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Simultaneous UV Spectrophotometric Estimation of Clotrimazole and

Beclomethasone Dipropionate in Their Combined Dosage Forms by
Q absorption Ratio, Dual Wavelength and First Order...

Article · February 2012

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 RESEARCH ARTICLE

STANDARD AND TEST SOLUTIONS

Simultaneous UV Spectrophotometric Estimation of Preparation of Standard Solution
Clotrimazole and Beclomethasone Dipropionate in The standard stock solutions containing
1000μg/mL each of CT and BD were
Their Combined Dosage Forms by QǦǦabsorption prepared separately by dissolving reference
standards in Methanol and diluting with the
Ratio, Dual Wavelength and First Order Derivative same diluent.
Method
Preparation of Test Solution
Komal R Dhudashia1*, Amit V Patel1, Chhaganbhai N Patel1 Take 1 ml of lotion in 10 ml volumetric flask
Abstracts: Development and validation of UV Spectrophotometric method for simultaneous and make up volume with methanol. Then
estimation of clotrimazole and beclomethasone dipropionate in their combined dosage form. extract the prepared solution with
First method is based on QǦabsorption Ratio method using two wavelengths, 260.6 nm (λmax cyclohexane using separating funnel. Discard
of CT) and 250.8 nm (Isoabsorptive point). The second method is the dual wavelength method, the cyclohexane layer and collect the
where 237 nm and 241 nm were selected as λ1 and λ2 for the determination of Clotrimazole methanol layer. From the methanol layer
and 259 nm and 264 nm were selected similarly for the determination of Beclomethasone. The take 3 ml and dilute up to 10 ml with
last method involves the use of First order derivative technique. Here 239 nm, the zero methanol. Take absorbance of this solution at
crossing point of Beclomethasone, was selected for the determination of Clotrimazole and both the selected wavelength.
260.8 nm, the zero crossing point of Clotrimazole, was selected for the determination of
Beclomethasone. Clotrimazole showed linearity in the range of 100Ǧ450μg/mL and METHODS
Beclomethasone showed linearity in the range of 6-34μg/mL in all the methods. All methods Q-absorption Ratio Method
were validated statistically and recovery studies were carried out. All methods were found to This method is applicable to the drugs that
be accurate, precise and reproducible. These methods were applied to the assay of the drugs in obey Beer’s law at all wavelengths and the
marketed formulation, which were found in therange of 98.0% to 100.0% of the labeled value ratios of absorbances at any two wavelengths
for both Clotrimazole and Beclomethasone. Three new, simple, accurate and precise UV are a constant value, independent of
spectrophotometric methods have been developed and validated for the simultaneous concentration or pathlength [4, 5, 6, 7]. Two
determination of Clotrimazole (CT) and Beclomethasone dipropionate (BD) in their combined wavelengths, 250.80 nm (Isoabsorptive
dosage forms. point) and 260.6 nm (λmax of CT) were
selected for the formation of QǦabsorbance
Key Words: Clotrimazole, Beclomethasone dipropionate, UVǦSpectrophotometric,
equation. The absorptivity coǦefficient of
QǦabsorption Ratio, dual wavelength, first order derivative.
each drug at both the wavelengths were
INTRODUCTION graphy (HPTLC)[17,18] and stability-indicating determined.
Clotrimazole is 1-[(2-chlorophenyl) HPLC method20. Similarly, a survey of the The concentration of individual
(diphenyl) methyl]-1h-imidazole8. It is a analytical literature for BD revealed methods components, CCT and CBD may be calculated
prescription drug indicated for the treatment based on HPLC for determination in using the following equations
and prophylaxis of fungal infections. pharmaceuticals[10,11,12,21]. According to
Clotrimazole interacts with yeast 14-α detailed survey of analytical literature none େ୘ ൌ ሺ୫ െ ୆ୈ Τେ୘ି ୆ୈ ሻ ൈ ͳΤƒšͳ
demethylase, a cytochrome p-450 enzyme of the reported analytical procedures …………………………. (1)
that converts lanosterol to ergosterol, an describes a simple and satisfactory UV ୆ୈ ൌ ሺ୫ െ େ୘ Ȁେ୘ െ ୆ୈ ሻ ൈ ͳΤƒ›ͳ
essential component of the membrane. In spectrophotometric method for ………………………….. (2)
this way, clotrimazole inhibits ergosterol simultaneous determination of CT and BD in
synthesis, resulting in increased cellular their combined dosage forms. So the ୫ ൌ ʹΤͳ
permeability [1, 2, 3]. Beclomethasone objective of this work was to develop simple, .................……………… (3)
dipropionate is 9a-chloro-11b–hydroxy-16b– precise and rapid spectrophotometric
methyl-3, 20-dioxopregna 1, 4-diene-17, 21- methods for combination drug products େ୘ ൌ ƒšʹΤƒšͳ Ƭ୆ୈ ൌ ƒ›ʹΤƒ›ͳ
diyldipropionate9.It is a synthetic containing CT and BD. ..............................…….. (4)
halogenated glucocorticoid with anti- where, A1 and A2 are absorbance of
inflammatory and vasoconstrictive effects, is EXPERIMENTAL sample solution at Isoabsorptive point
used for treating steroid-dependent asthma, Materials and Reagents (250.8 nm) and λmaxof CT (260.6 nm)
allergic or nonallergic rhinitis. The anti- Reference standards of Clotrimazole and respectively;
inflammatory actions of corticosteroids are Beclomethasone dipropionate were supplied ax1 and ax2 are the absorptivities of CT
thought to involve phospholipase A2 by Glenmark pharmaceuticals Ltd. at 250.8 and 260.6 nm respectively and ay1
inhibitory proteins, lipocortins, which Lotion formulation containing 10 mg of and ay2 are the absorptivities of BD at the
control the biosynthesis of potent mediators CT and 0.25 mg of BD from Glenmark two wavelengths respectively.
of inflammation such as prostaglandins and pharmaceuticals Ltd. (candid-B lotion).
leukotrienes [1, 2, 3]. Methanol (AR grade) was purchased Dual Wavelength Method
The chemicalstructures of CT and BD are from SD Fine Chemicals Pvt. Ltd., The utility of dual wavelength data
shown inFigure1. (A) (B) Ahmedabad, India. processing programme is to calculate the
A detailed survey of analytical literature Cyclohexane from SD Fine Chemicals Pvt. unknown concentration of a component of
for CT revealed several methods based on Ltd., Ahmedabad, India. interest present in a mixture containing both
varied techniques, viz, HPLC [10,11,12,19], the components of interest and an unwanted
Spectrophotometry[14,15,16], Spectrofluorimetry13, Instrumentation interfering component by the mechanism of
HighǦ Performance ThinǦ Layer Chromato- A doubleǦbeam Shimadzu UVǦVisible the absorbance difference between two
spectrophotometer, Model UVǦ1800 points on the mixture spectra. This is directly
x Spectophotometric Conditions: proportional to the concentration of the
1Department
x Mode: Spectrum components of interest, independent of the
of Quality Assurance, Shri
Sarvajanik Pharmacy College, Mehsana-384001, x Scan speed: Medium interfering components. The preǦrequisite
Gujarat, India. x Wavelength range:400-200nm for dual wavelength method is the selection
E-mail: komaldhudashia@gmail.com x Absorbance scale:0.00A-2.00A of two such wavelengths where the
*Corresponding author x Initial baseline correction: Methanol AR interfering component shows same

Inventi Impact: Pharm Ana & Qual Assur Vol. 2012, Issue 2 94 2012ppaqa295, CCC: $10 © Inventi Journals (P) Ltd
[E- ISSN 0976–755X, P- ISSN 2229–4198] Published on Web 15/04/2012, www.inventi.in
 RESEARCH ARTICLE

Table 1: Data showing linearity of the developed methods

Methods Q-Absorption ratio method Dual wavelength method First derivative spectroscopy method
Parameters CT BD CT BD CT BD
Linearity-range(μg/ml) 100 -450 6-34 100-450 6-34 100-450 6-34
Slope 0.002 0.0204 0.0029 0.0024 -0.0007 -0.0005
Intercept 0.005 0.0296 0.0514 0.0025 0.0089 0.0004
Correlation coǦefficient 0.9993 0.9993 0.9993 0.9992 0.9997 0.9995

Table 2: Data showing Accuracy of the developed methods

% Recovery (n=3) *
Amt of sample Amt.of drug added
First derivative Dual wavelength Q-Absorption ratio
(μg/ml) (μg/ml)
spectroscopy method method method
CT BD CT BD CT BD CT BD CT BD
300 7.5 100 6 98.6 98.3 99.6 99.1 100.3 100.6
300 7.5 120 7.5 99.6 98.6 99.8 98 101 99.2
300 7.5 140 9 99.5 98.8 100.0 98.3 100.2 100.4
*(n = number of repetition)

Table 3: Data showing Precision of the developed methods (n=3)*

Q-Absorption ratio method Dual wavelength method First derivative Spectroscopy
Methods (%RSD) (%RSD) method(%RSD)
CT BD CT BD CT BD
System Intraday 0.25 0.59 0.42 0.55 0.58 0.22
precision Intraday 0.53 0.50 0.27 1.13 0.44 1.03
*(n = number of repetition)

Table 4: Data showing Solution Stability of the developed methods

% Assay Difference – Sample solution % Assay Difference – Standard solution
Time
CT BD CT BD
Initial ---- ---- ---- ----
After 12 hours 0.46 0.98 0.26 0.67
After 24 hours 1.06 0.44 0.87 0.58
After 36 hours 0.50 0.66 0.47 0.94

Table 5: Data showing LOD and LOQ of the developed methods

Q-Absorption ratio method Dual wavelength method First derivative spectroscopy method
Methods
CT BD CT BD CT BD
LOD(μg/ml) 1.65 0.32 1.7 0.41 1.74 0.39
LOQ(μg/ml) 5 0.98 5.17 1.25 5.28 1.2

Table 6: Result of Analysis of formulation

Q-Absorption ratio method Dual wavelength method First derivative spectroscopy method
Methods
CT BD CT BD CT BD
%Assay 98.6 99.6 99.34 99.2 99 99.18
S.D.(n=3) 0.55 0.39 0.44 0.28 0.38 0.56
(n = number of repetition)

absorbance whereas the component of
interest shows significant difference in
absorbance with concentration [4, 5, 6, 7]. Based
on this criterion, two wavelengths 237 nm
and 241 nm were selected as λ1 and λ2 for
the estimation of CT. BD shows the same
absorbance at these wavelengths. Similarly,
wavelengths 259 nm and 264 nm were
selected as λ1 and λ2 for estimation of BD.
First Derivative Spectroscopy Method
First derivative spectroscopy on the basis of
zeroǦcrossing measurements involves
measurement of the absolute value of the
total derivative spectrum at an abscissa value
corresponding to them zeroǦcrossing
wavelength of the derivative spectrum of
another
Component[4,5,6,7]. In this method,
260.80nm was selected for the
determination of BD, which is the zero
crossing point of CT and 239 nm, the zero
crossing point of BD, was selected for the
determination of CT.
Method Validation
All the methods were validated as per ICH
guidelines for parameters like solution
stability, linearity, accuracy, precision, limit
of detection, limit of quantitation22.
RESULTS AND DISCUSSION
In the present work, three methods, namely,
QǦabsorption ratio method, dual wavelength
method and first derivative spectroscopy
method were developed for the simultaneous
spectroscopic estimation of CT and BD in
commercially available lotion dosage forms.
Methanol was used as the solvent since both
the drugs exhibit good solubility in it and no
interference due to excipients of the lotion
formulation were observed.
Q-absorption Ratio Method
As shown in FigureǦ2, the overlain spectra of
both drugs show a Reproducible IsoǦ
absorptive point at 250.8 nm. Thus
estimation of drugs by QǦabsorbance ratio
equation method was carried out at 250.8
nm (Isoabsorptive point) and 260.6nm
(λmaxof CT). The standard solutions of BD
and CT were prepared to determine the
absorptivity values of the subject analyte at
the two selected wavelengths. The method
showed good linearity in the range of
6Ǧ34μg/mL for BD and 100-450μg/mL for
CT.
Dual Wavelength Method
In this method, standard solutions of CT and
BD were scanned in the entire range from
200 nm to 400 nm. As shown in FigureǦ3, at
wavelengths of 237 nm and 241 nm, BD
shows same absorbance, and hence were

Inventi Impact: Pharm Ana & Qual Assur Vol. 2012, Issue 2 95 2012ppaqa295, CCC: $10 © Inventi Journals (P) Ltd
[E- ISSN 0976–755X, P- ISSN 2229–4198] Published on Web 15/04/2012, www.inventi.in
 RESEARCH ARTICLE
Figure 1: Chemical structure of (A) Clotrimazole and (B) Beclomethasone dipropionate [8,9]
Figure 2: Overlain zero order spectra of CT and BD (Q-Absorption ratio Method)
Figure 3: Overlain zero order spectra of CT and BD (Dual Wavelength Method)
selected for the estimation of CT. Similarly, at
wavelengths of 259 nm and 264 nm, CT
shows same absorbance, which were
selected for the estimation of BD. For
estimation of CT, difference in absorbances
at 237nm and 241nm were plotted against
the concentrations, while for the estimation
of BD, difference in absorbances at 259 nm
and 264 nm were plotted against the
concentrations to construct two separate
calibration curves for both RSV and EZE.
The method showed good linearity in the
range of 6Ǧ34μg/mL for BD and 100-
450μg/mL for CT.
Inventi Impact: Pharm Ana & Qual Assur Vol. 2012, Issue 2
[E- ISSN 0976–755X, P- ISSN 2229–4198]
against the respective concentrations of BD.
The method showed good linearity in the
range of 6Ǧ34μg/mL for BD and 100-
450μg/mL for CT.
Method Validation
The developed methods were validated for
parameters like solution stability,
linearity, precision, accuracy, LOD, LOQ.
the data for which are presented in the
Tables. The low value of R.S.D. value
indicates that all the methods are precise
and accurate.
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96 2012ppaqa295, CCC: $10 © Inventi Journals (P) Ltd
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 RESEARCH ARTICLE

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Inventi Impact: Pharm Ana & Qual Assur Vol. 2012, Issue 2 97 2012ppaqa295, CCC: $10 © Inventi Journals (P) Ltd
[E- ISSN 0976–755X, P- ISSN 2229–4198] Published on Web 15/04/2012, www.inventi.in

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