Sharma Ekta A et al.

IRJP 2012, 3 (5)

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 reveals RP-HPLC15-17 methods for determination of DES

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
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
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welfare”, controller and publication, Delhi, 2007, Vol. 2, 78-79.
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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) 2.30628 7.61850
Intercept (c) 4423.10 418.59
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 Amount added Amount Recovered % Recovery ± S.D
(ng/spot) (ng/spot) (ng/spot) (n=3) (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 Amount Recovered % Label claim ± S.D
(ng/spot) (ng/spot) (n=3)
Tablet AMB DES AMB DES AMB DES AMB DES
DYL - AX 75 5 3375 225 3456 223.1 102.4 ± 99.11 ± 0.12
0.84

AMB

DES

Figure 3: Photograph of developed HPTLC plate of AMB and DESs

AMB

DES

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

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