AASCIT Journal of Chemistry 2015; 2(2): 32-42 Published online April 10, 2015 (http://www.aascit.org/journal/chemistry) Keywords Betamethasone, Dexchlorpheniramine Maleate, HPTLC, Stability Indicating, Degradation Received: February 9, 2015 Revised: March 9, 2015 Accepted: March 10, 2015 Stability Indicating HPTLC Method Development for Determination of Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form Tadesse Haile Fereja 1, * , Ariaya Hymete 2 , Adnan A. Bekhit 2, 3 1 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 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 (R f = 0.32 ± 0.04) and for dexchlorpheniramine maleate (R f = 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 r 2 ± RSD = 0.997 ± 0.00102 for betamethasone and 100 - 800 ng/ul for dexchlorpheniramine maleate with r 2 ± 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% H 2 O 2 (R f = 0.36), 1N HCl (R f 0.19, 0.25 and 0.36), 1N NaOH (R f = 0.36) and thermal condition (R f 0.26 and 0.36). Dexchlorpheniramine maleate degraded with 30% H 2 O 2 showed additional peak at R f 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-2- yl) propan-1-amine (Z)-butanedioate (Figure 1 b) is a potent antihistamine used for the
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AASCIT Journal of Chemistry 2015; 2(2): 32-42
Published online April 10, 2015 (http://www.aascit.org/journal/chemistry)
Keywords Betamethasone,
Dexchlorpheniramine Maleate,
HPTLC,
Stability Indicating,
Degradation
Received: February 9, 2015
Revised: March 9, 2015
Accepted: March 10, 2015
Stability Indicating HPTLC Method Development for Determination of Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form
Tadesse Haile Fereja1, *
, Ariaya Hymete2, Adnan A. Bekhit
2, 3
1Department of Pharmacy, College of Medicine and Health Science, Ambo University, Ambo,
Ethiopia 2Department of Pharmaceutical Chemistry, School of Pharmacy, Addis Ababa University, Addis
Ababa, Ethiopia 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria,
Table 3. Recovery studies for betamethasone and dexchlorpheniramine maleate (n = 5)
Drug added to the analyte (%) Theoretical content(ng) Reference Added(ng) Recovery (%)±SD % RSD
(a)Betamethasone
0 50 99.70 ± 0.64 0.49
80 90 40 99.87 ± 0.53 0.27
100 100 50 100.44 ± 0.6 0.22
120 110 60 100.10 ± 0.42 0.19
(b)Dexchlorpheniramine maleate
0 400 99.90 ± 0.04 0.38
80 720 320 99.79 ± 0.02 0.21
100 800 400 99.73 ± 0.06 0.51
0.29 120 880 480 100.49 ± 0.03
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 r
2 = 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
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
respectively.
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)
Parameters Betamethasone a Dexchlorpheniramine maleate b
SD* RSD* t cal SD* RSD* t cal
Optimized solvent system 2.0:13.0:1.0 (16ml) 14.5 0.53 13.3 0.27
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
nature of the method.
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 39
A 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.
There was no degradation product for dexchlorpheniramine
maleate under the alkaline conditions.
A 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 41
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
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