VALIDATED RP – HPLC METHOD FOR DETERMINATION OF MONTELUKAST SODIUM AND LEVOCETIRIZINE IN BULK AND ITS PHARMACEUTICAL FORMULATIONS USING UV-VIS DETECTOR
RAVISANKAR.M*1, SUBASINI.U2, ANAND THANGADHURAI.S3, KARTHIKEYAN.S4, CHANDRA SEKAR.E.5
1.Swamy Vivekanandha College of pharmacy, dept of pharmaceutical analysis. Thiruchengode
2.Swamy Vivekanandha College of pharmacy, dept of pharmacognosy. Thiruchengode
3.Swamy Vivekanandha College of pharmacy, dept of pharmaceutical analysis. Thiruchengode
4.Kausikh therapeutics and private limited.chennai
5.Swamy Vivekanandha College of pharmacy, dept of pharmaceutical analysis. Thiruchengode
An isocratic reverse phase high performance liquid chromatographic method for estimation of montelukast sodium and levocetirizine in bulk dosage and in marketed formulations has been devised and validated. The chromatographic separation achieved on shodex c18-4E column (5µm, 250 mm x 4.6 mm) and acetonitrile: methanol: ammonium acetate buffer (PH- 5.5) in the ratio of 25:55:20 v/v. The flow rate was 1.0 ml/min and the UV detection was identified at 225nm.The retention times for montelukast sodium and levocetirizine was found to be 5.15 min and 3.12 min respectively. The linearity of montelukast sodium and levocetirizine is 10 -50µg/ml with the correlation co efficient 0.99 respectively. The validation parameters such as accuracy, precision, LOD, LOQ, Robustness, ruggedness were performed as per ICH guidelines. This method can be used for routine analysis of montelukast sodium and levocetirizine in bulk and marketed dosage forms.
Reference Id: PHARMATUTOR-ART-1256
Montelukast sodium is a selective orally active leukotriene, receptor antagonist that inhibits the cysteinyl leukotriene CysLT1 receptor. It is used for the treatment of asthma. Chemically it is a [R-(E)]-1-[[[1-[3-[2-(7-chloro-2quinolinyl) ethenyl]phenyl]-3-[2-(1-hydroxy-1-methyl ethyl) phenyl] propyl] thio] methyl] cyclopropaneacetic acid, monosodium salt. Montelukast sodium is a hygroscopic, optically active, white to off white powder Freely soluble in ethanol, methanol and water.
Levocetirizine is a H1 receptor antagonist. It is used to treat allergies and chronic hives that are due to unknown causes. Chemically it is a R-(+)-2-[2-[4-[(4-chlorophenyl) phenyl methyl] piperazin-1-yl] ethoxy] acetic acid .It is a white or almost white powder and freely soluble in water. Figure 1 and 2 shows the structure of montelukast sodium and levocetirizine respectively.
Literature survey shows that the simultaneous estimation of montelukast sodium and Levocetirizine by hplc has been reported[1-5]. The simultaneous estimation of montelukast sodium and levocetirizine by HPTLC also reported [5, 7-8]. UV methods for analysis of montelukast with levocetirizine [10-12] and the stability indicating hplc method for the montelukast sodium in tablets and in human plasma were studied and reported.TLC method for the analysis of these drugs in solid dosage form also reported. The aim of the present work to develop the simple, economical, accurate, reliable reverse phase HPLC method for the estimation of montelukast sodium and levocetirizine in bulk and combined dosage form. This method validated by as per ICH guidelines. Suitable statistical tests were performed on validation report.
MATERIALS AND METHODS
Instrumentation and chromatographic conditions
The HPLC system consisted of MERK HITACHI. It consisted of L-7100 model pump and L-7400 UV detector, version 4.1.Shodex C-18-4E column (250 x 4.6mm) 5µm particle.Rheodyne injector with 50 µl of fixed loop. The mobile phase is a mixture of ammonium acetate buffer: methanol: acetonitrile in the proportion of (20:55:25 v/v).Ammonium acetate buffer maintained with PH of 5.5, adjusted by acetic acid. The mobile phase was delivered at the flow rate of 1ml/min the detection was carried out at 225 nm.
Chemicals and reagents
The Reference standards are provided by the kausikh therapeutics and private limited (Chennai).Commercial tablets contains 10mg of montelukast sodium and 5mg of levocetirizine. It is obtained from sun pharma with the brand name montec-lc.All the reagents were used HPLC grade.
Stock solution of montelukast sodium (80µl) and levocetirizine (40µl) were prepared by 25 mg of montelukast and 25 mg of levocetirizine dissolved in mobile phase and diluted up to 25 ml with volumetric flask. From that concentration of 80 µg/ml montelukast and 40 µg/ml concentration of levocetirizine were prepared.
Average weight of tablet was calculated. Then the tablet were crushed to fine powder, dose equivalent to 25 mg was transferred to 25 ml volumetric flask, dissolved in a solvent and sonicated for 15 minutes. From this concentration of 80µg/ml montelukast and concentration of 40µg/ml levocetirizine were prepared.
The prepared sample and standard solutions were injected in to the column. Amount present and the percentage purity of the drug was calculated and listed in [Table 8] and Assay chromatograms showed in figure  and .
The developed method has been validated by as per ICH guidelines and the data’s were analyzed by statistically.
To check the accuracy of the proposed method, recovery studies were performed at 80 %, 100%.120% of the test concentration as per ICH guidelines. Three replicates of each concentration were injected in to the column.
Inter day and intraday precision was performed by six replicates of standard stock solution and area under curve (AUC) were recorded. It is evaluated by calculating standard deviation of resulting data.
Linearity To establish the linearity of the drug five different concentrations of the solutions were prepared from 10µg/ml – 50 µg/ml and injected. The calibration curves were obtained and the correlation co-efficient was calculated by statistically.
LOD and LOQ
The LOD and LOQ determined from slope of the calibration curve.LOD can be determine as signal to noise ratio usually 2:1 or 3:1.LOD can be calculated by the following formula LOD = 3.3 SD/S.
LOQ can be determined as a signal to noise ratio usually 10: 1.LOQ can be calculated by the following formula LOQ = 10 SD/S
To investigate the robustness of the developed method, experimental conditions were changed .The flow rate of mobile phase was 1ml/min. To study the effect of flow rate, flow was altered by 0.1 units from 0.9 to 1.1 ml/min. The effect of PH of mobile phase buffer was studied by varying PH ± 0.1 units and other mobile phase components were held constant as stated previously.
To establish the ruggedness of the method, ruggedness was carried out by using different day with different analysts.
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