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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 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 AMB and DES bulk powder was kindly gifted by Cadila Pharmaceuticals Ltd. Ahmedabad, Gujarat, India and Sun Pharmaceutical Ltd., Halol, Panchmahal, Gujarat, India respectively. The commercial fixed dose combination product Dyl Ax (AMB – 75 mg, DES – 5 mg) was procured from the local market which is manufactured by Ajanta Pharma Limited (APL). All chemicals and reagents were of analytical

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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. The proposed method was found to be simple, specific, accurate, and precise for the routine simultaneous estimation of two drugs. The linearity range for AMB and DES were found to be 1500 - 5250 ng/spot and 100 - 350 ng/spot respectively. Regression analysis data and summary of all validation parameters is given in Table 1. Precision was calculated as repeatability (% RSD) and intra and inter day variation (% RSD) for both the drugs. Accuracy was determined by calculating the recovery and the mean was determined. The LOD and LOQ were found to be 201.26 and 609.87 ng/spot respectively for AMB and 14.05 and 42.57 ng/spot respectively for DES indicates sensitivity of the proposed method. The peak purity of AMB and DES was assessed by comparing their respective spectra at the peak start, apex and peak end positions of the spot. The peak purity was found to be 0.9991 and 0.9989 for AMB and DES respectively. The method was successfully used to determine the amounts of AMB and DES present in tablets. The results obtained are in good agreement with the corresponding labelled amount. By observing the validation parameters, the method was found to be specific, accurate and precise. Hence the method can be employed for the routine analysis of these drugs in combinations.

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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.

5. Makarand Avhad, Dr. C.G. Bonde, “Development and validation of simultaneous UV spectrophotometric Method for the determination of Levofloxacin and Ambroxol in tablets”, International Journal of ChemTech Research. 2009; 1(4): 873-878.

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.

13. Satish Bondili, Sudarshan Reddy P, “Spectroscopic method for determination of Desloratadine in bulk and its tablet dosage forms”, International Journal of Pharmaceutical & Industrial Research, Vol.1, Issue 2, Apr-Jun 2011, pp 131-34.

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.

15. Lihe Liua, Meiling Qib, Peng Wangc, Haozhi Lia, “High-performance liquid chromatographic method for the bioequivalence evaluation of Desloratadine fumarate tablets in dogs”, Journal of Pharmaceutical and Biomedical Analysis Vol.34, Issue 5, 10 March 2004, pp 1013-19.

16. Dina T. El-Sherbinya, Nahed El-Enanyb, Fathalla F. Belalb, Steen H. Hansenc, “Simultaneous determination of Loratadine and Desloratadine in pharmaceutical preparations using liquid chromatography with a micro emulsion as eluent”, Journal of Pharmaceutical and Biomedical Analysis Vol.43, Issue 4, 12 March 2007, pp 1236-42.

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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 ± 0.42 120 % 250 300 551.00 100.18 ± 0.36

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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

DES