Stability Indicating HPTLC Method Development for

AASCIT Journal of Chemistry
2015; 2(2): 32-42
Published online April 10, 2015 (http://www.aascit.org/journal/chemistry)
Stability Indicating HPTLC Method
Development for Determination of
Betamethasone and
Dexchlorpheniramine Maleate in a
Tablet Dosage Form
Tadesse Haile Fereja1, *, Ariaya Hymete2, Adnan A. Bekhit2, 3
1
Keywords
Betamethasone,
Dexchlorpheniramine Maleate,
HPTLC,
Stability Indicating,
Degradation
Department of Pharmacy, College of Medicine and Health Science, Ambo University, Ambo,
Ethiopia
2
Department of Pharmaceutical Chemistry, School of Pharmacy, Addis Ababa University, Addis
Ababa, Ethiopia
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria,
Alexandria, 21521-Egypt
Email address
[email protected] (T. H. Fereja), [email protected] (A. Hymete),
[email protected] (A. A. Bekhit)
Citation
Received: February 9, 2015
Revised: March 9, 2015
Accepted: March 10, 2015
Tadesse Haile Fereja, Ariaya Hymete, Adnan A. Bekhit. Stability Indicating HPTLC Method
Development for Determination of Betamethasone and Dexchlorpheniramine Maleate in a Tablet
Dosage Form. AASCIT Journal of Chemistry. Vol. 2, No. 2, 2015, pp. 32-42.
Abstract
A simple, specific, precise, accurate, robust and stability-indicating HPTLC method for
determination of the two drugs in tablet dosage form was developed and validated. The
method employed HPTLC aluminum plates precoated with silica gel 60F- 254 as the
stationary phase. The solvent system consisted of ethyl acetate: methanol: ammonia (2 :
13 : 1 v/v/v). This system was found to give compact spots for betamethasone (Rf = 0.32
± 0.04) and for dexchlorpheniramine maleate (Rf = 0.76 ± 0.05). Densitometric analysis
of the drugs was carried out in the absorbance mode at 226 nm. Linearity was found over
the concentration range of 25 - 137.5 ng/ul with r2 ± RSD = 0.997 ± 0.00102 for
betamethasone and 100 - 800 ng/ul for dexchlorpheniramine maleate with r2 ± RSD =
0.999 ± 0.141, respectively. LOD for the method was found to be 2.49ng and 19.7ng for
betamethasone and dexchlorpheniramine maleate, respectively. LOQ was found to be
7.54 ng for betamethasone and 59.70 ng for dexchlorpheniramine maleate. Extra peaks
were observed for betamethasone treated with 30% H2O2 (Rf = 0.36), 1N HCl (Rf 0.19,
0.25 and 0.36), 1N NaOH (Rf = 0.36) and thermal condition (Rf 0.26 and 0.36).
Dexchlorpheniramine maleate degraded with 30% H2O2 showed additional peak at Rf
value of 0.67. Statistical analysis proved that the method is reproducible and selective for
the simultaneous estimation of betamethasone and dexchlorpheniramine maleate. As the
method could effectively separate the drugs from their degradation products, it can be
employed as a stability indicating.
1. Introduction
Betamethasone 9 α -fluoro-16 ß -methyl-11 ß, 17 α 21-trihydroxy-1, 4-pregnadiene- 3,
20-dione (Figure 1 a) is a synthetic glucocorticoid that suppress the activity of
endogenous mediators of inflammation including prostaglandins, kinins, and histamine
[1]. Dexchlorpheniramine maleate (3S)-3-(4-chlorophenyl)-N, N-dimethyl-3-(pyridin-2yl) propan-1-amine (Z)-butanedioate (Figure 1 b) is a potent antihistamine used for the
AASCIT Journal of Chemistry 2015; 2(2): 32-42
treatment of several allergies and skin irritation. It readily
crosses the blood brain barrier and one of its collateral effects
O
33
is the induction of sedation [2].
Cl
O
OH
H
H3C
CH3
OH
OH
20
N
1
CH3
16
H
H
N
O
H
O
H
CH3
CH3
H
F
H
O
7
O
4
A
B
Figure 1. Structure of betamethasone [A]; dexchlorpheniramine maleate [B].
Various methods have been reported for the determination
of betamethasone in a single dosage form and in combination
with other drugs. Quantification of betamethasone in human
plasma by liquid chromatography–tandem mass spectrometry
using atmospheric pressure photo ionization in negative
mode has been reported [3]. Betamethasone and
betamethasone 17-monopropionate determinations were
reported in human plasma by liquid chromatography–
positive/negative electrospray ionization tandem mass
spectrometry [4]. Analysis of corticosteroids including
betamethasone in hair has been reported using liquid
chromatography– electrospray ionization mass spectrometry
[5]. Coupled-column liquid chromatographic–tandem mass
spectrometric method has been reported for the determination
of betamethasone in urine sample [6]. HPLC method has
been reported for determinations of betamethasone in
combination with cyanocobalamin and diclofenac sodium in
pharmaceutical formulations [7] and betamethasone
dipropionate and salicylic acid in pharmaceutical
preparations [8]. Ion-paired RP-HPLC assay for
simultaneous measurement of betamethasone and
betamethasone sodium phosphate in plasma was developed
and reported [9]. RP-HPLC method has been described for
the concurrent assay of betamethasone dipropionate and
tolnaftate in combined semisolid formulation [10].
Determination of betamethasone and dexamethasone in
plasma has been reported using fluorogenic derivatization
and liquid chromatography [11]. Simultaneous analysis of
betamethasone valerate and miconazole nitrate in cream [12]
and determination of betamethasone in tablet dosage form
[13] by densitometric method has also been reported.
Thermal degradation of betamethasone sodium phosphate
under solid state and determination of the degradation
products using LC–MS- NMR methods has been described
[14]. A stability indicating RP-HPLC method has been
described for simultaneous determination of salicylic acid,
betamethasone dipropionate, and their related compounds in
Diprosalic Lotion® [15], for assay of betamethasone and
estimation of its related compounds [16] and separation of
betamethasone from low level dexamethasone and other
related compounds [17].
A method has been described for the determination of
dexchlorpheniramine maleate [18] in human plasma by
HPLC-MS. RP-HPLC with aqueous-organic and micellarorganic mobile phases has been reported to determine the
correlation between hydrophobicity and retention data of
several antihistamines including dexchlorpheniramine [19].
HPLC method has been reported for determination of optical
purity of dexchlorpheniramine maleate with circular
dichroism (CD) detector [20]. The use of the first and second
derivatives of the ratio of the emission spectra with a zerocrossing technique has been reported for determination of
mixture of dexamethasone, dexchlorpheniramine maleate and
fluphenazine hydrochloride [21]. There is no reported
method for the simultaneous determination of this
combination of drugs.
According to ICH guidelines, stress testing is necessary to
elucidate the inherent stability of the active substance.
Susceptibility of the active substance to oxidation, hydrolysis,
and photolysis has to be tested [22]. The hydrolytic
degradation of a new drug in acidic and alkaline conditions
can be studied by exposing the drug in 0.1 N HCl / NaOH for
1-7 days [23, 24]. If reasonable degradation is seen, testing
can be stopped at this point. However, in case no degradation
is seen under these conditions, the drug should be exposed to
acid/alkali of higher strengths and for longer duration.
Alternatively, if total degradation is seen after subjecting the
drug to initial conditions, acid/alkali strength can be
decreased along with decrease in the reaction temperature. To
test for oxidation, it is suggested to use hydrogen peroxide in
the concentration range of 3–30% [23]. The photochemical
degradation should be carried out by exposure to direct
sunlight for 24 h [24]. Thermal degradation can be study by
exposing the drug substance in solid state to temperature of
≥ 70 o C [24].
Nowadays, HPTLC has become a routine analytical
technique due to its advantages of reliability in quantification
of analytes at micro and even in nanogram levels and cost
34
Tadesse Haile Fereja et al.: Stability Indicating HPTLC Method Development for Determination of
Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form
effectiveness. Due to the lower sample capacity of the
HPTLC layer, the amount of sample applied to the layer is
reduced. A further advantage is that the compact starting
spots allow an increase in the number of samples which may
be applied to the HPTLC plate. Simultaneous assay of
several components in a multicomponent formulation is
possible [25, 26]. As there is no official or reported analytical
method for the simultaneous determination of the two drugs,
the objective of this study was to develop and validate a
stability indicating HPTLC-densitometry method for
simultaneous determination of betamethasone and
dexchlorpheniramine maleate combination in commercial
tablet dosage form.
2. Experimental
2.1. Materials
2.1.1. Chemicals
Analytical grade methanol (Sigma- Aldrich, Germany) and
ethyl acetate (BDH, England) were purchased from
Pharmaceuticals Fund and Supply Agency, Ethiopia.
Celestamine®
tablet
(0.25mg
betamethasone/2mg
dexchlorpheniramine maleate, Sharing-Plough, France) was
purchased from pharmacy retail outlet in Addis Ababa.
Working standards of betamethasone (98.7% purity) and
dexchlorpheniramine maleate (99.7% purity) (SharingPlough, France) were obtained from Food, Medicine and
Health Care Control Authority FMHACA, Ethiopia.
2.2.2. Preparation of Test Solutions
30 Celestamine® tablets whose mean weight was
determined were finely powdered. Powder equivalent to 6.25
mg of betamethasone and 50 mg of dexchlorpheniramine
maleate was transferred into a 50 ml volumetric flask
containing 25 ml methanol, sonicated for 30 min and diluted
to volume with methanol. The resulting solution was filtered
using Whatman No 42 filter paper. Supernatant containing
1mg/ml of dexchlorpheniramine maleate and 0.125 mg/ml of
betamethasone was used as stock solution.
2.2.3. Sample Application and
Chromatogram Development
The sample was applied in the form of band with a length
of 6 mm using Camag Linomat 5 sample applicator and 100
µl Camag syringe, 10 mm from the bottom and 10 mm from
the side edges of the plate. The band was dried with the aid
of an online nitrogen gas. 20 x10 cm twin trough Camag
chamber containing 16 ml of the selected solvent was used
for the development. Ascending mode of development was
employed throughout the course of the experiment. The
optimized chamber saturation time for mobile phase was 15
min at room temperature (25◦C ± 2) at relative humidity of
60 % ± 5. The length of chromatogram run was 8 cm that
was achieved in about 30 min. Subsequent to development;
chromatograms were dried in a current of warm air with the
help of hair dryer for 20 min.
2.2. Method
2.2.4. Selection of Mobile Phase
The mobile phase composition is generally selected by a
controlled process of trial and error. The mobile phase should
be chosen taking into consideration the chemical properties
of analytes and the sorbent layer. After these experiments the
solvents giving the most appropriate separations are chosen
for further optimization of the mobile phase. At the
optimization level mixtures of solvents from different
selectivity groups are investigated by adjusting strength, if
required. At this level addition of small amounts of acidic or
basic modifier can be considered and tested whenever this
appears promising.
2.2.1. Preparation of Standard Solutions
2.3. Validation of the Developed Method
(i) Stock Standard Solution
10mg of betamethasone and 80mg dexchlorpheniramine
maleate were weighed and transferred to 100 ml volumetric
flask and dissolved in 50 ml methanol. Then, it was diluted to
the volume with methanol to obtain a concentration of 0.1
mg/ml and 0.8 mg/ml of betamethasone and
dexchlorpheniramine maleate respectively. The solution was
kept at room temperature until used.
2.3.1. Determination of Linearity
Linearity relationship between peak area of the spots and
concentration of the drugs were evaluated over a range of
concentrations (ng/band). Fifteen levels of standard solutions
were prepared as described in section 2.2.1.2. From these
serial dilutions, solutions with concentrations of 0.004 mg/ml
to 0.056 mg/ml with a variation of 0.004 mg/ml for
betamethasone and between 0.032 mg/ml to 0.48 mg/ml with
a variation of 0.032 mg/ml for dexchlorpheniramine maleate
were obtained. 3 µl of these solutions were applied to the
plates to get 12.5-187.5 ng/band of betamethasone and 1001500 ng/band of dexchlorpheniramine maleate.
2.1.2. Instruments
Micro syringe (Linomat syringe 659.004), linomat 5
applicator, twin trough chamber 20x10cm, UV chamber, TLC
scanner III, winCATS version 1.4.0 software (all from
Camag, Muttenz, Switzerland), precoated aluminum backed
HPTLC plates (silica gel 60 F-254, 20 x 20 cm with 200 µm,
thickness, Merck, Germany) and hair dryer (Philip lady 1000,
Type HP 4312, Hong Kong) were used in the study.
(ii) Calibration Standard Solutions
Fifteen different concentration levels of calibration
standard solutions were freshly prepared by diluting 1-15 ml
of the stock standard solution with 1ml interval to 25 ml in
appropriate volumetric flasks using methanol.
2.3.2. Precision Studies
Repeatability and intermediate precision were studied by
taking a concentration within the linearity range at three
AASCIT Journal of Chemistry 2015; 2(2): 32-42
different levels; 50, 75, 100 ng/spot for betamethasone and
400, 600, 800 ng/spot for dexchlorpheniramine maleate. The
solutions were spotted in replicate of six to the plate and the
relative standard deviation (RSD) at each level was
calculated.
2.3.3. Accuracy
In this study, accuracy was checked by standard addition
method which involves the spiking of equal amount of the
sample with at least three different amount of the reference
standard, and then measuring the response for both spiked
and non-spiked preparations.
For the developed method, recovery study was carried out
by applying the method on drug sample to which known
amount of betamethasone and dexchlorpheniramine maleate
corresponding to 80, 100 and 120 % of label claim had been
added.
2.3.4. Robustness
By introducing small changes in the mobile phase
composition, the effects on the results were examined.
Mobile phases having different composition like ethyl
acetate–methanol–ammonia (1.8: 13.0: 1.0 + 0.1v/v/v); (2.2:
13.0: 1.0 + 0.1v/v/v); (2.0: 11.7: 1.0 + 0.1v/v/v); (2.0: 14.3:
1.0, + 0.1v/v/v); (2.0: 13.0: 0.9 + 0.1 v/v/v) and (2.0: 13.0:
1.1 + 0.1v/v/v) were tried and chromatograms were run. The
volume of mobile phase (16ml) was varied in the range of ±
5%. Time from spotting to chromatography was varied from
10 + 5 minutes and from chromatography to scanning was
varied from 20 + 5 minutes. Robustness of the method was
checked by analyst variation. For all robustness studies, three
different concentration levels 50, 75, 100 ng/spot and 400,
600,
800
ng/spot
for
betamethasone
and
dexchlorpheniramine maleate, respectively were used.
2.3.5. Detection and Quantitation Limits
The detection and quantitation limits of the developed
method were calculated using the following formulae LOD =
3.3 × SD/S and LOQ = 10 × SD/S where ‘SD’ is the standard
deviation of the y-intercepts of the regression lines
constructed as described in section 2. 3. 1 for the blank
response and ‘S’ is the slope of the calibration plot.
2.3.6. Specificity
Specificity of the method was ascertained by analyzing
standard drug and sample. The spot for betamethasone and
dexchlorpheniramine maleate in sample was confirmed by
comparing the Rf and the UV spectra of the spot with that of
standard. The peak purity of betamethasone and
dexchlorpheniramine maleate was assessed by comparing the
spectra at three different levels, i.e., peak start (S), peak apex
(M) and peak end (E) positions of the spot.
2.4. Sample Solution Stability Study
Solutions having three different concentrations (50, 75 and
100 ng/spot for betamethasone) and (400, 600 and 800 ng/spot
for dexchlorpheniramine maleate) were prepared from
reference solution as described in section 2.2.1.2 and stored at
35
room temperature for 2.0, 4.0, 6.0, 12.0 and 24 hrs.
2.5. Analysis of Dosage Form
For
determination
of
betamethasone
and
dexchlorpheniramine maleate in commercial tablet dosage
form, sample solution prepared as described in section 2.2.2
were used. From this solution, 1 ml was transferred to a 10ml
volumetric flask and diluted to volume with methanol. 6 µl
from the diluted solution was spotted in triplicate.
Betamethasone and dexchlorpheniramine maleate content
was determined from peak area of densitogram. The
calibration curves, constructed as described in section 2.3.1
were used for determination of the actual contents of the two
substances in the dosage form.
2.6. Stress Degradation Studies
All degradation studies were carried out at a drug
concentration of 1mg/ml. Acid (1N) and alkaline (1N)
hydrolysis and oxidation by 3 and 30 % H2O2 was done by
refluxing the drug solutions in water bath at 70oC for 6 hrs.
Thermal degradation studies were carried out by exposing the
powdered drugs to dry heat at 105o C for 24 hrs. Photo
degradation studies were performed by exposing the
powdered drugs to direct sun light for 3 days. Samples for
each treatment were subjected to HPTLC analysis at a
concentration of 1000 ng/spot.
3. Results and Discussion
3.1. Method Development
Well resolved and sharp peaks for betamethasone (Rf =
0.32 ± 0.04) and dexchlorpheniramine maleate (Rf = 0.76 ±
0.05) were obtained [Figure 2], when ethyl acetate: methanol:
ammonia (2: 13: 1 v/v/v) was used as a mobile phase. The
solvent system composed of dichloromethane: methanol:
ammonia (3: 13: 1 v/v/v) showed good resolution for the two
spots but the spot for dexchlorpheniramine maleate appeared
near the solvent front while propanol: methanol: ammonia (6:
14: 1 v/v/v) showed small migration distance for
betamethasone from the application point. Good separation
was observed in the solvent system methanol: ethanol:
ammonia (12: 4: 1 v/v/v) but a tailed spot was observed for
betamethasone.
Quantitative
determinations
of
betamethasone and dexchlorpheniramine maleate were
performed by scanning the spots of the two substances at 226
nm [Figure 3]. The selected and optimized mobile phase was
let to saturate the chamber for 20minutes. The plate was then
allowed to dry for 20minutes using hair dryer.
3.2. Linearity and Calibration Curves
Linearity was found over the concentration range of 25 137.5 ng/spot with r2 ± RSD of 0.997 ± 0.00102 for
betamethasone and 100-800 ng/spot with r2 ± RSD of 0.999 ±
0.141 for dexchlorpheniramine maleate. Peak area and
concentration were subjected to least square linear regression
36
Tadesse Haile Fereja et al.: Stability Indicating HPTLC Method Development for Determination of
Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form
analysis to obtain the calibration equation and correlation
coefficients [Table 1].
Figure 2. Typical densitogram of betamethasone [1] and dexchlorpheniramine maleate [2].
Table 1. Linear regression data for the calibration curves (n = 5)
Parameters
Linearity range
r ± RSD
Slope ± RSD
Intercept ± RSD
Confidence limit of slope a
Confidence limit of intercept a
a
Betamethasone
25 - 137.5 ng/spot
0.998 ± 0 .00102
15.17 ± 0. 14
271.0 ± 2.5
15.17 ± 0.133
271.0 ± 0.21
Dexchlorpheniramine maleate
100-800 ng/spot
0.999 ± 0.0141
2.303 ± 0.836
309.7 ± 4.44
2.303 ± 0.037
309.7 ± 26.45
95% confidence limit.
Figure 3. The UV spectral display of dexchlorpheniramine maleate [1] and betamethasone [2].
AASCIT Journal of Chemistry 2015; 2(2): 32-42
37
3.3. Method Validation
repeatability of the method.
3.3.1. Precision
Repeatability and intermediate precision of the developed
method were expressed in terms of RSD of the peak area.
The results showed that intra- and inter day variations of the
peak areas at three different concentration levels of 50, 75,
and 100 ng/spot for betamethasone and 400, 600, and 800
ng/spot for dexchlorpheniramine maleate [Table 2] were
within the acceptable range. The low RSD values indicate the
3.3.2. Recovery Studies
The proposed method when used for extraction and
subsequent
estimation
of
betamethasone
and
dexchlorpheniramine maleate from pharmaceutical dosage
forms after spiking with 80, 100 and 120 % of additional
drug afforded a recovery rate of 99.70–100.44 % for
betamethasone and 99.73-100.49 % for dexchlorpheniramine
maleate (Table 3).
Table 2. Intra- and inter-day precision of HPTLC method (n = 6)
Compound
Betamethasone
Dexchlorpheniramine maleate
Amount (ng/band)
50
75
100
400
600
800
Intra-day precision
SD
5.0
2.3
8.8
7.3
9.9
9.6
RSD
1.10
0.17
0.80
0.99
1.11
1.07
Inter-day precision
SD
14.3
12.4
11.9
17.0
15.1
1.3
RSD
1.13
1.10
0.9
1.09
1.06
0.06
Table 3. Recovery studies for betamethasone and dexchlorpheniramine maleate (n = 5)
Drug added to the analyte (%)
(a)Betamethasone
0
80
100
120
(b)Dexchlorpheniramine maleate
0
80
100
120
Theoretical content(ng)
Reference Added(ng)
Recovery (%)±SD
% RSD
50
90
100
110
40
50
60
99.70 ± 0.64
99.87 ± 0.53
100.44 ± 0.6
100.10 ± 0.42
0.49
0.27
0.22
0.19
400
720
800
880
320
400
480
99.90 ± 0.04
99.79 ± 0.02
99.73 ± 0.06
100.49 ± 0.03
0.38
0.21
0.51
0.29
3.3.3. Specificity
The results for betamethasone were found to be peak start,
peak apex [r2 = 0.9999], and peak apex and peak end positions
[r2 = 0.9997] while for dexchlorpheniramine maleate the
results were found to be peak start, peak apex [r2 = 0.9999],
and peak apex and peak end positions [r2 = 0.9997]. Good
correlation i.e. r2 = 0.9996 and r2 = 0.9997 was also obtained
between standard and sample spectra of betamethasone and
dexchlorpheniramine maleate, respectively.
Figure 4. UV spectra comparison of the spots of the standards [2 & 3] and dosage form [1& 4] for betamethasone [*] and dexchlorpheniramine maleate [**].
38
Tadesse Haile Fereja et al.: Stability Indicating HPTLC Method Development for Determination of
Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form
respectively.
3.3.4. Limit of Detection and Quantification
The limit of detection and quantification of the developed
method were calculated as in section 3.4.4 and it was found
to be 2.49 and 7.54 ng/spot respectively for betamethasone.
For dexchlorpheniramine maleate the limit of detection and
quantification were found to be 19.51 and 60.70 ng/spot
3.3.5. Robustness
Standard deviation of peak areas was calculated for each
parameter and RSD was found to be less than 2 %. The low
RSD values as shown in Table 4 indicate robustness of the
method.
Table 4. Results for robustness study of the method (p = 0.05, n = 6; tstat = 4.3)
Betamethasone a
Dexchlorpheniramine maleate b
Parameters
Optimized solvent system
Mobile phase composition (v/v/v)
Volume of mobile phase (v/v/v)
Time from spotting to chromatography
Time from chromatography to scanning
a
SD*
RSD*
2.0:13.0:1.0 (16ml)
14.5
0.53
1.8:13.0:1.0
28.7
0.85
2.2:13.0:1.0
14.1
0.41
2.0:11.7:1.0
13.2
2.0:14.3:1.0
23.0
2.0:13.0:0.9
t cal
SD*
RSD*
13.3
0.27
t cal
0.25
17.4
0.54
0.95
0.98
14.0
0.30
0.55
0.31
0.86
24.1
0.30
0.46
0.29
0.63
12.5
0.18
0.26
12.3
0.22
0.79
22.9
0.21
0.00
2.0:13.0:1.1
22.1
0.21
0.20
23.0
0.22
0.39
2.1:13.6:1.1(16.8ml)
22.1
0.21
0.17
11.5
0.11
0.03
1.9:12.4:0.9(15.2ml)
32.4
0.24
0.20
13.1
0.22
0.49
Optimized time (10min)
12.1
0.41
12.8
0.23
15min
23.0
0.22
0.63
13.0
0.21
0.03
20min
24.7
0.34
0.74
21.1
0.34
0.13
Optimized time (20min)
16.5
0.12
0.43
25.6
0.2
0.29
25min
23.2
0.31
0.63
24.1
0.18
0.46
30min
12.3
0.21
0.42
13.2
0.23
0.51
Principal analyst
16.7
0.67
0.62
29.5
0.42
0.12
Analyst one
22.1
0.53
0.69
14.3
0.16
0.55
Analyst two
35.4
0.25
0.43
12.3
0.17
0.11
b
50 ng/band
400 ng/band
*
SD and RSD calculated from peak areas of densitograms.
3.3.6. Application of the Method for
Marketed Formulation
Analysis of the marketed formulations was performed in
triplicate and results are presented in Table 5. The drug content
was found to be 98.20 % of the claimed value (RSD = 0.228)
and 101.60 % (RSD = 0.164) for betamethasone and
dexchlorpheniramine maleate, respectively. The low % RSD
values indicated suitability of the developed method for
routine simultaneous determination of betamethasone and
dexchlorpheniramine maleate in pharmaceutical dosage forms.
Table 5. Data for analysis of the marketed dosage form (Celestamine®) using the developed method
Substance
Average peak area ± SD
% RSD
Amount recovered ± SD
% Recovery ± SD
a
1382.17 ± 3.15
0.23
73.69ng ± 0.21
98.20% ± 0.21
Dexchlorpheniramine maleate b
1684.13 ± 2.77
0.16
609.78ng ±1.26
101.60% ±1.26
Betamethasone
a = 75 ng/band, b = 600 ng/band
3.4. Forced Degradation Studies
nature of the method.
Densitograms obtained for the drugs treated with acid,
base, hydrogen peroxide, and heat contained well resolved
spots for the pure drugs and the degradation products. The Rf
values of
the parent drugs (betamethasone and
dexchlorpheniramine maleate) were not significantly
changed from the original position in the presence of the
degradation products, which showed the stability-indicating
3.4.1. Degradation Under Acidic Conditions
Betamethasone was degraded under acidic condition and
showed three degradation products at Rf of 0.19, 0.25 and 0.36
in addition to the one for the original compound at Rf = 0.32 as
shown in Figure 5. No degradation product was observed
under acidic condition for dexchlorpheniramine maleate.
AASCIT Journal of Chemistry 2015; 2(2): 32-42
A
39
B
Figure 5. Densitogram for betamethasone [A] and synthetic mixture [B] under acid induced degradation, mobile phase, ethyl acetate: methanol: ammonia 2:
13: 1, v/v/v, scanned at 226 nm.
3.4.2. Degradation Under Alkaline Conditions
Betamethasone was degraded in alkaline condition and
showed an additional spot at Rf = 0.36 as shown in Figure 6.
A
There was no degradation product for dexchlorpheniramine
maleate under the alkaline conditions.
B
Figure 6. Densitogram for betamethasone [A] and synthetic mixture [B] under alkaline induced degradation, mobile phase, ethyl acetate: methanol: ammonia
2: 13: 1, v/v/v, scanned at 226 nm.
3.4.3. Degradation Studies Under Oxidative
Condition
Betamethasone and dexchlorpheniramine maleate when
exposed to 3 % H2O2 showed no degradation product. When
the drug solutions were exposed to 30 % H2O2,
betamethasone showed one degradation product at Rf = 0.36
and dexchlorpheniramine maleate had one additional peak
that appeared at Rf = 0.67 [Figure 7].
40
Tadesse Haile Fereja et al.: Stability Indicating HPTLC Method Development for Determination of
Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form
A
B
C
Figure 7. Densitogram for betamethasone [A], dexchlorpheniramine maleate [B] and synthetic mixture [C] under oxidative degradation condition by 30%
H2O2, mobile phase, ethyl acetate: methanol: ammonia 2:13:1, v/v/v, scanned at 226 nm
3.4.4. Thermal Degradation
Betamethasone was degraded when subjected to heat for
24 hrs and degradation products appeared at Rf = 0.26 and Rf
= 0.36 as shown in Figure 8. No degradation was observed
under thermal stress for dexchlorpheniramine maleate.
Figure 8. Densitogram for betamethasone under thermal induced degradation. (Mobile phase, ethyl acetate: methanol: ammonia 2:13:1, v/v/v, scanned at
226nm)
AASCIT Journal of Chemistry 2015; 2(2): 32-42
3.4.5. Photolytic Conditions
The photo degradation study conducted showed no
degradation product for the two drug substances. The drugs
were found to be stable on exposure to day light continuously
for three days.
4. Conclusion
The developed HPTLC technique is rapid, precise, specific,
robust, accurate and stability-indicating. Statistical analysis
proved that the method is reproducible and selective for
simultaneous
determination
of
betamethasone
and
dexchlorpheniramine maleate in pharmaceutical formulations.
As the method could effectively separate the drugs from their
degradation products it can be employed as a stability
indicating one.
Acknowledgments
One of the authors (T. H.) would like to acknowledge the
Graduated Studies and Research Office of Addis Ababa
University for sponsoring this research. We also acknowledge
the Food, Medicine and Health Care Control Authority of
Ethiopia (FMHCCA), for the laboratory facilities.
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