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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 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,

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 (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-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 33

treatment of several allergies and skin irritation. It readily

crosses the blood brain barrier and one of its collateral effects

is the induction of sedation [2].

O

OH

CH3

CH3 H

HF

HOH

H

O

N

H

N

CH3

CH3

Cl

OH

H3C

OH

OH

O

O

1

4

16

20

7

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 micellar-

organic 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 zero-

crossing 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

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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) (Sharing-

Plough, France) were obtained from Food, Medicine and

Health Care Control Authority FMHACA, Ethiopia.

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.

2.2. Method

2.2.1. Preparation of Standard Solutions

(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.

(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.

2.2.2. 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.

2.2.4. 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.

2.3. Validation of the Developed Method

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 100-

1500 ng/band of dexchlorpheniramine maleate.

2.3.2. Precision Studies

Repeatability and intermediate precision were studied by

taking a concentration within the linearity range at three

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AASCIT Journal of Chemistry 2015; 2(2): 32-42 35

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

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

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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 Betamethasone Dexchlorpheniramine maleate

Linearity range 25 - 137.5 ng/spot 100-800 ng/spot

r ± RSD 0.998 ± 0 .00102 0.999 ± 0.0141

Slope ± RSD 15.17 ± 0. 14 2.303 ± 0.836

Intercept ± RSD 271.0 ± 2.5 309.7 ± 4.44

Confidence limit of slope a 15.17 ± 0.133 2.303 ± 0.037

Confidence limit of intercept a 271.0 ± 0.21 309.7 ± 26.45

a 95% confidence limit.

Figure 3. The UV spectral display of dexchlorpheniramine maleate [1] and betamethasone [2].

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AASCIT Journal of Chemistry 2015; 2(2): 32-42 37

3.3. Method Validation

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

repeatability of the method.

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 Amount (ng/band) Intra-day precision Inter-day precision

SD RSD SD RSD

Betamethasone

50 5.0 1.10 14.3 1.13

75 2.3 0.17 12.4 1.10

100 8.8 0.80 11.9 0.9

Dexchlorpheniramine maleate

400 7.3 0.99 17.0 1.09

600 9.9 1.11 15.1 1.06

800 9.6 1.07 1.3 0.06

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 [**].

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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

Mobile phase composition (v/v/v)

1.8:13.0:1.0 28.7 0.85 0.25 17.4 0.54 0.95

2.2:13.0:1.0 14.1 0.41 0.98 14.0 0.30 0.55

2.0:11.7:1.0 13.2 0.31 0.86 24.1 0.30 0.46

2.0:14.3:1.0 23.0 0.29 0.63 12.5 0.18 0.26

2.0:13.0:0.9 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

Volume of mobile phase (v/v/v) 1.9:12.4:0.9(15.2ml) 32.4 0.24 0.20 13.1 0.22 0.49

Time from spotting to chromatography

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

Time from chromatography to scanning

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

a 50 ng/band

b 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

Betamethasone 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

a = 75 ng/band, b = 600 ng/band

3.4. Forced Degradation Studies

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.

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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].

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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)

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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

Ethiopia (FMHCCA), for the laboratory facilities.

References

[1] McGraw-Hill (2003). Basic & Clinical Pharmacology. 9th edition. pp 1457-1458.

[2] Montoro J., Sastre J., Bartra J., del Cuvillo A., Dávila I., Jáuregui I., Mullol J. and Valero A. (2006). Effect of H1 antihistamines upon the central nervous system. J Investig Allergol Clin Immunol; 16: 24-28.

[3] Alberto S. P., Lina S. O. B., Gustavo D. M., Jorge J. G. and Gilberto D. N. (2005). Quantification of betamethasone in human plasma by liquid chromatography–tandem mass spectrometry using atmospheric pressure photoionization in negative mode. J Chrom B; 828: 27–32.

[4] Jian-Jun Z., Li D., Da-Wei X., Zhu-Yu B., Hong-Wei F., Li L., and Guang-Ji W. (2008). Determination of betamethasone and betamethasone 17-monopropionate in human plasma by liquid chromatography–positive/negative electrospray ionization tandem mass spectrometry. J Chrom B; 873: 159–164.

[5] Fabien B., Yvan G., Michel A. and Gilbert P. (2000). Analysis of corticosteroids in hair by liquid chromatography–electrospray ionization mass spectrometry. J Chrom B; 740: 227– 236.

[6] Aldo P., Giorgio M. B. and Maria M. (1998). Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine. J Chrom B; 713: 339–352.

[7] Gonza´lez L., Yuln G. and Volonte M. G. (1999). Determination of cyanocobalamin, betamethasone, and diclofenac sodium in pharmaceutical formulations, by high performance liquid chromatography. J Pharmaceu Biomed Anal; 20: 487–492.

[8] Kedor-Hackmann E. R. M., Gianotto E. A. S. and Santoro M. I. R. M. (1998). Determination of betamethasone dipropionate and salicylic acid in pharmaceutical preparations by high-performance liquid chromatography. Drug Devel and Indu Pharm; 24: 553-555.

[9] Mahesh N. S., Matthias S., Peter W. N. and William J. J. (2004). Stabilization and HPLC analysis of betamethasone sodium phosphate in plasma. J Pharmac Sci; 93: 726-732.

[10] Chandra N. S. and Sanjib B. (2009). A validated simultaneous RP-HPLC method for determination of betamethasone dipropionate and tolnaftate in combined semisolid formulation. Intern J Chem Tech Res; 1: 671-674.

[11] Shou-Mei W., Hsin-Lung W. and Su-Hwei C. (1995). Determination of betamethasone and dexamethasone in plasma by fluorogenic derivatization and liquid chromatography. Analytica Chimica Acta; 307: 103-107.

[12] Gunawan I., Sonja W. and Sesylia S. (1999). Simultaneous densitometric determination of betamethasone valerate and miconazole nitrate in cream, and its validation. J Liq Chrom & Rel Technol; 22: 143–152.

[13] Lestyo W. and Gunawan I. (2003). HPTLC determination of Betamethasone in tablets and its validation. J Lliq Chrom & Related Tech; 26: 2709–2717.

[14] Min L., Xin W., Bin C., Tze-Ming C. and Abu R. (2009). Forced degradation of betamethasone sodium phosphate under solid state: Formation, characterization, and mechanistic study of all four 17, 20-diastereomers of betamethasone 17-deoxy-20- hydroxyl 21-oic acid. J Pharm Scie; 98: 894-904.

[15] Minshan S., Wilmer A. G., Yu-Chien W., Qinglin T., Robert J. M. and Abu M. R. (2009). Development and validation of a stability-indicating HPLC method for simultaneous determination of salicylic acid, betamethasone dipropionate and their related compounds in Diprosalic Lotion. J Pharmaceu Biomed Anal; 50: 356–361.

[16] Qiang F., Minshan S., Dwight C., Robert M. and Abu M. R. (2010). Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds. J Pharmaceu Biomed Anal; 51: 617–625.

[17] Yuan X., Kang P. X.and Abu M. R. (2009). Development and validation of a stability- indicating RP-HPLC method to separate low levels of dexamethasone and other related compounds from betamethasone. J Pharmaceu Biomed Anal; 49: 646–654.

[18] Aravindaraj J. R., Gopinath R., Rajan S., Mahesh K. S., Nanjan M. J. and Suresh B. (2007). Development and application of a validated liquid chromatography-mass spectrometry method for the determination of dexchlorpheniramine maleate in human plasma. Iranian J Pharmac Sci Summer; 3: 161-170.

[19] Mayte Gil-A., M-a Celia G.A.-C. and Josep E.-R. (2000). Correlation between hydrophobicity and retention data of several antihistamines in reversed-phase liquid chromatography with aqueous-organic and micellar-organic mobile phases. Analytica Chimica Acta; 421: 45–55.

[20] Elena B., Viviana C., Gianluca G. and Anna F. (2001). Determination of optical purity by nonenantioselective LC using CD detection. J Pharmaceu Biomed Anal; 26: 837–848.

Page 11: Stability Indicating HPTLC Method Development for ...article.aascit.org/file/pdf/9780719.pdf · Stability Indicating HPTLC Method Development for Determination of ... Stability Indicating

42 Tadesse Haile Fereja et al.: Stability Indicating HPTLC Method Development for Determination of

Betamethasone and Dexchlorpheniramine Maleate in a Tablet Dosage Form

[21] Fawzy A. El-Y., Hassan H. H. and Sulaf A. A. (2006). New spectrofluorometric application for the determination of ternary mixtures of drugs. Analytica Chimica Acta; 580: 39–46.

[22] ICH Topic Q 1 A (R2) (2003). Stability testing of new drug substances and products.

[23] Monika B. and Saranjit S. (2002). Development of validated stability-indicating assay methods—critical review. J Pharmaceu Biomed Anal; 28: 1011–1040.

[24] Paradkar A.R., Himani A. and Neeraj K. (2003). Stability-

indicating HPTLC determination of tizanidine hydrochloride in bulk drug and pharmaceutical formulations. J Pharmaceu Biomed Anal; 33: 545-552.

[25] Ali J., Ali N., Sultana Y., Baboota S. and Faiyaz S. (2007). Development and validation of a stability-indicating HPTLC method for analysis of anti tubercular drugs. Acta Chromatographica; 18: 168-179.

[26] Sapna N. M. and Pradeep R. V. (2001). Stability indicating HPTLC method for the simultaneous determination of pseudoephedrine and cetirizine in pharmaceutical formulations. J Pharmaceu Biomed Anal; 25: 663–667.