 26.66 26.4 26.69 26.37
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7. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
Figure-4: Standard solution system suitability chromatograms
Table-5: Linearity Results.
Linearity solutions area
Active Ingredient Linearity Linearity Linearity Linearity Linearity
Linearity level- Co-relation
Name level-2 level-3 level-4 level-5 level-6
1 (25%) Coefficient
(50%) (75%) (100%) (125%) (150%)
Pseudoephedrine
Bambuterol
3929981 8152866 12291125 16516368 20666550 24676668 0.999970
Levocetirizine
410793 870869 1322585 1795780 2232422 2769734 0.999639
Montelukast
481666 1001290 1513533 2028715 2535195 3048322 0.999994
680955 1425061 2167601 2915401 3657614 4401669 1.000000
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8. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
Figure-5: Overlaid Linearity chromatograms
A gradient RP-HPLC method developed and validated for the simultaneous determination of Pseudoephedrine,
CONCLUSION
Bambuterol, Levocetirizine and Montelukast in both bulk and tablet dosage form. The validation results reveals that, method
have good precision and accuracy, which proves the reliability of the proposed method. The retention time of
Pseudoephedrine is 4.2min, Bambuterol is 10.9min, Levocetirizine is 15.3min and Montelukast is 21.1min. linearity results
were found to be linear for Pseudoephedrine is 0.9999, Bambuterol is 0.999, Levocetirizine is 0.9999 and Montelukast is
1.000. The short runtime and low solvent consumption are advantageous for applying in quality control analysis.
Sweetman SC, editor. Martindale: The Complete Drug Reference. 33rd ed. London: Pharmaceutical Press; 2002. p. 768.
REFERENCES
Budavari S, editor. The Merck Index. 12th ed. Whitehouse Station, NJ: Merck & Co Inc; 1996. p. 1070.
1.
Morrow JD, Roberts LJ. In: Goodman and Gilman's The Pharmacological Basis of Therapeutics. 10th ed. Hardman JG,
2.
Limbird LE, Gilman AG, editors. New York: McGraw-Hill; 2001. p. 669.
3.
Al-Rawithi S, Al-Gazlan S, Al-Ahmadi W, Alshowaier I, Yusuf A, Raines D. Expedient liquid chromatographic method with
fluorescence detection for montelukast sodium in microsamples of plasma. J Chromatogr B Biomed Sci Appl.
4.
2001;754:527–31
Ochiai H, Uchiyama N, Takano T, Hara K, Kamei T. Determination of montelukast sodium in human plasma by column-
switching high performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Appl.
5.
1998;713:409–14.
Chauhan B, Shubha Rani, Nivsarkar M, Padh H. New liquid liquid extraction method for determination for montelukast in
small volume human plasma samples using HPLC with fluorescence detector. Indian J Pharm Sci. 2006;68:517–20.
6.
Liu L, Cheng H, Zhao JJ, Rogers JD. Determination of montelukast (MK-0476) and S-enatiomer in human plasma by
stereoselective high performance liquid chromatography with column switching. J Pharm Biomed Anal. 1997;15:631–8.
7.
Radhakrishna T, Narasaraju A, Ramakrishna M, Satyanarayana A. Simultaneous determination of montelukast and
loratadine by HPLC and derivative Spectrophotometric methods. J Pharm Biomed Anal. 2003;31:359–68
8.
International Journal of Science Innovations and Discoveries, Volume 2, Issue 3, May-June 2012
384

9. Hanimi Reddy Bapatu et al., IJSID, 2012, 2 (3), 377-385
Alsarra I. 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;12:136–43.
9.
10. Sweetman SC, editor. Martindale: The Complete Drug Reference. 33rd ed. London: Pharmaceutical Press; 2002. p. 761.
11. Budavari S, editor. The Merck Index. 12th ed. Whitehouse Station, NJ: Merck & Co Inc; 1996. p. 163.
12. The British Pharmacopoeia. 3rd ed. Vol. 1. London: Stationary Office Books; 2001. pp. 174–5.
13. Zhang Y. Determination bambuterol hydrochloride tablets by. Jiangsu Pharm Clin Res. 2001;9:13–4.
14. Bartolincic A, Druskovic V, Sporec A, Vinkovic V. Development and validation of HPLC methods for the enantioselective
analysis of bambuterol and albuterol. J Pharm Biomed Anal. 2005;36:1003–10
15. Wannerberg O, Persson B. Liquid chromatographic methods for the determination of bambuterol hydrochloride and
related compounds. J Chromatogr A. 1988;435:199–203.
16. Harris R, Hodgkinson P, Larsson T, Muruganatham A. Quantification of bambuterol hydrochloride in a formulated product
using solid-state NMR. J Pharm Biomed Anal. 2005;38:858–64.
17. Chauhn B, Rani Shubha, Nivasarkar M, Padh H, Indian Journal of Pharmaceutical Sciences, 2006, 68 (4), 517-520.
18. Alsarra I, Khalil NY, Sultan M, Al-Ashaban R, Pharmazie, 2005, 60 (11), 823-826.
19. Smith GA, Rawls CM, Kunka RL, Pharm. Res, 2004, 21 (9), 1539-1544.
20. Arayne MS et al., Simultaneous quantitation of cefpirome and cetirizine or levocetirizine in pharmaceutical formulations
and human plasma by RP-HPLC, Journal of Ana. Chem, 2008, 63, 881-887.
21. D. W. Empey, G. A. Young, E. Letley, G. C. John, P.Smith, K. A. McDonnell, L. R. Bagg, D. T.Hughes, Br.J. Clin. Pharmacol., 9, 351
(1980).
22. H. Richardson, B.A. Bidlingmeyer, J. Pharm. Sci. 73 (1984) 1480.
23. A. Yacobi, Z.M. Look, C.M. Lai, J. Pharm. Sci. 67 (1978)1668
24. M. K. Chao, I. J. Holcomb, S. A. Fusari, J. Pharm. Sci.,68, 1463 (1979)
25. B. R. Thomas, X. Fang, P. Shen, S. Ghodbane, J. Pharm.Biomed. Anal., 12, 85 (1994)
26. M. D. Paciolla, S. A. Jansen, S. A. Martellucci, A. A.Osei, J. Pharm. Biomed. Anal., 26, 143 (2001).
27. The United States Pharmacopeia 24, United States Pharmacopeial Convention, Inc, Rockville, 2000, p. 32.
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