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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
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8407
Research Article
DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF AMBROXOL
HYDROCHLORIDE AND DESLORATADINE HYDROCHLORIDE IN THEIR COMBINED
TABLET DOSAGE FORM
Sharma Ekta A.*, Shah Nehal J.
Quality Assurance Department, Indubhai Patel College of Pharmacy and Research Centre, Dharmaj, Petlad-Khambhat road, Dhramaj, Anand, Gujarat, India
Article Received on: 09/03/12 Revised on: 20/04/12 Approved for publication: 02/05/12
*Email: ektasharma120289@gmail.com
ABSTRACT
The present manuscript describes new, simple, accurate, and precise high performance thin layer chromatography method for the simultaneous determination of Ambroxol Hydrochloride and Desloratadine Hydrochloride in combined tablet dosage form. Chromatographic separation of the drugs was performed on aluminium plates pre coated with silica gel 60 F254 as the stationary phase and the solvent system consisted of Chloroform: Ethyl Acetate: Methanol: Tri
ethylamine (6: 4.5: 2.5: 0.8, v/v/v/v). Densitometric evaluation of the separated zones was performed at 245 nm. The two drugs were satisfactorily resolved with Rf values 0.72 and 0.26 for Ambroxol Hydrochloride and Desloratadine Hydrochloride, respectively. The linear regression data for the calibration plots
showed good relationship with r2 = 0.99886 from 1500 – 5250 ng/spot for Ambroxol Hydrochloride and r2 = 0.99875 from 100 – 350 ng/spot for Desloratadine
Hydrochloride. The methods were validated for precision, accuracy, and recovery. The percentage recovery for Ambroxol Hydrochloride was found to be 99.92 – 100.20 % and 99.73 – 100.18 % for Desloratadine Hydrochloride. The limits of detection and quantification were 201.26 and 609.87 ng/spot per spot for Ambroxol Hydrochloride and 14.05 and 42.57 ng/spot per spot for Desloratadine Hydrochloride, respectively.
Keywords: Ambroxol Hydrochloride, Desloratadine Hydrochloride, High Performance Thin Layer Chromatography Method, Validation
INTRODUCTION
Ambroxol Hydrochloride (AMB) is chemically trans–4–(2–
Amino-3, 5–dibromobenzylamino)- cyclohexanol1 is a secretolytic agent used in the treatment of tracheobronchitis, emphysema with bronchitis pneumoconiosis, chronic inflammatory pulmonary conditions, bronchiectasis, bronchitis with bronchospasm asthma2. It is official in Indian Pharmacopoeia (IP) and British Pharmacopoeia (BP). IP1 describes High Performance Liquid Chromatography (HPLC) method and BP3 describes HPLC, Spectrophotometric and Thin Layer Chromatography (TLC) method. Literature survey also reveals Spectrophotometric4-5, HPLC6-7, Ultra Performance Liquid Chromatography (UPLC) 8 and HPTLC9 methods for determination of AMB with other drugs.
Figure 1: Structure of Ambroxol hydrochloride
Desloratadine Hydrochloride (DES) is chemically 8-chloro-6, 11-dihydro-11-(4-piperdinylidene) - 5H benzo [5, 6] cyclohepta [1, 2-b] pyridine10 is a second generation antihistaminic drug. It is used for the relief of symptoms of seasonal allergic rhinitis, perennial (non-seasonal) allergic rhinitis and for the symptomatic treatment of pruritus and urticaria (hives) associated with chronic idiopathic urticaria11. Desloratadine is not official in any pharmacopoeia. Literature survey reveals HPTLC12 and Spectrophotometric13-14 methods for the determination of DES. Literature survey also
reveals RP-HPLC15-17 methods for determination of DES with other drugs.
Figure 2: Structure of Desloratadine Hydrochloride
The combined dosage forms of AMB and DES are available in the market (DYL – AX) for the prophylaxis and treatment of chronic asthma and chronic bronchitis. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of AMB and DES in their combined dosage forms. Literature survey does not reveal any simple chromatography or other method for simultaneous estimation of AMB and DES in combined dosage forms. The present communication describes simple, specific, rapid, accurate and precise chromatographic method based on High Performance Thin Layer Chromatographic method for simultaneous estimation of both drugs in their combined tablet dosage forms.
MATERIALS AND METHODS
Reagents and Materials
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grade and were purchased from Thermo fisher scientific Pvt. Ltd, Mumbai, India.
Instrumentation
CAMAG HPTLC instrument (Camag Muttenz, Switzerland) was used in this method. CAMAG HPTLC is equipped with CAMAG TLC scanner-3, Linnomate V Automatic sample applicator controlled by WIN CATS software (1.4.3 version). Aluminium packed silica Gel 60 F254 HPTLC plates (100 X
100 mm, layer thickness 0.2mm, E.MERCK). Linear ascending development was carried out in a 20 cm × 10 cm twin trough glass chamber (Camag Muttenz, Switzerland). The source of radiation used was deuterium lamp emitting a continuous UV spectrum between 190 to 400 nm.
Optimized chromatographic condition
Stationary phase: Pre-coated silica gel 60 F254 Aluminium
Plates (10x10cm)
Mobile phase: Chloroform: Ethyl Acetate: Methanol: Tri ethylamine (6: 4.5: 2.5: 0.8, v/v/v/v)
Chamber saturation: 20 minutes Development distance: 70mm Development time: 15 minutes Relative temperature: 25 ±2˚C Scanning Speed: 20 mm/sec Detection wavelength: 245 nm AMB Rf: 0.72
DES Rf: 0.26
Preparation of standard stock solutions
An accurately weighed quantity of AMB (100 mg) and DES (100 mg) were transferred to a separate 100 ml volumetric flask and 50 ml methanol is added to both volumetric flask and sonicated for 10 minutes. Volume was adjusted up to the mark with methanol to obtain standard solution having concentration of AMB (1000 ng/μl) and DES (1000 ng/μl). 37.5 ml of AMB (1000 ng/μl) and 2.5 ml of DES (1000 ng/μl) aliquot were transferred to a separate 50 ml volumetric flask and diluted up to concentration of AMB (750 ng/μl) and DES (50 ng/μl) with methanol.
VALIDATION OF THE PROPOSED METHOD
The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines18.
Linearity and range
From the mixed standard stock solution 750 ng/μl of AMB and 50 ng/μl of DES, 2 to 7 μl solution spotted on HPTLC plate to obtain final concentration 1500 – 5250 ng/spot for AMB and 100 – 350 ng/spot for DES. Each concentration was applied six times to the HPTLC plate. The plate was then developed using the previously described mobile phase and the peak areas were plotted against the corresponding concentrations to obtain the calibration curves.
Precision
The precision of the method was verified by repeatability and intermediate precision studies.
Repeatability
Repeatability studies were performed by analysis of all concentrations (1500, 2250, 3000, 3750, 4500 and 5250 ng/spot for AMB and 100, 150, 200, 250, 300 and 350 ng/spot for DES) of the drug in six times on the same day.
Intermediate precision
The intermediate precision of the method was checked by intra day and inter day study. The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of AMB and
DES (3000, 3750, 4500 ng/spot for AMB and 200, 250, 300 ng/spot for DES). The result was reported in terms of relative standard deviation (% RSD).
Specificity
The specificity of the method was determined by analyzing standard drug and test samples. The spot for AMB and DES in the samples was confirmed by comparing the Rf and
spectrum of the spot with that of a standard. The peak purity of AMB and DES was determined by comparing the spectrum at three different regions of the spot i.e. peak start (S), peak apex (M) and peak end (E).
Accuracy
Accuracy of the method was carried out by applying the method to drug sample (AMB and DES combination tablet) to which know amount of AMB and DES standard powder corresponding to 80, 100 and 120% of label claim had been added (standard addition method), mixed and the powder was extracted and analyzed by running chromatogram in optimized mobile phase.
ANALYSIS OF AMB AND DES IN COMBINED
TABLET DOSAGE FORM
Twenty Tablets were weighed and powdered. The powder equivalent to 75 mg of AMB and 5 mg of DES was transferred to a 100 ml volumetric flask. Methanol (50 ml) was added to it and sonicated for 20 min. The solution was filtered through Whatman filter paper (0.45µ) and the volume was adjusted up to the mark with methanol. This solution is expected to contain 750 ng/μl of AMB and 50 ng/μl of DES. 4.5 µl of the prepared sample was applied on pre-washed TLC plate, developed in the above mobile phase, dried in air and photo metrically analyzed by running chromatogram in optimized mobile phase. From the peak area obtained in the chromatogram, the amounts of both the drugs were calculated.
RESULTS AND DISCUSSION
The results of validation studies on simultaneous estimation method developed for AMB and DES in the current study involving Chloroform: Ethyl Acetate: Methanol: Tri ethylamine (6: 4.5: 2.5: 0.8, v/v/v/v) as the mobile phase for HPTLC are given below.
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CONCLUSION
Introducing HPTLC into pharmaceutical analysis represents a major step in terms of quality assurance. Today HPTLC is rapidly becoming a routine analytical technique due to its advantages of low operating costs, high sample throughput and the need for minimum sample preparation. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase-unlike HPLC - thus reducing the analysis time and cost per analysis. The developed HPTLC technique is precise, specific and accurate. Statistical analysis proves that the method is suitable for the analysis of AMB and DES in pharmaceutical formulation without any interference from the excipients. The common excipients and other additives are usually present in the tablet dosage form do not interfere in the analysis of AMB and DES in method, hence it can be conveniently adopted for routine quality control.
ACKNOWLEDGMENT
The authors are thankful to Cadila Pharmaceuticals Ltd. Ahmedabad, Gujarat, India and Baroque Pharmaceutical Ltd., Khambhat, Anand, Gujarat, India for providing gift sample of AMB and DES, respectively for research. The authors are highly thankful to Indubhai Patel college of Pharmacy and Research centre, Dharmaj, Gujarat, India for providing all the facilities to carry out the work.
REFERENCES
1. Indian pharmacopoeia, Govt. of India, “ministry of health and family welfare”, controller and publication, Delhi, 2007, Vol. 2, 78-79. 2. Ambroxol – Drug information online, Drugs.com, October, 2011,
Available from : http://www.drugs.com/international/ambroxol.html 3. British Pharmacopoeia, Government British Pharmacopoeial
Commission, 2011, volume I and II, monograph 1489.
4. Prasanthi N. L., Mohan Ch. Krishna, Manikiran S.S., Rao N. Rama, “Estimation of Ambroxol hydrochloride and Guiaphensin in tablet dosage form by simultaneous equation method”, International Journal of Research in Ayurveda & Pharmacy. 2010; 1(1): 140-146.
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6. Mukesh Maithani, Richa Raturi, Vertika Gautam, Dharmendra Kumar, Anand Gaurav and Ranjit Singh, “Simultaneous estimation of Ambroxol hydrochloride and Cetirizine hydrochloride in tablet dosage form by
RP-HPLC method”, International Journal of Comprehensive Pharmacy.2010;1(2): 1-3.
7. Maarit Heinanen, Coral Barbas, “Validation of an HPLC method for the quantification of Ambroxol hydrochloride and Benzoic acid in a syrup as pharmaceutical form stress test for stability evaluation”, Journal of Pharmaceutical and Biomedical Analysis. 2001; 24:1005-10.
8. Rakshit Kanubhai Trivedi, Mukesh C. Patel, Sushant B. Jadhav, “A Rapid, Stability Indicating RP-UPLC method for simultaneous determination of Ambroxol hydrochloride, Cetirizine hydrochloride and antimicrobial preservatives in liquid pharmaceutical formulation”,
Scientific Pharma.2011;79: 525–43.
9. Shashikant B. Bagade, Narendra M. Gowekar And Avinash V. Kasture, “Simultaneous HPTLC Estimation of Ambroxol HCl and Cetirizine HCl in Their Combined Dose Tablet”, Asian Journal of Chemistry. 2007; 19(2): 1487-1493.
10. Desloratadine – Drug Information Online –Drugs.com, October, 2011 Available from: http://www.drugs.com/pro/desloratadine.html
11. Desloratadine – Drug Bank – Open data drug and drug target database Available from : http://www.drugbank.ca/drugs/DB00967
12. Endang Sumarlik, Hoshi Berniati Tampubolon, Mochammad Yuwono, Gunawan Indrayanto,” Densitometric determination of Desloratadine in tablets, and validation of method”, JPC- Journal of Planar Chromatography Modern TLC, Vol 18, Issue No 101, February 2005, pp 19-22.
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14. Gehad G. Mohamed, Fekria M. Abou Attia, Nahla S. Ismail and Neveen S. Ibrahim, “Analytical uses of charge-transfer complexes: determination of dosage forms of Desloratadine”, Acta Pharmaceutica Sciencia, Vol. 53, 2011, pp 89-98.
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Table 1: Regression analysis data and summary of validation parameters for the proposed method Parameters High Performance Thin Layer Chromatography method
AMB DES
Concentration Range (ng/spot) 1500 – 5250 100 – 350 Slope (m)
Intercept (c)
2.30628 4423.10
7.61850 418.59 Correlation Coefficient (r2) 0.99886 0.99875
Accuracy (% recovery) (n = 3) 99.92 – 100.20 % 99.73 – 100.18 % Repeatability (%RSD) (n = 6) 0.09 % 0.38 %
Intraday (n = 3) (%RSD) 0.71 – 0.73 % 0.70 – 1.23 % Interday(n = 3) (%RSD) 0.96 – 1.08 % 1.15 – 1.49 %
LOD (ng/spot) 201.26 14.05
LOQ (ng/spot) 609.87 42.57
Table 2: Recovery data of proposed method Drug Level Amount taken
(ng/spot)
Amount added (ng/spot)
Amount Recovered (ng/spot) (n=3)
% Recovery ± S.D (n=3)
AMB 0 % 3750 0 3749.00 99.97 ± 0.10
80 % 3750 3000 6750.67 100.01 ± 0.07
100 % 3750 3750 7515.67 100.20 ± 0.08
120 % 3750 4500 8244.00 99.92 ± 0.09
DES 0 % 250 0 249.33 99.73 ± 0.61
80 % 250 200 450.33 100.07 ± 0.46
100 % 250 250 499.67 99.93 ± 0.42
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Table 3: Analysis of AMB and DES by proposed method Label claim (mg) Amount taken
(ng/spot)
Amount Recovered (ng/spot) (n=3)
% Label claim ± S.D
Tablet AMB DES AMB DES AMB DES AMB DES
DYL - AX 75 5 3375 225 3456 223.1 102.4 ± 0.84
99.11 ± 0.12
Figure 3: Photograph of developed HPTLC plate of AMB and DESs
Figure 4: Overlain view of all tracks of AMB and DES at 245nm
Figure 5: Densitogram of AMB (3375 ng /spot ) and DES (225ng / spot)
Source of support: Nil, Conflict of interest: None Declared
AMB
DES
AMB
