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International Journal Of Pharma Professional’s Research
Research Article
ANALYTICAL METHOD DEVELOPMENT &
VALIDATION FOR SIMULTANEOUS ESTIMATION OF AMBROXOL HCL AND MONTELUKAST SODIUM IN ORAL SYRUP BY RP-HPLC METHOD
Sravani*, M. madhu , vineet kumar, Dipankar karmakar, Achhrish goel
ISSN NO:0976-6723
Annamacharya college of pharmacy Arvind Remedies Limited, Chennai Abstract
The present work describes a simple, precise and accurate RP-HPLC method for estimation of Montelukast sodium and Ambroxol HCL in Oral syrup dosage form. The separation was achieved by using Sun fire column (C18) and mixture of methanol; buffer (Ammonium acetate was taken in HPLC grade water and adjusted with pH 6.0 with orthophosphoric acid) in the ratio 80:20 as mobile phase, at a flow rate of 1.5 ml/min. Detection was carried out at 283 nm. The retention time of Montelukast sodium & Ambroxol Hcl were found to be 3.982 & 9.662 min. The accuracy and reliability of the proposed method was ascertained by evaluating various validation parameters like linearity, precision, accuracy and specificity. The proposed method provides an accurate and precise quality control tool for routine analysis of Montelukast sodium & Ambroxol Hcl in Oral syrup dosage form.
Keywords: - : Montelukast sodium, Ambroxol HCL, Validation Introduction
In the mid-1970’s High pressure liquid chromatography (HPLC) was developed and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970’s, new and innovating methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. In the 1980's HPLC was commonly used for the separation of numerous chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques.
Computers and automation aid convenience to HPLC.
Improvements in type of columns and their reproducibility were made such terms like micro- columns, affinity columns, and fast HPLC began to immerge. A vast undertaking in the development of the micro-columns, and other specialized columns was been achieving from the past decade.
Ambroxol hydrochloride is chemically, 1 ({[2 – Amino –3, 5 dibromo phenyl] methyl} amino) cyclohexanol monohydrochloride which is a semi synthetic derivative of vasicine from the Indian shrub
“Adhatoda vasica”. [2] It is an expectoration improver and a mucolytic agent used in the treatment of bronchial asthama and chronic bronchitis . Ambroxol hydrochloride has also been reported to have a cough suppressing effect and anti- inflammatory action. Recently the inhibition of nitric oxide dependent activation of soluble guanylate cyclase was suggested one of the molecular mechanism of the therapeutic action of ambroxol hydrochloride, also used in pulmonary alveolar proteinosis in pulmonary distress and infant respiratory distress syndrome. [3]
Montelukast sodium 2-[1-[(R)-[3-[2(E)-(7- chloroquinolin-2-yl) vinyl] phenyl] -3-[2- (1- hydroxy-1-methylethyl) phenyl] propyl - sulfanylmethyl] cyclopropyl] acetic acid sodium salt is a fast acting and potent cysteinyl leukotriene recept or antagonist which is being used in the treatment of asthma & to relive symptoms of seasonal allergies.[4]
It is white colored powder and it is freely soluble in ethanol, methanol, and water and practically insoluble in acetonitrile. Molecular weight of Montelukast Sodium is 608.2 g/mol and formula is C35H35ClNO3S.Na. [5]
MATERIALS AND METHODS Instrumentation
The liquid chromatographic system consisted of Waters 2 6 9 5 HPLC model (e series) containing Separator module, Photodiode array detector waters 2996 and variable wave length programmable UV/visible detector waters 2489 and rheodyne injector (7725i) with 100µl fixed loop.
Chromatographic analysis was performed using Sun fire C-18 column with 250mm x 4.6mm internal diameter and 5µm particle size. Sartorius electronic balance (CPA224S) was used for weighing purpose.
Reagents and Materials
Methanol of HPLC grade was purchased from Loba chemie, Mumbai, India and HPLC grade water was obtained by double distillation and purification through mille-Q water purification system. Ambroxol HCL & Montelukast Sodium standards are purchased from Shilpa Medicare lmt., Rayachur, India.
Preparation of Standard Stock Solution
Standard solution A: Weighed 20 mg of Montelukast working reference standard and transferred into 50ml of volumetric flask. 10ml of Methanol was added and sonicated for 5 minutes.
Then made up to volume with Diluent (methanol &
water in ratio 1:1) and mix well.
Standard solution B: Weighed 30 mg of Ambroxol working reference standard and transferred into 200ml of volumetric flask. 20ml of Solution A was added and sonicated for 5 minutes. Then made up to volume with Diluent (methanol & water in ratio 1:1) and mix well.
Preparation of mobile phase
A mixture of methanol, buffer in the ratio 80:20 were filtered and degassed.
Preparation of buffer
0.3854g of Ammonium acetate (10 ml ) of HPLC grade was taken in 500ml of HPLC grade water. Mixed well.
The pH was adjusted to 6.0 with Ortho phosphoric acid. Filter through 0.45µ membrane filter.
Chromatographic Conditions
The mobile phase consisting of methanol, buffer were filtered through 0.45µ Ultipor N66 Nylon 6,6 membrane solvent filter, degassed and were pumped from the solvent reservoir in the ratio of 80:20,v/v and was pumped into the column. The flow rate of mobile phase was maintained at 1.5ml/min and detection wavelength was set at 230 nm with a run time of 1 5 min. The volume of injection loop was 20µl prior to injection of the drug solution the column was equilibrated for at least 30 minutes with the mobile phase flowing through the system. The column and the HPLC system were kept in ambient temperature.
Sample preparation
Weighed 5000 mg of sample and it was transferred into 100ml of volumetric flask. 20ml of diluent was added and shaken until sonicated for 5 minutes.
Allowed to cool at room temperature then made up to volume with diluent.
EXPERIMENTAL WORK:
Optimised conditions:
Column: SUNFIRE C18 (4.6 ×250mm) , 5 µm Elution: Isocratic
Buffer : Ammonium acetate Buffer pH : 6.0
Mobile Phase: Methanol : Buffer ( 80 : 20 ) v/v Diluent: Methanol : Water (50 : 50) v/v Flow rate: 1.5 ml/min
λmax : 230 nm
Column oven temp: Ambient Injection volume: 20 µl Runtime: 15 minutes
Figure no 3: - Chromatogram showing the optimized method of assay of Ambroxol HCL &
Montelukast Sodium
Table No.1 Showing Specificity results
Method Validation Specificity
The evaluation of the specificity of the method was determined against placebo. The interference of the excipients of the claimed placebo present in pharmaceutical dosage form was derived from placebo solution. Further the specificity of the method toward the drug was established by means of checking the interference of the degradation products in the drug quantification for assay during the forced degradation study. Prepare sample solution in triplicate, sample solution spiked with known impurities in triplicate and analyse as per testing procedure above mentioned. Calculate the percentage difference between the mean assay of spiked and unspiked results with respect to the unspiked result.
Linearity
Linearity test solutions for the assay method were prepared at five concentration levels from 80 to 120
% of assay analyte concentration (80, 90, 100, 110, and 120 μg/ml. The peak areas versus concentration data were evaluated by linear regression analysis.
Precision
The precision of the assay method was evaluated in terms of repeatability by carrying out six independent assays of Ambroxol & Montelukast test sample preparation and calculated the % RSD of assay (intraday). Intermediate precision of the method was checked by performing same procedure on the different day (interday) by different person under the same experimental condition.
Accuracy
An accuracy study was performed by adding known amounts of Ambroxol & Montelukast to the placebo preparation. The actual and measured concentrations were compared. Prepare sample solution by spiking the respective standard with placebo at 75%, 100%
and 125% of standard working concentration in triplicate alternatively 90%, 100% and 110% is also acceptable. Analyse as per testing procedure.
Calculate the percentage recovery and percentage relative standard deviation (% RSD) at each level.
Robustness of method
The robustness of study was carried out to evaluate the influence of small but deliberate variations in the chromatographic conditions. The factors chosen for this study were the flow rate(1.5 ml/min), mobile phase composition [methanol-buffer (48: 52 and 52:
48, v/v)], buffer pH (0.2 pH) and using different lot of Liquid chromatographic column.
Result and Discussion: - System suitability
A system suitability test of the chromatographic system was performed before each validation run.
Five replicate injections of standard preparation were injected and asymmetry, theoretical plate and % RSD of peak area were determined for same. For all system suitability injections, asymmetry was less than 2.0, theoretical plate was not less than 2000 and
% RSD of peak area was less than 2.0 found.
Figure no 4: - Chromatogram of standard preparation
Figure no 5: - Chromatogram of Test preparation Specificity
The system suitability for specificity was carried out to determine whether there is any interference of any impurities in retention time of analytical peak. The study was performed by injecting blank. The chromatograms are shown in Fig. No.6-8 and it was tabulated in Table No. 1
Figure no 6 : - Chromatogram showing blank (diluent preparation)
Figure no 7 : - Chromatogram showing placebo
Figure no 8 : - Chromatogram showing sample Table No.2 Showing Specificity results
The specificity test was performed for Ambroxol HCL & Montelukast Sodium. It was found that there was no interference of impurities in retention time of analytical peak.
Linearity: -
Table No. 3: - Showing linearity results for Ambroxol HCL from level -1 to level-5
Table No. 4: - Showing linearity results for Montelukast from level -1to level-5
Ambroxol HCL r2 = 0.999
Montelukast Sodium r2 = 0.999
The linearity study was performed for concentration range of 120µg-180µg and 36µg-48µg of Ambroxol HCL
& Montelukast Sodium and the correlation coefficient was found to be 0.999 and 0.999 (NLT 0.999).
LOQ and LOD values: -
Table no 5: - Showing value of LOQ and LOD for Ambroxol HCL and Montelukast sodium.
Precision
The result of repeatability and intermediate precision study are shown in Table 1. The developed method was found to be precise as the %RSD values for the repeatability and intermediate precision studies were < 0.2 % and < 1.3 %, respectively, which confirm that method was precise.
Table No. 6: - Showing % RSD results for Ambroxol HCL
Table No. 7: - Showing % RSD results for Montelukast Sodium
Accuracy: -
The HPLC area responses for accuracy determination are depicted in Table 8,9. The results shown that best recoveries (75-125 %) of the spiked drug were obtained at each added concentration, indicating that the method was accurate.
Table No. 8: - Showing accuracy results for Ambroxol HCL
Table No. 9: - Showing accuracy results for Montelukast Sodium
The accuracy study was performed for % recovery of Ambroxol HCL & Montelukast Sodium. The % recovery was found to be 99.3 % to 99.8 % and 99.3 % to 100.3 % respectively (NLT 98% and NMT 102%).
Robustness of method
The result of robustness study of the developed assay method was performed. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. System suitability parameters were also found satisfactory; hence the analytical method would be concluded as robust.
REFERENCES:
1.Mukhopadhyay et al “HPLC Analysis on Separation of BSA from Dilute Solution”.
International journal of pharmaceutical engineering.
Page no 9-10(1).
2. Sateesh et al “Method Development and Validation of Ambroxol Hydrochloride and Loratadine by RP- HPLC in Tablet Dosage Form.
3. European Pharmacopoeia 5.0, Monographs, Page no 965-966.
4. Rao et al “A novel approach to sustained montelukast sodium release: Differentially coated mini-tablets in HPMC capsules” Int J Pharm Biomed Res, 2(2), 90-97.
5. Garh et al “Determination Of Montelukast Sodium In Oral Granules Dosage Forms By A Simple And Accurate Uv Spectrophotometric Methods”
.International Journal of Pharmaceutical Sciences Review and Research. Volume 7, Issue 2.
6. Devi et al “Chronomodulated drug delivery system of Montelukast Sodium” Der Pharmacia Lettre, 2(5):316-329.
Correspondence Address:
Sravani
Annamacharya college of pharmacy Phone: +919703999366
E-mail- [email protected]
