Application of three novel spectrophotometric methods manipulating ratio spectra for resolving a pharmaceutical mixture of Chlorphenoxamine hydrochloride and Caffeine

Abstract

Three spectrophotometric methods are presented for the determination of a binary mixture of Chlorphenoxaminehydrochloride (CPX) and Caffeine (CAF) in laboratory prepared mixture and pharmaceutical dosage form without prior separation. Method (I)is an extended ratio subtraction method (EXRSM) coupled with ratio subtraction method (RSM), which depends on subtraction of the plateau values from the ratio spectrum. Method (II)is a ratio difference spectrophotometric method (RDSM), which depends on the difference in value between two different wavelengths of the ratio spectrum. Method (III) is a mean centering of ratio spectra (MCR). Mathematical explanation of the three methods is illustrated. Calibration curves of the three methods are linear over the concentration ranges of 4-24 μgml-1and 2-24 μgml-1for CPX and CAF, respectively.The three methods proved to be simple, specific, accurate and precise. Solvent used is double distilled water. The three methods are validated as per the ICH guidelines where accuracy, precision, repeatability and robustness are found to be within the acceptable limits.

Full text

Research Article
APPLICATION OF THREE NOVEL SPECTROPHOTOMETRIC METHODS MANIPULATING RATIO
SPECTRA FOR RESOLVING A PHARMACEUTICAL MIXTURE OF CHLORPHENOXAMINE
HYDROCHLORIDE AND CAFFEINE
AYMAN M. MOHSENa, HAYAM M. LOTFYb, AMR M. BADAWEYb, HESHAM SALEMa AND SONIA Z. ELKHATEEBb
aAnalytical Chemistry Department, Faculty of Pharmacy, MSA University, 6th October City, Giza, Egypt,
bAnalytical Chemistry Department, Faculty of Pharmacy, Cairo University, KasrElinist., 11562, Cairo, Egypt.
Email: dr.aymanmohsen@gmail.com
Received: 22 Dec 2012, Revised and Accepted: 29 Jan 2013
ABSTRACT
Three spectrophotometric methods are presented for the determination of a binary mixture of Chlorphenoxamine hydrochloride (CPX) and Caffeine
(CAF) in laboratory prepared mixture and pharmaceutical dosage form without prior separation. Method (I) is an extended ratio subtraction
method (EXRSM) coupled with ratio subtraction method (RSM), which depends on subtraction of the plateau values from the ratio spectrum.
Method (II)is a ratio difference spectrophotometric method (RDSM), which depends on the difference in value between two different wavelengths
of the ratio spectrum. Method (III) is a mean centering of ratio spectra (MCR). Mathematical explanation of the three methods is illustrated.
Calibration curves of the three methods are linear over the concentration ranges of 4-24 µgml-1 and 2-24 µgml-1 for CPX and CAF, respectively. The
three methods proved to be simple, specific, accurate and precise. Solvent used is double distilled water. The three methods are validated as per the
ICH guidelines where accuracy, precision, repeatability and robustness are found to be within the acceptable limits.
Keywords: Chlorphenoxamine HCl, Caffeine, Extended ratio subtraction method, Ratio subtraction method, Mean centering ratio spectra, Mean
centering, Ratio difference, Ratio spectra
INTRODUCTION
Chlorphenoxamine hydrochloride (CPX),2- [1-(4-Chlorophenyl)-1–
phenylethoxy] -N, N-dimethylethanamine.HCL, is a histamine H1-
receptor antagonist with antimuscarinic properties [1] and Caffeine
(CAF), 3, 7-Dihydro-1, 3, 7-trimethyl-1 H- purine-2, 6-dione, is a
xanthine derivative with CNS stimulant activity [1, 2]. Combination
of the two drugs is available as Allergex Caffeine tablets produced by
the Egyptian International Pharmaceutical Industries Co. (EIPICO).
CPX provides symptomatic relief in allergic conditions, and CAF is
added to counteract the sedative effects of CPX. The chemical
structures of the two investigated drugs are shown in Fig. 1.
The literature in hand describes several articles dealing with the
simultaneous determination of the components of this pharmaceutical
mixture. It includes colorimetry [3], derivative spectrophotometry
[4,5], chemometry [4], TLC spectrodensitometry [6] and HPLC [7].
On the other hand, literature survey reveals that no ratio spectra UV-
spectroscopic methods [8-10] were reported for this simultaneous
determination of this combination.
Thus, the purpose of this study was to solve the problem of
overlapping spectra of both drugs by developing rapid, simple and
precise spectrophotometric methods for their simultaneous
determination.
Fig. 1: The Chemical Structure of Chlorphenoxamine Hydrochloride [1] and Caffeine [2].
Theory of the proposed methods
Extended ratio subtraction method (EXRSM)
Extended ratio subtraction method (EXRSM) starts with the ratio
subtraction method (RSM) [9] which depends on that, if you have a
mixture of two drugs X and Y having overlapped spectra, you can
determine X by dividing the spectrum of the mixture (X+Y) by a
known concentration of Y as a divisor Y'. The division will give a new
curve that represents X/Y' + constant. Measure the value of this
constant X/Y' in the plateau region. If we subtract this constant
value, then multiply the obtained curve after subtraction by Y' (the
divisor), then we can obtain the zero order absorption spectrum
(D0) of X, (original spectrum of X). This can be summarized in the
following equations:
(X+Y)/Y' = X/Y' + Y/Y' = X/Y' + constant
X/Y' + constant – constant = X/Y'
X/Y' x Y' = X
The concentration of X is calculated using the regression equation
representing the linear relationship between the absorbance at its
λmax versus the corresponding concentration of X.
To determine the second component Y, an extension of the already
developed method has been established as a new approach in which
Y could be determined by dividing the obtained D0 spectrum of X by
a known concentration of X as a divisor X' to get the value of the
constant X/X'. Dividing the spectrum of the mixture X+Y by the same
divisor X', the division will give a new curve that represents X/X' +
Y/X' where X/X' is the previously obtained constant, then multiply
the obtained curve after subtraction by X' (the divisor), therefore we
can obtain the zero order absorption spectrum D0 of Y (original
spectrum of Y).
Y/X' + X/X' – X/X' = Y/X' x X' = Y
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The concentration of Y is calculated by using the regression equation
representing the relationship between the absorbance at its λmax
versus the corresponding concentration of Y.
Ratio difference spectrophotometric method (RDSM)
This method is based on that the amplitude difference between two
points on the ratio spectra of a mixture is directly proportional to the
concentration of the component of interest. The independence of the
interfering component is the major advantage of this method [10].
This method depends on that, if you have a mixture of two drugs X
and Y having overlapped spectra, you can determine X by dividing
the spectrum of the mixture by a known concentration of Y as a
divisor Y'. The division will give a new curve that represents
(X+Y)/Y' i.e. X/Y' + Y/Y', where Y/Y' is a constant. By selecting two
wavelengths λ1 and λ2 on the obtained ratio spectrum and
subtracting the amplitudes at these two points, the constant Y/Y'
will be cancelled along with any other instrumental error or any
interference from the sample matrix. This can be summarized as the
following:
(X+Y)/Y' = X/Y' + Y/Y'= X/Y' + constant
Suppose the amplitudes at the two wavelengths are P1 and P2 at λ1
and λ2 respectively; by subtracting the two amplitudes the
interfering Y shows no interference; then:
P1 – P2 = (X/Y')1 + constant – {(X/Y')2 + constant}
P1 – P2 = (X/Y')1 – (X/Y')2
where; P1 is the peak amplitudes of the ratio spectrum at λ1, P2 is
the peak amplitudes of the ratio spectrum at λ2.
The concentration of X is calculated by using the regression equation
representing the linear relationship between the differences of the
ratio spectra amplitudes at the two selected wavelengths versus the
corresponding concentration of drug X.
Similarly, Y could be determined by the same procedure using a
known X as a divisor X`.
Mean centering of ratio spectra spectrophotometric method
(MCR)
This is a well-established spectrophotometric method in which both
binary and ternary mixtures could be determined without previous
separation. In this method the ratio spectra are obtained after which
the constant is removed by mean centering of the ratio spectra [11].
To explain the mean centering expression, consider a three-
dimensional vector [12]:
[5]
y = [1]
[3]
We center or mean center (MC) this column by subtracting the mean
of three numbers
3
calling: y' = 3
3
5 3 +2
MC(y) = y –y' = 1 - 3 = -2
3 3 0
It could be proved that if the vector y is multiplied by n (a constant
number), the mean centered vector is also multiplied by n and also if
a constant number is added to the vector y, the mean center of this
vector is not changed.
Consider a mixture of two compounds X and Y. If there is no
interaction among the compounds and Beer’s law is obeyed for each
compound, it can be written:
Am = αxCx + αyCy (1)
where Am is the vector of the absorbance of the mixture, αx and αy
are the molar absorptivity vectors of X and Y and Cx and Cy are the
concentrations of X, Y and Z, respectively.
If Eq. (1) is divided by αy corresponding to the spectrum of a
standard solution of Y in binary mixture, the first ratio spectrum is
obtained in the form of Eq. (2) (for possibility of dividing operation,
the zero values of αy should not be used in the divisor):
B = Am/αy = αx Cx/αy + Cy (2)
If Eq. (2) is mean centered (MC), since the mean centering of a
constant (Cy) is zero, Eq. (3) would be obtained:
MC(B) = MC [αxCα/αy] (3)
Eq. (3) is the mathematical foundation of binary mixture analysis
that permits the determination of concentration of each of the active
compounds in the solution (X in these equations) without interfering
from the other compound of the binary system (Y in these
equations). As Eq. (3) shows there is a linear relation between the
amount of MC(B) and the concentration of X in the solution.
A calibration curve could be constructed by plotting MC(B) against
concentration of X in the standard solutions of X or in the standard
binary mixtures. For more sensitivity the amount of MC(B)
corresponding to maximum or minimum wavelength should be
measured.
Calibration graphs for Y could also be constructed as described for X.
MATERIALS AND METHODS
Instrumentation
Spectrophotometric analysis was carried out on a Shimadzu 1601
double beam spectrophotometer with a fixed slit width (2 nm) using
a pair of 1 cm matched quartz cells. The spectrophotometer is
connected to an IBM PC with an HP inkjet printer. The bundle
software, UV-Probe spectroscopy software version 2.1 (Shimadzu,
Kyoto, Japan), was used to process absorption.
Software
Microsoft Excel was used for handling data and processing
calculations.
Materials
Pure samples
Pure drug samples of CPX and CAF were kindly supplied by EIPICO
Pharmaceuticals, 10th of Ramadan City, Egypt. Their purity was
checked and found to be 100.62 ± 0.61 and 99.20 ± 0.78 % according
to the manufacturer method of analysis [3] and the BP [2], for CPX
and CAF, respectively.
Solvents
Double distilled water.
Pharmaceutical dosage form
Allergex Caffeine tablets (EIPICO Pharmaceuticals) Batch No.
1106021, labeled to contain 20 mg CPX and 50 mg CAF per tablet
were purchased from local pharmacies.
Stock and working standard solutions
Stock standard solutions
CPX and CAF stock standard solutions (both are 1 mg ml-1), prepared
by dissolving 100 mg of CPX and CAF, respectively, in a few
milliliters of double distilled water into a 100-ml volumetric flasks
and then completing to volume with the same solvent.
Working standard solutions
CPX and CAF working standard solution (both 0.1 mg ml-1), prepared by
transferring 10 ml of each of CPX and CAF stock solutions, into a 100-ml
volumetric flask and complete to volume with the same solvent.

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Procedure
Spectral characteristics and wavelengths selection
The absorption spectra of 16 µg ml-1 of CPX and 16 µg ml-1 of CAF
were recorded over the spectral wavelength range 200–320 nm
using double distilled water as blank.
Linearity and construction of calibration curves
Accurately measured aliquots equivalent to 50-600 µg CPX and 25-
600 µg CAF are transferred from their working standard solution
(both are 0.1 mg ml-1) into two series of 25-ml volumetric flasks and
complete to volume with double distilled water. The spectra of the
prepared standard solutions are scanned from 200 to 320 nm and
stored in the computer.
For extended ratio subtraction method (EXRSM) coupled with
ratio subtraction method (RSM)
Calibration curves are constructed relating the absorbance of zero
order spectra of CPX at 222.4 nm and CAF at 273.0 nm versus the
corresponding concentrations and regression equations are
computed.
For ratio difference spectrophotometric method (RDSM)
The stored spectra of CPX are divided by the spectrum of 12 µg ml-
1CAF while CAF spectra are divided by the spectrum of 12 µg ml-1
CPX.
Calibration curves of CPX and CAF are constructed by plotting the
difference between the amplitudes of ratio spectra at 226.3 and
235.0 nm for CPX and 209.1 and 226.7 nm for CAF, versus the
corresponding concentrations and the regression equations are
computed.
For mean centering of ratio spectra method (MCR)
The scanned spectra are exported to Microsoft Excel for subsequent
calculations, and then the spectra of CPX are divided by the
normalized spectrum of CAF, the obtained ratio spectrum is mean
centered. The same procedure is applied to CAF.
The Calibration curves for CPX and CAF are constructed by plotting
the mean centered values at 226.8 and 274.0 nm, respectively,
versus the corresponding concentrations and the regression
equations are computed.
Application of extended ratio subtraction, ratio difference and
mean centering of ratio spectra for the determination of CPX
and CAF in laboratory prepared mixtures
Solutions containing different ratios of CPX and CAF were prepared
by transferring accurately measured aliquots from their standard
working solutions into a series of 25-ml volumetric flasks and the
volume was completed to the mark with double distilled water. The
final concentration ranges were 4 - 24 µg ml-1 for CPX and 2 - 24 µg
ml-1 for CAF. Zero order absorption spectra of these different
laboratory prepared mixtures were recorded from 200 to 320 nm
using double distilled water as blank and the procedure under
linearity for each method was then followed. Concentrations of CPX
and CAF in the prepared samples were calculated from the
corresponding computed regression equations.
Application to pharmaceutical preparation
To determine the content of CPX and CAF in commercial tablets
(each tablet labeled to contain 20 mg CPX and 50 mg CAF), 20
tablets were weighed and finely powdered. A portion of powder
equivalent to one tablet was weighed accurately and transferred to a
100-ml beaker. 50 ml of double distilled water was added, stirred
using a magnetic stirrer for 15 min and filtered through 0.5μm
Whatman filter paper into a 100-ml volumetric flask. The residue
was washed three times each with 10 ml of double distilled water
and the solution was completed to the mark with the same solvent.
From the above prepared solution, further dilutions were prepared
in the obtained linearity ranges using the same solvent. The general
procedure described above under each method was followed to
determine the concentration of both drugs in the prepared dosage
form solution. The analysis was done in triplicates. Concentrations
of CPX and CAF in the prepared samples were calculated from the
corresponding computed regression equations.
RESULTS AND DISCUSSION
This paper describes the application of three recently developed
spectrophotometric ratio-spectra methods for the simultaneous
determination of CPX and CAF. The zero order absorption spectra of
pure drugs show overlapping which hinders their direct
determination as shown in Fig. 2.
Extended ratio subtraction method (EXRSM) coupled with ratio
subtraction method (RSM)
Extended ratio subtraction method (EXRSM) starts after the
application of the ratio subtraction method (RSM) [9]. The RSM
depends on that, when a mixture of CPX and CAF, where the
spectrum of CAF is more extended (Fig. 2), the determination of CPX
in the mixture could be done by scanning the zero order absorption
spectra of the laboratory-prepared mixtures (CPX and CAF), dividing
them by a carefully chosen concentration of standard CAF` (12 µg ml-1)
as a divisor. This chosen concentration of the divisor gives the best
regression over the proposed concentration range. This will produce
new ratio spectra which represent CPX/CAF’ + constant as shown in
Fig. 3a and 3b. Next, subtraction of the values of these constants
CAF/CAF’ in the plateau region (280-290 nm) is done, as shown in
Fig. 4a and 4b. This is followed by multiplication of the obtained
spectra by the divisor CAF` (12 µgml-1) as shown in Fig. 5a and 5b,
which corresponds to the original spectra of CPX. These obtained
spectra are used for the direct determination of CPX at 222.4 nm and
calculation of the concentration from the corresponding regression
equation (obtained by plotting the absorbance values of the zero
order curves of CPX at 222.4 nm against the corresponding
concentrations).
The determination of CAF could be done by the extended ratio
subtraction by dividing these obtained spectra of CPX by a carefully
chosen concentration of standard CPX` (12µgml-1) producing ratio
spectra that represent the constants CPX/CPX ’in plateau (210-240
nm) as shown in Fig. 6. The previously scanned zero order
absorption spectra of the laboratory-prepared mixtures (CPX and
CAF) were divided by standard CPX` (12 µg ml-1) as a divisor
producing new ratio spectra which represent CAF/CPX’ + constant as
shown in Fig. 7. Then subtraction of these obtained constants
CPX/CPX’ as shown in Fig. 8, which is followed by multiplication of
the obtained spectra by the divisor CPX` (12 µg ml-1) as shown in Fig.
9. Finally, the original spectra of CAF (Fig. 9) could be obtained
which are used for direct determination of CAF at 273.0 nm and
calculation of the concentration from the corresponding regression
equation (obtained by plotting the absorbance values of the zero
order curves of CAF at 273.0 nm against the corresponding
concentrations).
The extended ratio subtraction method has an advantage that the
extended drug in the mixture could be determined at its λmax which could
not be achieved by the previously established ratio subtraction method
[9] which had determined unextended drug only. Therefore, the two
methods are considered to be complementary to each other since the
two components of interest in the mixture could be determined.
Ratio difference spectrophotometric method (RDSM)
The most striking feature of the ratio difference method is its
simplicity, rapidity and accuracy [10]. This is a newly developed
method having the ability for solving severely overlapped spectra
without prior separation; meanwhile it doesn’t require any
sophisticated apparatus or expensive computer programs.
The utilization of ratio difference method is to calculate the
unknown concentration of a component of interest present in a
mixture containing both the component and an interfering
component.
The only requirement in the ratio differe nce metho d is the
contr ibution of the two overlapped spectra at the two selected
wavelengths λ 1 and λ2 where the ratio s pectrum of the

Mohsen et al.
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481
interfering co mpone nt shows the same am plitude (constant)
whereas the component of interest shows significant difference
in these two amplitude values at these two selected wave lengths
with concentration. Similarly, another two wavelengths are
selected for the estimation of the second component. Thus, the
overlapped spectra of the cite d drugs suggested t hat a ratio
difference meth od was a suitable method for simultaneous
determination of CPX and CAF.
Fig. 2: Zero order absorption spectra of 20 μg ml-1 (- - - - -), 100 μg ml-1 ( ) of Chlorphenoxamine hydrochloride and 20 μg ml-1 of
Caffeine (……..).
Fig. 3a: Ratio Spectra of some laboratory prepared mixture of CPX (4-24 μg ml-1) and CAF (4 μg ml-1) using 12 μg ml-1 of CAF as a divisor.
Fig. 3b: Ratio Spectra of some laboratory prepared mixtures of CPX and CAF using 12 μg ml-1 of CAF as a divisor.
Fig. 4a: Ratio spectra of some laboratory prepared mixtures of CPX (4-24 μg ml-1) and CAF (4 μg ml-1) using 12 μg ml-1 of CAF as a divisor
after subtraction of the constant.

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Fig. 4b: Ratio spectra of some laboratory prepared mixtures of CPX and CAF using 12 μg ml-1 of CAF as a divisor after subtraction of the
constant.
Fig. 5a: The zero order absorption spectra of CPX (4-24 μg ml-1) (X) obtained by the proposed ratio subtraction method for the analysis of
laboratory prepared mixtures after multiplication by the divisor CAF` (Y`).
Fig. 5b: The zero order absorption spectra of different concentrations of CPX (X) obtained by the proposed ratio subtraction method for
the analysis of laboratory prepared mixtures after multiplication by the divisor CAF` (Y`).
Fig. 6: Ratio spectra of obtained spectra of CPX (X) using 12 μg ml-1 of CPX` (X`) as a divisor.

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Fig. 7: Ratio spectra of laboratory prepared mixtures of CPX (X) and CAF (Y) using 12 μg ml-1 of CPX` (X`) as a divisor.
Fig. 8: Ratio spectra of laboratory prepared mixtures of CPX (X) and CAF (Y) using 12 μg ml-1 of CPX` (X`) as a divisor after subtraction of
the constant.
Fig. 9: The zero order absorption spectra of CAF (Y) obtained after multiplication by the divisor using the proposed extended ratio
subtraction method for the analysis of laboratory prepared mixtures after multiplication by the divisor CPX` (X`).
Fig. 10: Ratio Spectra of some laboratory prepared mixture of CPX (4-24 μg ml-1) and CAF 4 μg ml-1 using 12 μg ml-1 of CAF as a divisor.

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Fig. 11: Ratio Spectra of some mixtures of CPX and CAF using 12 μg ml-1 of CAF as a divisor.
Fig. 12: Ratio Spectra of a mixture of CAF (4-24 μg ml-1) and CPX (4 μg ml-1) using (12 μg ml-1) of CPX as a divisor.
Ratio difference method starts by scanning the zero order
absorption spectra of the laboratory-prepared mixtures (CPX and
CAF). For determination of CPX, divide the previously scanned ratio
spectra by a carefully chosen concentration of standard CAF' (12 µg
ml-1) as a divisor to produce new ratio spectra which represent
CPX/CAF' + constant as shown in Fig. 10 and 11. The amplitudes at
226.3 nm and 235 nm were selected. The amplitudes at these two
wavelengths were subtracted, so the constant CAF/CAF' will be
cancelled. The concentration of CPX was calculated using the
corresponding regression equation (obtained by plotting the
difference in the amplitude at 226.3 nm and 235.0 nm of the ratio
spectra of CPX/CAF' against the corresponding concentrations).
Similarly, the two selected wavelengths for the estimation of CAF
using standard CPX` (12 µg ml-1) as a divisor were 209.1 nm and
226.7 nm as shown in Fig. 12.
Mean centering of the ratio spectra method (MCR)
The developed MCR method depends on the mean centering of ratio
spectra, it eliminates the derivative steps and therefore signal-to-
noise ratio is enhanced [11].
In order to optimize the developed MCR method, different
parameters were tested. Since the wavelength range taken has a
great effect on the obtained mean centered ratio spectra, different
wavelength ranges were tested and the best results were obtained
when using the wavelength range from 200 to 300 nm and 200 to
280 nm for CPX and CAF, respectively. Effect of divisor
concentration on the selectivity of the method has been tested.
Different concentrations each of CPX and CAF were tested. It was
found that the divisor concentration had no significant effect on the
specificity of CPX and CAF determination, therefore, normalized
spectrum each of CPX and CAF was used as a divisor.
As shown in Fig. 2, the absorption spectra of CPX and CAF are
overlapped. So, the absorption spectra of the standard solutions of
the CPX with different concentrations were recorded in the
wavelength range of 200-300 nm and divided by the normalized
spectrum of the CAF and the obtained ratio spectra were mean
centered. (Fig. 13) The concentration of CPX was determined by
measuring the amplitude at 226.8 nm corresponding to a maximum
wavelength as shown in the same figure.
Similarly, for determination of CAF, the absorption spectra of the
standard solution of CAF with different concentrations were
recorded in the wavelength range of 200-280 nm and divided by the
normalized spectrum of the CPX and the obtained ratio spectra were
mean centered. (Fig. 14) The concentration of CAF was determined
by measuring the amplitude at 274.0 nm corresponding to a
maximum wavelength as shown in the same figure.
Fig. 13: Mean centered ratio spectra of CPX (4-24 μg ml-1).

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Fig. 14: Mean centered ratio spectra of CAF (2-24 μg ml-1).
Method Validation
Validation of the proposed methods was assessed according to ICH
guidelines [13] as shown in tables 1-3.
Linearity
The linearity of the methods was evaluated by analyzing six
concentrations of CPX and seven concentrations of CAF ranging
among 4-24 µg ml-1 and 2-24 µg ml-1, respectively. Each
concentration was repeated three times. The assay was performed
according to the experimental conditions previously mentioned. The
linear regression equations are summarized in Table 1.
Range
The calibration range was established through consideration of the
practical range necessary according to adherence to Beer’s law and
the concentration of CPX and CAF present in the pharmaceutical
preparations to give accurate, precise and linear results. Assay
parameters are declared in Table 2.
Accuracy
The accuracy of the results was checked by applying the proposed
methods for determination of different blind samples of CPX and CAF.
The concentrations were obtained from the corresponding regression
equations, from which the percentage recoveries suggested good
accuracy of the proposed methods. Results are shown in Table 2.
Selectivity
Selectivity of the methods was achieved by the analysis of different
laboratory prepared mixtures of CPX and CAF within the linearity
range, including the ratio present in the pharmaceutical dosage
form. Satisfactory results were obtained as shown in Table 3.
Precision
Repeatability
Three concentrations of CPX (8, 12 and 20 µg ml-1) and CAF (8, 12
and 20 µg ml-1) were analyzed three times intra-daily using the
proposed methods. The relative standard deviations were calculated
(Table 2).
Reproducibility (Intermediate precision)
The previous procedures were repe ated inter-daily on three
different days for the analysis of the three chosen
conce ntrations. The re lative standard deviations were calculated
(Tabl e 2).
Table 1: Linearity studies and regression equations of the proposed methods
Drug
Method
Regression equation
Correlation coefficient (r)
Chlorphenoxamine
HCl
RSM
Ya = 0.0416x + 0.0027
0.9999
RDSM
Yb = 0.0552x + 0.0049
0.9999
MCR
Yc = 0.1315x + 0.0203
0.9997
Caffeine
EXRSM
Ya = 0.0538x + 0.0132
0.9999
RDSM
Yb = 0.1092x + 0.0862
0.9997
MCR
Yc = 0.5950x + 0.0450
0.9998
Where RSM is the ratio subtraction method, EXRSM is the extended ratio subtraction method, RDSM is the ratio difference spectrophotometric
method and MCR is the mean centering of ratio spectra method.
Ya: Absorbance of the drug at its λmax; Yb: difference between the amplitudes of the ratio spectra at the two selected wavelengths; Yc: the mean
centered values at the specified wavelength and x is the corresponding Concentration
Table 2: Assay Parameters and method validation obtained by applying the proposed methods
Parameter
Chlorphenoxamine HCl
Caffeine
RSM
RDSM
MCR
EXRSM
RDSM
MCR
Range µg ml-1
4-24
4-24
4-24
2-24
2-24
2-24
Slope
0.0416
0.0552
0.1315
0.0538
0.1092
0.5950
Intercept
0.0027
0.0049
0.0203
0.0132
0.0862
0.045
Corr. Coef. (r)
0.9999
0.9999
0.9997
0.9999
0.9997
0.9998
Accuracy
99.91 ± 1.073
99.41± 0.726
99.88 ± 1.245
99.05 ± 0.440
100.07 ± 1.250
99.62 ± 1.527
Repeatabilitya
99.80 ± 0.699
99.58 ± 0.813
99.85 ± 0.450
100.09 ± 0.565
99.71 ± 0.561
100.24± 0.458
RSD% a
0.700
0.816
0.451
0.565
0.562
0.457
Intermediate
Precision b
100.14 ± 0.639
99.23 ± 0.843
99.94 ± 0.340
99.98 ± 0.520
99.94 ± 0.494
100.16 ± 0.361
RSD% b
0.637
0.849
0.34
0.521
0.494
0.36
a: Intra-day (n=3), average of three concentrations of CPX (8, 12& 20 µg ml-1) repeated 3 times within the same day.
b: Inter-day (n=3), average of three concentrations of CPX (8, 12& 20 µg ml-1) repeated 3 times in three consecutive days.

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Table 3: Determination of Chlorphenoxamine HCl and Caffeine in their binary laboratory mixtures by the proposed methods
Ratios
Chlorphenoxamine HCl
(Recovery % ± SD)
Caffeine (Recovery % ± SD)
CPX : CAF
Conc.
µg ml-1
RSM at
RDSM at
MCR at
EXRSM at
RDSM at
MCR at
222.4 nm
226.3 - 235 nm
226.8 nm
273.0 nm
209.1 - 226.7 nm
274.0 nm
2 : 1
8 : 4
100.56 ± 0.762
99.27 ± 0.514
99.17 ± 0.614
99.58 ± 0.415
100.51 ± 0.465
98.93 ± 0.521
5 : 1
20 : 4
99.58 ± 0.527
100.68 ± 0.814
99.87 ± 0.479
99.62 ± 0.361
99.87 ± 0.642
99.62 ± 0.432
2 : 5*
4 : 20
100.19 ± 0.375
100.46 ± 0.671
100.62 ± 0.378
99.87 ± 0.396
99.53 ± 0.613
99.39 ± 0.512
1 : 2
4 : 8
99.28 ± 0.625
99.80 ± 0.527
99.93 ± 0.510
100.24 ± 0.484
100.23 ± 0.341
99.81 ± 0.316
1 : 5
4 : 20
99.81 ± 0.719
100.37 ± 0.351
99.83 ± 0.476
99.68 ± 0.591
100.19 ± 0.354
99.28 ± 0.418
*: The Ratio in Allergex Caffeine tablets.
All calculations were done in triplicates.
Table 4: Determination of Chlorphenoxamine HCl and Caffeine in Allergex Caffeine tablets by the proposed methods and application of
the standard addition technique
Product
Claimed
(µg ml-1)
Standard addition
Recovery (Mean ± SD%)
Allergex Caffeine
(B.N.: 1106021)
Added
( µg ml-1)
Found
( µg ml-1)
Recovery %
Proposed method
Standard addition
RSM & EXRSM
CPX
4
4
3.94
98.50
100.72± 1.218
100.13± 1.410
8
8.07
100.88
12
12.12
101.00
CAF
10
4
4.01
100.25
99.14 ± 0.40
99.53 ± 0.632
8
7.94
99.25
12
11.89
99.08
RDSM
CPX
4
4
3.97
99.25
100.16± 0.54
99.07 ± 0.387
8
7.89
98.63
12
11.92
99.33
CAF
10
4
3.99
99.75
99.72 ± 0.814
99.49 ± 0.357
8
7.97
99.63
12
11.89
99.08
MCR
CPX
4
4
4.06
101.50
99.76 ± 1.051
100.65± 1.161
8
8.09
101.13
12
11.92
99.33
CAF
10
4
3.98
99.50
98.91 ± 1.342
99.99 ± 1.462
8
8.13
101.63
12
11.86
98.83
Table 5: Statistical comparison for the results obtained by the proposed methods and the reference methods for the determination of
Chlorphenoxamine HCl and Caffeine in pure powder form
Parameter
CPX
CAF
Manufacturer
Method*
RSM
RDSM
MCR
BP**
Method
EXRSM
RDSM
MCR
Mean
100.62
99.91
99.41
99.88
99.20
99.05
100.07
99.62
S.D.
0.605
1.073
0.726
1.245
0.783
0.440
1.250
1.527
N
5
5
5
5
5
5
5
5
Variance
0.366
1.151
0.528
1.550
0.613
0.194
1.563
2.332
student t (2.306)
0.24
0.022
0.276
0.732
0.229
0.602
F (6.388)
3.145
1.442
4.237
3.159
2.550
3.804
*: Manufacturer method is a non-aqueous potentiometric titration method, obtained by personal communication with EIPICO [14].
**: BP method is a non-aqueous potentiometric titration method.
Figures in parenthesis are the corresponding tabulated values at P = 0.05.
Stability
CPX and CAF working solutions in double distilled water showed no
spectrophotometric changes up to 2 weeks when stored at room
temperature.
Application of the method in Assay of tablets
The proposed spectrophotometric ratio-spectra methods were
applied for the determination of CPX and CAF in their combined
pharmaceutical formulation (Allergex Caffeine tablets). The validity
of the methods was assessed by applying the standard addition
technique (Table 4). It shows that the developed methods are
accurate and specific for determination of the cited drugs in
presence of dosage form excipients.
Statistical Analysis
Results obtained by the proposed methods for the determination of
pure samples of CPX and CAF are statistically compared to those
obtained by the official methods. The calculated t and F values were
found to be less than their corresponding theoretical ones
confirming good accuracy and excellent precision (Table 5).

Mohsen et al.
Int J Pharm Pharm Sci, Vol 5, Suppl 1, 478-487
487
CONCLUSION
From the previous discussion, it could be concluded that the
proposed procedures are simple and do not require sophisticated
techniques or instruments. They are also sensitive and selective and
could be used for routine analysis of CPX and CAF in their available
dosage form without prior separation. It is noteworthy to mention
that using double distilled water as a solvent, besides being cheap; it
is extremely safe to the environment. The methods are also suitable
and valid for application in laboratories lacking liquid
chromatographic instruments. Moreover, using Microsoft Excel, for
manipulation of the spectral data during handling the three
proposed methods, eliminates the need for using specific expensive
software. Excel, can be downloaded for free or comes pre-installed
on any new laptop or desktop as a part of the Microsoft® Office
Starter: reduced-functionality Word &Excel.
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