INTRODUCTION:

Montelukast Sodium is a leukotriene antagonist, and it is used as anti-asthmatic and also in the treatment of allergic rhinitis and chemically it is 1-[[[(1R)-1-[3-1(E)-2-7chloro2quinolinyl)ether]phenyl] 3[2(1hydroxy1 methylethyl)phenyl]propyl] thio] methyl]cycly propancacetic acid^1-9, Literature described cited for the quantification of, montelukast Sodium HPLC and derivative spectrophotometric methods spectrofluorometric methods^10-14 for its determination in pharmaceutical determination alone and with other combination. Levocetirizine hydrochloride. It is active Isomer (R-form) of cetirizine. It selectively inhibits Histamine H₁-receptors chemically it is [2-[4-[(4-chlorophenyl)-phenylmethyl]-1-piperazinyl] ethoxy] acetic acid, Literature described cited for the quantification of levocetirizine hydrochloride RP-HPLC, UV Spectrophotometric methods^15-20 for its determination in pharmaceutical determination alone and with other combination. The present paper describes a simple, rapid, accurate and reproducible method for the simultaneous estimation of montelukast sodium and levocetrizine hydrochloride in tablet formulation by RP-HPLC method.

EXPERIMENT:

Materials:

Samples of montelukast sodium and levoceterizine hydrochloride were gift sample from Ranbaxy laboratories ltd, Gurgaon, India. Tablets of Romilast 10mg (Ranbaxy), Levosiz 10mg (systopic) was procured from the local market. Methanol (HPLC Grade), Acetonitrile (HPLC Grade) Water (HPLC grade)

Chromatographic System and Conditions:

Analysis was performed with a Shimadzu Model – LC-10AT VP Pump SPD-10A and UV-Visible detectors shimadzu, and symmetry C₁₈250X4.6 mm, 5µm, Phenomenex ODS analytical column was used for the separation. The mobile phase was delivered through the column at a flow rate of 1.0 ml/min. Column was operated at ambient temperature (~22^oC). The sample injection volume was 20µl. The UV detector was set at a wavelength of 232nm. Ax – 200 Digital Balance, Sonica ultra sonic cleaner- model 2200 MH solutions

Mobile phase preparation:

The mobile phase consisted of a mixture of acetonitrile, methonal, water in the ratio 40:40:20(v/v). The mobile phase was fillter through at 0.45µm nylon membreane filter and degassed prior to use.

Standard preparation:

Standard solution of montelukast sodium and levocetrizine hydrochloride were prepared in mobile phase. The working concentrations for both the drugs were 0.050mg/ml. The volumetric flask containing montelukast sodium were wrapped with black paper and stored in the dark. A typical chromatogram of two compounds is shown in the (Fig.1 and 2)

Fig. 1: Typical standard chromatogram of Levocetrizine hydrochloride.

Fig . 2: Typical standard chromatogram of Montelukast sodium

Table 1: System Precision:

S. No	Area (Levo)	Area (Mont)	Acceptance Criteria
1	6179453	8220967	RSD should be NMT 1.0% or half of the system suitability
2	6166547	8139107
3	6289139	8289445
4	6187405	8183174
5	6188715	8155277
6	6196423	8159013
Average	6201280	8191164
STDEV	40365	55933
%RSD	0.6509	0.6829

Preparation of montelukast sodium and levocetrizine hydrochloride samples from Romilast and Levosiz tablets:

Twenty tablets of Romilast (each tablets containing 10mg of montelukast sodium) and Levosiz (each tablets containing 10mg of levoceterizine hydrochloride) in each case were weighed and ground to powder separately. Accurately weighed powder samples equivalent to 50mg of montelukast sodium and 50mg of levoceterizine hydrochloride were transfer to a 100ml volumetric flask.About 50ml of solvent consisting a mixture of acetonitrile, methanol and water in the ratio 40:40:20 (v/v) was added to the flask and placed in an ultrasonic bath at room temperature for 15min. The solution was cooled and made up to volume and then centrifuged using Biofuse –13 centrifuger. The supernatant was taken into a clean dry flask, tightly capped and wrapped with black paper and placed in the dark. Different sample solutions of required concentrations were prepared using supernatant

RESULTS AND DISCUSSION:

Method development and Optimisation:

To investigate the appropriate wavelength for simultaneous determination of montelukast sodium and levocetrizine hydrochloride, solutions of these compounds in the mobile phase were scanned by UV–visible spectrophotometry (shimadzu, uv-1700, pharmaspec. Cuvetts: 1 cm matched quartz cells) in the range 210–400 nm. From the overlaid UV spectra, suitable wavelength considered for monitoring the drugs was 232 nm (Fig. 3).

Fig. 3: Overlaid UV spectra of Montelukast sodium and Levocetrizine hydrochloride

Table 2: Method Precision:

Injection Number	Area responses
Injection Number	Levo	Mont
1	99.65	100.36
2	99.44	99.36
3	101.42	101.19
4	99.78	99.9
5	99.78	99.56
6	99.92	99.61
Average	99.9983	99.9967
STDEV	0.7149	0.6798
%RSD	0.7150	0.6799

For HPLC analysis, initially various mobile phases and stationary phases were tried in attempts to obtain the best separation and resolution between montelukast sodium and levocetrizine hydrochloride. The mobile phase consisting of acetonitrile, methonal, water in the ratio 40:40:20(v/v) was found to be an appropriate mobile phase allowing adequat separation of two drugs using a symmetry C₁₈ 250 mm column at a flow rate of 1.0ml/min. A typical chromatogram of separation of two compounds is shown in (Fig.4)

Table 3: Accuracy data Evolution:

S. No	Sample	Sample	Wt of Sample (mg/ml)	Amount of STD added per ml (mg)	Amount of STD recovery per ml (mg)	Percentage Recovery
1	Levocetrizine hydrochloride	Sample +80%	0.0502	0.00803	0.00801	99.75
2		Sample +100%	0.0502	0.01002	0.01001	99.90
3		Sample +120%	0.0502	0.01203	0.01207	100.33
4	Montelukast sodium	Sample +80%	0.0503	0.00803	0.00804	100.12
5		Sample +100%	0.0503	0.01002	0.01001	99.90
6		Sample +120%	0.0503	0.01203	0.01213	100.83

Fig. 4: HPLC Chromatogram of Montelukast sodium and Levocetrizine hydrochloride

Method validation:

The developed method was validated for simultaneous assay determination of montelukast sodium and levocetrizine hydrochloride using following parameters.

Linearity:

Linearity was checked by preparing standard solution of both montelukast sodium and levocetrizine hydrochloride at four different concentration levels in the same volumetric flasks using their respective stock solutions. The calibration curves for montelukast sodium and levocetrizine hydrochloride were drawn in the concentration range of 0.030-0.060 mg/ ml, respectively (Fig. 5).

Fig. 5: Linearity of Levocetrizine hydrochloride and Montelukast sodium

Precision and Accuracy:

System precision:

The system precision was evaluated by measuring the peak response of montelukast sodium and levocetrizine hydrochloride solution prepared as per the proposed method and chromatograms were recorded Tabel.1.

Method Precision:

The method precision was determined by preparing the sample of a single batch of formulation six times and analyzing as the proposed method Table.2.

Accuracy:

A study of accuracy was conducted. Drug assay was performed in triplicate by spiking with equivalent amount of montelukast sodium and levocetrizine hydrochloride raw material into each volumetric flask for each spike level to get the concentration equivalent to 80%, 100%, and 120% of the labeled amount as per the test method. The average % recovery of montelukast sodium and levocetrizine hydrochloride was found to be 100.28% and 99.99% (within the limits). The results were summarized in Table. 3

Table 4: Analyst to Analyst variation:

Injection Number	Assay performed by Analyst - I		Assay performed by Analyst - II
Injection Number	Levo	Mont	Levo	Mont
1	101.1	100.4	100.8	100.3
2	100.4	99.8	100.3	99.5
3	99.7	99.9	100	100.1
4	99.5	100.1	99.3	100.2
5	100.1	100	99.7	99.9
6	100.2	100.1	100.1	99.8
Average	100.17	100.05	100.03	99.97
STDEV	0.5646	0.2074	0.5125	0.2944
%RSD	0.5636	0.2073	0.5123	0.2945

Ruggedness:

The ruggedness of an analytical method is the of reproducibility of test results obtained by the analysis of the same sample under a variety of conditions, such as different laboratories, different analysis, different instruments, different list of reagents, different elapsed assay times, different assay temperatures, difference days etc., Table 4-6.

CONCLUSIONS:

RP-HPLC method was developed and validated for the simultaneous determination of montelukast sodium and levocetirizine hydrochloride in pharmaceutical dosage forms. RP-HPLC method developed using methanol: acetonitrile: water in the ratio of 40:40:20 as a mobile phase.

Table 5: Effect of variation in mobile flow rate

System Suitability Parameter	Observed Value - Levocetrizine			Observed Value - Montelukast			Acceptance criteria
System Suitability Parameter	0.6ml/min	0.7ml/min	0.8ml/min	0.6ml/min0.	0.7ml/min	0.8ml/min	Acceptance criteria
RSD of Montelukast sodium and Levocetrizine hydrochloride peak area from six replicate injection of standard preparation	0.1%	0.1%	0.1%	0.1%	0.1%	0.1%	NMT 2.0%

Table 6: Effect of variation in column temperature

System Suitability Parameter	Observed Value - Levocetrazine			Observed Value - Montelukast			Acceptance criteria
System Suitability Parameter	28^°C	30^°C	32^°C	28^°C	30^°C	32^°C	Acceptance criteria
RSD of Montelukast sodium and Levocetrizine hydrochloride peak area from six replicate injection of standard preparation	0.1%	0.1%	0.1%	0.1%	0.1%	0.1%	NMT 2.0%

It is a simple mobile phase. Mobile phase preparation is very simple and utilizes less time. The developed method was accurate and precise, which was evident for the analytical data and recovery studies. Therefore this method can be extended to routine analysis of montelukast sodium and levocetirizine hydrochloride in tablet dosage forms.

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Received on 15.07.2010 Modified on 02.08.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1069-1072