Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Novel spectrophotometric methods for simultaneous determination

of timolol and dorzolamide in their binary mixture
Hayam Mahmoud Lotfy ⇑, Maha A. Hegazy, Mamdouh R. Rezk, Yasmin Rostom Omran
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, 11562 Cairo, Egypt

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Two novel methods namely; Zero-order spectra of 40 lg/mL of DOR (—) and TIM (. . .), separately in methanol, and binary of a mixture
absorbance subtraction (AS) and of DOR and TIM, 20 lg/mL of each (- - -) and their ratio spectra using the spectrum of TIM (1 lg/mL) as a
amplitude modulation (AM) methods divisor.
were developed.
 Six recently well established
spectrophotometric methods (SRS,
RD, RS, EXRS, CM and MCR) were
applied.
 The proposed methods are very
simple, accurate, precise.
 They do not require any sophisticated
apparatus or computer programs.

a r t i c l e i n f o a b s t r a c t

Article history: Two smart and novel spectrophotometric methods namely; absorbance subtraction (AS) and amplitude
Received 2 December 2013 modulation (AM) were developed and validated for the determination of a binary mixture of timolol
Received in revised form 30 January 2014 maleate (TIM) and dorzolamide hydrochloride (DOR) in presence of benzalkonium chloride without prior
Accepted 2 February 2014
separation, using unified regression equation. Additionally, simple, specific, accurate and precise spectro-
Available online 15 February 2014
photometric methods manipulating ratio spectra were developed and validated for simultaneous deter-
mination of the binary mixture namely; simultaneous ratio subtraction (SRS), ratio difference (RD), ratio
Keywords:
subtraction (RS) coupled with extended ratio subtraction (EXRS), constant multiplication method (CM)
Absorbance subtraction
Amplitude modulation
and mean centering of ratio spectra (MCR). The proposed spectrophotometric procedures do not require
Dorzolamide hydrochloride any separation steps. Accuracy, precision and linearity ranges of the proposed methods were determined
Ratio spectra and timolol maleate and the specificity was assessed by analyzing synthetic mixtures of both drugs. They were applied to
their pharmaceutical formulation and the results obtained were statistically compared to that of a
reported spectrophotometric method. The statistical comparison showed that there is no significant dif-
ference between the proposed methods and the reported one regarding both accuracy and precision.
Ó 2014 Published by Elsevier B.V.

⇑ Corresponding author. Tel.: +20 1112887066.

E-mail address: hayamlotfyhm@hotmail.com (H.M. Lotfy).

http://dx.doi.org/10.1016/j.saa.2014.02.005
1386-1425/Ó 2014 Published by Elsevier B.V.
198 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207
Introduction
Timolol maleate (TIM), (S)-1-[(1,1-dimethyl)amino]-3-[[4-(4-mor-
pholinyl9-1,2,5-thiadiazol-3-yl]oxy]-2-propanol is a nonspecific-adren-
ergic blocker (Fig. 1a). It was the first-blocker to be used as an
antiglaucoma agent [1]. None of the newer-blockers were found to be
more effective than timolol [13]. Dorzolamide hydrochloride (DOR),
(4S,6S)-4-ethylamino-5,6-dihydro-6-methyl-4H-thieno (2,3-b)thiopy-
ran-2-sulfonamide-7,7-dioxide monohydrochloride (Fig. 1b), is a human
carbonic anhydrase-II inhibitor used in eye drops to treat increased pres-
sure in the eye caused by open-angle glaucoma and to treat a condition
called ocular hypertension [1]. Both drugs have been co-formulated
and widely used to reduce intraocular pressure in open-angle glaucoma
and for treating ocular hypertension.
Only few methods have been reported for the determination of
DOR based on HPLC assay with ultraviolet detection in human ser-
um and urine [2–4] and capillary electrophoresis [5]. Several ana-
lytical procedures have been reported for determination of TIM,
including GC [6], HPLC in plasma [7,8], in pharmaceuticals [9–11]
and HPTLC [12].
To the best of my knowledge, very few spectrophotometric
methods [13,14], capillary electrophoretic method [15] and few
HPLC methods [16,17] were described for the simultaneous deter-
mination of both drugs in eye drops.
The main problem of spectrophotometric multicomponent anal-
ysis is the simultaneous determination of two or more compounds
in the same mixtures without preliminary separation. Several uni-
variate spectrophotometric methods have been used for resolving
mixtures with overlapping spectra such as derivative spectropho-
tometry [18], H-point standard addition method for binary [19]
and ternary [20] mixtures, the ratio derivative spectrophotometry
for binary [21] and derivative ratio spectra-zero crossing for ternary
mixtures [22–27], the double divisor-ratio spectra derivative meth-
od for determination of ternary mixtures [18,26,28]. Multivariate
spectrophotometric methods also were reported as partial least
squares regression [29], principal component regression [28] and
multiple linear regression analysis [30].
The aim of this work is to develop and conduct two novel methods
namely; absorbance subtraction (AS) and amplitude modulation
(AM) methods for resolving those binary mixtures with spectral
interfering problems, either drugs in mixture or drug and its degrada-
Fig. 1a. Chemical structure of timolol maleate.
Fig. 1b. Chemical structure of dorzolamide hydrochloride.
tion product without preliminary separation using unified regression
equation, in addition to a comparative study was done between the
two novel methods (AS and AM) and six recently well established
spectrophotometric methods (SRS, RD, RS, EXRS, CM and MCR) in
terms of specificity and validation. These two novel methods are con-
sidered as new approach of the isosbestic point method either in zero
order absorption spectrum i.e. isoabsorptive point [27,31–37] or in
the ratio spectrum [38] at which the total concentration of both com-
ponents in the mixture was calculated, by using smart mathematical
techniques utilizing the absorption factor [39,40] in zero order
absorption spectra or constants in the ratio spectra which could be
adapted to isosbestic point analysis for separate quantitative estima-
tion of each drug in their mixture using unified regression equation.
The proposed methods are very simple, accurate, precise and do not
require any sophisticated apparatus or computer programs.
Theoretical background
Absorbance subtraction method (AS)
This method is based on the same principles as the absorption
factor method [39,40]. The method could be applied for the analy-
sis of a mixture of two drugs X and Y having overlapped spectra
intersect at isoabsorptive point and Y is extended over X, while X
does not show any contribution at another wavelength (k2). In this
method the isoabsorptive point kiso could be used for separate
quantitative estimation of each X and Y in their mixture (X + Y).
The determination can be done using mathematically calculated
factor of one of these components. By simple manipulation step,
we can get the absorbance value corresponding to X and Y, sepa-
rately. So, the concentration of each component could be obtained
via the isoabsorptive point regression equation without any need
for a complementary method.
The absorbance values corresponding to X and Y at kiso were cal-
culated by using absorbance factor which is a constant for pure Y
representing the average of the ratio between the absorbance val-
ues of different concentrations of pure Y at k1 (Aiso) to those at k2
(A2) i.e {Aiso/A2}
abs1
Absorbance of Y in the mixture at kiso ¼  absk2 ðX þ YÞ
abs2
Absorbance of X in the mixture at kiso
abs1
¼ abskiso ðX þ YÞ   absk2 ðX þ YÞ
abs2
where abskiso, absk2 is the absorbance of Y at kiso and k2; abs1
abs2
is the
absorbance factor and abskiso(X + Y) and absk2(X + Y) are the absor-
bance of the mixture at these wavelengths .
The concentration of each X and Y, separately, is calculated
using the isobsorptive point unified regression equation (obtained
by plotting the absorbance values of the zero order spectra of
either X or Y at isobsorptive point (kiso) against the corresponding
concentrations X or Y respectively).
Amplitude modulation method (AM)
The amplitude modulation method is a novel ratio spectrum
manipulating method using normalized spectrum of the divisor
obtained by dividing certain spectrum of Y0 component by its con-
centration. For a mixture of X and Y, where Y is more extended than
X; X and Y shows isoabsorptive point at the zero spectrum and con-
sequently retained as an isosbestic point at the ratio spectrum.
At isoabsorptive point kiso
½Am  ¼ ½AX  þ ½AY 
 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207
Am, AX, AY are the absorbance values of the mixture, component X
and component Y, respectively at isoabsorptive point.
½Am  ¼ ½aX C X  þ ½aY C Y 
Dividing the previous equation with spectrum of Y0 as a divisor
to get the isosbestic point of the ratio spectrum (at the same wave-
length of the isoabsorptive point in zero order spectra), so the fol-
lowing equation was obtained:
½Am =½aY C Y 0  ¼ ½aX C X =½aY C Y 0  þ ½aY C Y =½aY C Y 0 
Pm ¼ PX þ PY
where Pm, PX and PY are the amplitudes of the mixture, component X
and component Y, respectively at that isosbestic point.
The amplitude of constant (PY) [aYCY]/[aYCY0 ] can be measured
directly from the spectrum in the region where Y is extended since
the constant is a straight line parallel to the wavelength axis.
Upon using the normalized spectrum of Y0 , the following equa-
tion was obtained:
Pm ¼ PX þ PY
Pm ¼ ½aX C X =½aY  þ ½aY C Y =½aY 
Pm ¼ ½aXC X =½aY  þ constant
PY ¼ ½aY C Y =½aY 
PY ¼ ½C Y 
The recorded amplitude of the constant is modulated to concen-
tration, so it represents the concentration of Y [CY]. (CRecorded of Y).
For determination of X, the measured value of the constant is
subtracted from the ratio spectrum then the amplitude at kiso
was recorded:
PX ¼ Pm  PY
PX ¼ ½aX C X =½aY  þ constant  constant
PX ¼ ½aX C X =½aY 
At that isosbestic point aX = aY
PX ¼ ½C X 
This obtained amplitude of ratio spectrum is modulated to con-
centration and it represents concentration of X [CX]. (CRecorded of X).
To eliminate any error, this recorded concentration of X and Y
could be corrected to the actual concentration by using the follow-
ing unified regression equation at that isosbestic point:
C Recorded ¼ slope C  intercept
The slope is found to be around unity and intercept around zero.
Where CRecorded represents the recorded amplitude correspond-
ing to the concentrations of X or Y that obtained from the ratio
spectrum using normalized spectrum of Y0 as a divisor and C repre-
sents the corresponding concentration of X or Y.
Simultaneous ratio subtraction, simultaneous ratio subtraction
coupled with constant multiplication and ratio subtraction coupled
with extended ratio subtraction methods
These methods are applied for the analysis of a mixture of two
drugs X and Y having overlapped spectra and one of them is ex-
tended, one can determine X by dividing the spectrum of the mix-
ture by a known concentration of Y as a divisor (Y0 ). The division
will give a new curve that represents YX0 þ constant. If we measure
this constant which is parallel to the wavelength axis in the region
199
where (Y) is extended, then a new curve is obtained after subtrac-
tion of the constant. This can be summarized as the following:
XþY X Y X
¼ 0 þ 0 ¼ 0 þ constant ð1Þ
Y0 Y Y Y
X X
þ constant  constant ¼ 0 ð2Þ
Y0 Y
Simultaneous ratio subtraction (SRS)
The concentration of X is calculated using the regression equa-
tion representing correlation between the amplitudes of ratio spec-
tra YX0 and the corresponding concentration of X in lg/mL.
The concentration of the second component (Y) is calculated via
measured constant value (CV) using the regression equation repre-
senting correlation between the amplitudes of ratio spectra YY0 and
the corresponding concentration of Y in lg/mL [39].
Simultaneous ratio subtraction coupled with constant multiplication
(SRS–CM)
The concentration of X is determined as previously detailed in
SRS, while the concentration of Y can be determined using constant
multiplication method (CM) by multiplying the previously
measured constant value YY0 in SRS [39] by the divisor Y0 [41,42]
to get zero order spectrum of Y then using the regression equation
representing correlation between the absorbance values of the zero
order spectra of Y at its kmax against the corresponding
concentrations.
Y
Y¼  Y0; ð3Þ
Y0
Ratio subtraction (RS) coupled with extended ratio subtraction (EXRS)
The mixture of X and Y could be analyzed using well established
ratio subtraction method [43] where zero order spectrum of com-
ponent X could be obtained by multiplying the obtained ratio spec-
trum in Eq. (2) by the divisor Y0
X
 Y0 ¼ X ð4Þ
Y0
Another extension of the already developed method has been
established as a new approach namely extended ratio subtraction
[39,42] to get the zero order spectrum of second component (Y),
by dividing the obtained D0 spectrum of X in Eq. (3) by a known
concentration of X as a divisor (X0 ) to get the constant XX0 for each
concentration in the mixtures then follow the same procedure of
the ratio subtraction by dividing each mixture using a known con-
centration of X as a divisor then subtracting the corresponding con-
stant XX0 and multiplying by (X0 )
Y
 X0 ¼ Y ð5Þ
X0
The concentrations of X or Y in the mixture were calculated
from the corresponding regression equations (obtained by plotting
the absorbance values of the zero order spectra of each drug at its
kmax against its corresponding concentration).
Ratio difference spectrophotometric method (RD)
Ratio difference spectrophotometric method [39,44] was re-
cently developed for analyzing a mixture of two drugs X and Y hav-
ing overlapped spectra depend on the amplitude difference
between two wavelengths k1 and k2 in the ratio spectra where
the interfering substance should be contributed at these chosen
wavelengths and subtracting the recorded amplitudes at these
200 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207
two points, the constant will be cancelled along with any other
instrumental error. This can be summarized as the following:
   
X X
P1  P2 ¼ 1  2
Y0 Y0
where P1 is the peak amplitude of the ratio spectrum at k1, P2 is the
peak amplitude of the ratio spectrum at k2.
The concentration of X is calculated using the regression equa-
tion representing the linear correlation between the differences of
ratio spectra amplitudes at the two selected wavelengths using Y
as a divisor (Y0 ) to the corresponding concentrations of drug (X).
Similarly, the concentration of Y could be determined by the
same procedure using a known concentration of X as a divisor (X0 ).
Mean centering of ratio spectra spectrophotometric method (MCR)
This is a well established spectrophotometric method in which
binary mixtures of X and Y 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 spec-
tra [44–47].
Experimental
Chemicals and reagents
Timolol maleate (TIM) was kindly supplied by EIPICO, Cairo,
Egypt. BN: TMM-021341. Its purity was found to be 99.96
± 0.51% according to the reported method [14].
Dorzolamide hydrochloride (DOR) was kindly supplied by
Merck Sharp & Dohm Company, USA. Batch number HC-B12-02-
001148102291. Its purity was found to be 100.25 ± 0.46% accord-
ing to the reported method [14].
CosoptÒ eye drops bottles were manufactured by Global Napi
Pharmaceuticals-Egypt. BN:21182/2001, each 1 mL is claimed to
contain 20 mg dorzolamide and 5 mg timolol.
Methanol was E. Merck, Darmstadt, Germany.
Instruments and software
Spectrophotometric measurements were carried out on Shima-
dzu 1650 UV-PC spectrophotometer, using 1.00 cm quartz cells.
Scans were carried out in the range from 200 to 400 nm at
0.1 nm intervals.
For MCR computations, MatlabÒ 7 was used along with PLS_
toolbox.
Spectral characteristics
The absorption spectra of 40 lg/mL TIM and DOR, separately,
and that of a laboratory mixture containing equal concentrations
of both drugs (each, 20 lg/mL) in methanol were scanned over
the range 200–400 nm as shown in (Fig. 3)
Solutions and calibrations
Stock solutions of TIM and DOR (each, 1 mg/mL), calculated
equivalent to their base, were prepared by dissolving the com-
pounds in methanol then completing in 100-mL volumetric flasks.
Aliquots of the prepared stock solutions were further diluted with
methanol to a final volume of 100-mL. The diluted solutions were
used as the working solutions for TIM and DOR (each, 100 lg/mL).
Standard solutions containing 5–60 lg/mL of TIM and 5–40 lg/mL
of DOR were prepared separately in methanol. The absorption
spectra of the resulting solution were measured in the range of
200–400 nm and stored in the computer.
Absorbance Subtraction method (AS)
The scanned spectra of 5–40 lg/mL TIM were measured at
272.8 nm and 315 nm. The absorbance factor was calculated which
is the ratio of the absorbance at these two wavelengths. Calibration
curve was constructed relating the absorbance of the zero order
spectra of TIM at 272.8 nm versus the corresponding concentra-
tions of TIM and the regression equation was computed.
Amplitude modulation method (AM)
The stored spectra of 5–40 lg/mL TIM were divided by the nor-
malized spectrum of TIM. The obtained ratio spectra of TIM were
measured at 272.8 nm and 315 nm. Calibration curve was con-
structed relating the recorded concentrations of TIM at 272.8 nm
versus the corresponding concentrations of TIM and the regression
equation was computed.
Simultaneous ratio subtraction methods (SRS)
The stored spectra were divided by the absorption spectrum of
standard solution of TIM (30 lg/mL), where the obtained ratio
spectra were recorded. Calibration curves relating the amplitudes
of the ratio spectra of TIM representing constant value at plateau
region 315–325 nm versus the corresponding concentrations of
TIM, the absorbance of zero order absorption spectra at 297.5 nm
versus the corresponding concentrations of TIM and the ampli-
tudes of the ratio spectra at 250 nm versus the corresponding con-
centrations of DOR were constructed and the regression equations
were computed.
Ratio difference spectrophotometric method (RD)
The scanned spectra of TIM and DOR solutions were divided by
the absorption spectra of standard solution of DOR (40 lg/mL) and
TIM (30 lg/mL), respectively. The obtained ratio spectra were re-
corded. Calibration curves for TIM and DOR were constructed by
plotting the difference between the amplitudes of the obtained ra-
tio spectra at 250–290 nm and 250–280 nm versus the corre-
sponding concentrations of TIM and DOR, respectively; and the
regression equations were computed.
Ratio subtraction methods coupled with extended ratio subtraction
methods (RS–EXRS)
The stored spectra of the resulting solutions were divided by
the absorption spectrum of standard solution of TIM0 (30 lg/mL)
and standard solution of DOR0 (40 lg/mL). The obtained ratio spec-
tra were recorded. Calibration curve relating the absorbance of the
zero order spectra of TIM at 297.5 nm and DOR at 252.2 nm versus
their corresponding concentrations were constructed and the
regression equation was computed.
Mean centering of ratio spectra method (MCR)
The scanned spectra were exported to Matlab for subsequent
calculation, then the spectra of DOR were divided by the absorp-
tion spectrum of standard solution of TIM (30 lg/mL), the obtained
ratio spectrum was then mean centered. Also the spectra of TIM
were divided by the absorption spectrum of standard solution of
DOR (40 lg/mL) and the obtained ratio spectra were mean cen-
tered. The calibration curves for TIM and DOR were constructed,
each by plotting the mean centered values at 250 nm and
249.9 nm, respectively versus their corresponding concentrations
and the regression equations were computed.
 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207
Analysis of laboratory prepared mixtures
For preparation of laboratory mixtures, into a series of 10-mL
volumetric flasks, aliquots equivalent to 50–600 lg of TIM and
50–400 lg of DOR were accurately transferred from their working
solutions (each, 100 lg/mL) with different ratios of the two drugs
and the volume was completed with methanol. The spectra of
the prepared mixtures were scanned from 200 to 400 nm and
stored in the computer.
Absorbance subtraction method (AS)
The absorbance of TIM in the mixture was calculated using the
absorbance factor equation, and then the absorbance of DOR was
calculated after subtraction of TIM interference. The concentra-
tions of TIM and DOR were calculated from the computed unified
regression equation at 272.8 nm.
Amplitude modulation method (AM)
The absorption spectra of different laboratory prepared mix-
tures were divided by the normalized spectrum of TIM. The ratio
spectra were recorded at 272.8 nm and 315 nm. The concentra-
tions of both drugs were calculated from the computed unified
regression equation at 272.8 nm.
Simultaneous Ratio Subtraction Method (SRS), Ratio Subtraction
coupled with extended ratio subtraction (RS–EXRS)
The stored zero order absorption spectra of the laboratory pre-
pared mixtures were divided by the absorption spectrum of stan-
dard TIM0 (30 lg/mL), then the amplitudes in the plateau region
at k 315–325 nm (the constant) was recorded and subtracted from
the obtained ratio spectra respectively. The amplitude of the ob-
tained spectra were recorded at 250 nm and used for the determi-
nation of DOR using its corresponding regression equation of SRS
or via ratio subtraction method by multiplying the obtained ratio
spectra by TIM0 (30 lg/mL) to get the zero spectra of DOR and
the concentration of DOR is calculated using the corresponding
regression equation at its kmax and consequently, the constants of
DOR in different mixtures could be obtained using standard solu-
tion of DOR0 (40 lg/mL) as a divisor. The concentration of TIM in
each mixture is calculated using the corresponding regression
equation of the constant value at k 315–325 nm. Zero order
absorption spectra of TIM could be obtained via constant multipli-
cation by multiplying the obtained constant value by the divisor
TIM0 (30 lg/mL) Or via EXRS by the same procedures as RS using
DOR0 (40 lg/mL) as a divisor. The concentration of TIM in each
mixture is calculated using the corresponding regression equation
at its kmax.
Ratio difference spectrophotometric method (RD)
The stored spectra of different laboratory prepared mixtures
were divided by the absorption spectra of standard TIM (30 lg/
mL) and standard DOR (40 lg/mL). The ratio spectra were recorded
at 250 nm and 290 nm for TIM and at 250 nm and 280 nm for DOR.
The concentrations of the drugs were calculated from the com-
puted regression equations.
Mean Centring Method (MCR)
The spectra of the prepared solutions of the two series were re-
corded from 200 to 400 nm and stored in the computer then pro-
ceed as under the proposed method. The concentration of each
drug was calculated using the specified regression equation.
201
Application to pharmaceutical formulation
CosoptÒ eye drops bottle each 1 mL is claimed to contain
22.26 mg dorzolamide hydrochloride equivalent to 20 mg dorzola-
mide and 6.83 mg timolol maleate equivalent to 5 mg timolol.
For AS and AM methods, from eye drops bottle 1 mL was trans-
ferred into 50-mL volumetric flask then the volume was completed
with methanol to obtain a final concentration of 100 lg/mL of TIM
and 400 lg/mL of DOR. Aliquots equivalent to 50 lg of TIM and
200 lg of DOR were transferred into 10-mL volumetric flasks.
The volume was completed with methanol to obtain final concen-
tration 5 lg/mL of TIM and 20 lg/mL of DOR.
For SRS, RD, RS, EXRS, CM and MCR methods, Aliquots equiva-
lent to 100 lg of TIM and 400 lg of DOR were transferred into
10-mL volumetric flasks. The volume was completed with metha-
nol to obtain final concentration 10 lg/mL of TIM and 40 lg/mL of
DOR.
The proposed methods were applied for the analysis of the
studied drugs in their pharmaceutical formulation using the proce-
dures mentioned under analysis of laboratory prepared mixtures
for each method and the concentrations of the cited drugs were
calculated from the corresponding regression equations.
Results and discussion
The main task of this work is to establish novel, simple, sensi-
tive and accurate analytical methods for simultaneous determina-
tion of TIM and DOR in their bulk powders and pharmaceutical
dosage form with satisfactory precision and accuracy. As well, to
construct a statistical comparison between the ability of the pro-
posed methods to determine both drugs in their pure form, labora-
tory prepared mixture and in their pharmaceutical formulations.
By scanning the absorption spectra of TIM and DOR in metha-
nol, severely overlapped spectral bands were observed in the
wavelength region of 200–300 nm which hinders their direct
determination (Figs. 2a and 2b), so different methods were applied
Fig. 2a. Zero-order spectra of 20 lg/mL of DOR (—) and 5 lg/mL of TIM (. . .),
separately in methanol, in presence of 5 lg/mL of benzalkonium chloride (h  h) as
preservative.
202 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207
Fig. 2b. Zero-order spectra of 40 lg/mL of DOR (—) and TIM (. . .), separately in
methanol, and binary of a mixture of DOR and TIM, 20 lg/mL of each (- - -) in
presence of 5 lg/mL of benzalkonium chloride (h  h) preservative.
for achieving best resolution and quantitative determination of
each drug without any interference from the other.
All spectral measurements were done without interference of
benzalkonium chloride (preservative present in CosoptÒ eye drops)
which did not show any absorption and its contribution to the
absorption of the mixture above 220 nm was considered to be neg-
ligible at a concentration up to 100 lg/mL, so the ternary mixture
in range (220–400 nm) acts as a binary mixture of TIM and DOR
(Fig. 2a).
Absorbance subtraction method (AS)
TIM and DOR present in their dosage form in ratio (1:4); in
which their absorption spectra in methanol are severely
overlapped in the wavelength region of 200–300 nm (Fig. 2a) and
intersect at isoabsorptive point 272.8 nm. This could be confirmed
experimentally by recording the absorbance spectra of 40 lg/mL of
TIM and DOR, separately, and that of a mixture containing equal
concentrations of TIM and DOR (each, 20 lg/mL) as shown in
(Fig. 2b). In this figure, one can observe that, the mixture and the
pure drugs show an isoabsorptive point at 272.8 nm. These data
permit one to conclude that the mixture of drugs act as a single
component and give the same absorbance value as pure drug at
their isoabsorptive point.
The absorption spectra of the standard solutions of TIM with
different concentrations were recorded in the wavelength range
of 200–400 nm. The value of absorbance factor of pure TIM repre-
senting the average of the ratio between absorbance at two wave-
lengths, one of them showing interference from DOR (kiso) while
DOR has no contribution at the other wavelength (k2) was found
to be 0.833. Quantitative estimation of DOR in mixture (DOR + -
TIM) was carried out by subtracting the absorbance due to TIM
at kiso using the following equations:
abs1
Absorption of TIM at kiso 272:8 nm ¼ abs315 
abs2
Fig. 3. Ratio spectra of 40 lg/mL of DOR (—) and TIM (. . .), separately in methanol,
and binary of a mixture of DOR and TIM, 20 lg/mL of each (- - -) using the
normalized spectrum of TIM as a divisor.
Absorption of DOR at kiso 272:8 nm
abs1
¼ abs272:8 ðTIM þ DORÞ   abs315
abs2
where abs272.8 and abs315 are absorption of TIM in the mixture
at 272.8 nm (isoabsorptive point) nm and 315 nm (no interference)
and abs1 is the absorbance factor is absorption ratio of pure TIM at
abs2
272.8 nm/315 nm. The concentrations of TIM and DOR were calcu-
lated using the corresponding unified regression equation (ob-
tained by plotting the absorbance of the zero order spectra of
TIM or DOR at kiso 272.8 nm against the corresponding
concentrations).
The absorbance subtraction method has advantage that the two
drugs in the mixture can be determined using unified regression
equation at kiso in contrary to the previously established
isoabsorptive point method [27,31–38] which determines total
concentration of two drugs while one of the drugs is measured
by using conventional spectrophotometric method as a comple-
mentary method.
Amplitude modulation method (AM)
As shown in (Fig. 2b), the absorption spectra of timolol (TIM)
and dorzolamide (DOR) in methanol shows isoabsorptive point at
272.8 nm (aTIM = aDOR) which is the same place in the ratio spec-
trum of TIM using the normalized spectrum of TIM as a divisor ob-
tained by dividing the whole spectrum of any concentration by its
corresponding concentration to give a new spectrum represents
the absorptivity of the analyte of interest versus all the measured
wavelengths (Fig. 3). The amplitude modulation method was ap-
plied to solve the mixture of TIM and DOR of overlapping spectra
by dividing the spectrum of the mixture by 1 lg/mL TIM as a divi-
sor. The division will give a new curve that represents
DOR
TIM0
þ constant (Fig. 4). The constant value will be measured in pla-
teau (315–325 nm), after subtraction of the measured value the
constant; the obtained ratio spectrum TIM DOR
0 is used for calculation
the concentration of DOR in the mixture.
 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207
Fig. 4. Ratio spectra of Timolol (TIM) 5 lg/mL and Dorzolamide (DOR) 20 lg/mL
(. . .) and their laboratory prepared mixture with the same ratio (1:4) (- - -) using the
spectrum of TIM (—) as a divisor.
The concentration of TIM and DOR in the mixture could be ob-
tained from the corresponding regression equation (obtained by
plotting the absorbance values of the isosbestic point of ratio spec-
tra of TIM or DOR at 272.8 nm against the corresponding
concentrations).
The advantages of amplitude modulation method over other
mathematical techniques utilizing the constants that it reduces
the manipulation steps and only one divisor is needed in order to
determine each component in the mixture separately. By using
the normalized spectrum of the divisor which represent the
absorption spectrum, therefore the results will not affected by
the use of different concentrations of divisor and it has advantage
over the isoabsorptive point at zero order that measure the total
concentration therefore there is always a need of other conven-
tional method as complementary one to measure one of the com-
ponents in the mixture (Fig. 2b).
This method has advantages over the newly developed absor-
bance subtraction using unified regression equation at isoabsorp-
tive point that the obtained values at the ratio spectrum will
represent the concentration of each component and it will cancel
all error upon the determination of absorbance factor (Aiso/A2) if
A2 which showing no contribution of interfering component has
lower absorbance value. As well as, the manipulation steps are re-
duced by eliminating of the absorbance factor calculation step
since the value of the constant is the same at the two selected
wavelengths.
Simultaneous ratio subtraction method (SRS)
The ratio subtraction method was applied to solve the mixture of
TIM and DOR of overlapping spectra by dividing the spectrum of the
mixture by a known concentration of TIM (30 lg/mL) as a divisor.
DOR
The division will give a new curve that represents TIM 0 þ constant
(Fig. 5). The constant will be measured at plateau (315–325 nm).
DOR
If we subtract this constant, the obtained ratio spectrum TIM 0 is used
for calculation the concentration of DOR in the mixture from the
corresponding regression equation (obtained by plotting the ampli-
203
Fig. 5. Division spectra of laboratory prepared mixture of Dorzolamide (DOR) 15,
20, 30, 40 lg/mL and Timolol (TIM) 10 lg/mL using spectrum of TIM (30 lg/mL) as
a divisor.
tudes values of the ratio spectra of DOR at 250 nm against the cor-
responding concentrations).
The concentration of TIM in the mixture could be obtained
either via constant value step by recording the constant value
and substituting in equation representing the constant values
against the corresponding concentration of TIM or via constant
multiplication step by multiplying the obtained constant of the
laboratory mixture by TIM (the divisor), to get the original zero or-
der spectra of TIM (Y) in the mixture, which is used for direct deter-
mination of TIM at 297.5 nm (Fig. 6) and calculation of the
concentration from the corresponding regression equation (ob-
tained by plotting the absorbance values of the zero order curves
of TIM at 297.5 nm against the corresponding concentrations).
The advantages of simultaneous ratio subtraction method [39]
that only one divisor was needed and minimum manipulation
steps in order to determine both components in the mixture
therefore, it is very simple, rapid, accurate and the two drugs in
the mixture could be determined in contrary to the previously
established ratio subtraction method [43] which determines one
drug only.
Ratio difference spectrophotometric method (RD)
In the RD spectrophotometry, the absorption spectrum of the
mixture is obtained and divided by the absorption spectrum of
the standard solution of one of the components and the ratio spec-
TIM
trum is obtained represents DOR þ constant or DORTIM
þ constant. Ratio
difference spectrophotometric method (RD) was applied to solve
the problem of the overlapped absorption spectra of the cited
TIM

TIM




drugs DOR 1  DOR 2 or DOR
TIM
1  DOR
TIM
2, where the interfering sub-
stance was cancelled and subsequently shows no interference.
The overlapped spectra of the two drugs suggested that a RD
method was a suitable method for simultaneous determination
of TIM and DOR, respectively.
The two main steps affecting the ratio difference method is the
choice of the divisors and the two selected wavelengths. The se-
lected divisors should compromise between minimal noise and
204 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207

Fig. 8. Ratio spectra of TIM 20 lg/mL (—), DOR 40 lg/mL (. . .) and their binary
Fig. 6. The obtained zero order absorption spectra of TIM after applying the mixture 20 lg/mL TIM and 40 lg/mL DOR (- - -) using 30 lg/mL of TIM as a divisor
proposed method to the laboratory mixture of Dorzolamide (DOR) 15, 20, 30, 40 lg/ showing the two selected wavelengths (250 and 280 nm).
mL and Timolol (TIM) 10 lg/mL.

Fig. 9. The obtained zero order absorption spectra of Dorzolamide (DOR) after
applying the proposed method to the laboratory mixture of Dorzolamide (DOR) 20,
Fig. 7. Ratio spectra of TIM 20 lg/mL, 40 lg/mL of DOR (. . .) and their binary
30, 40 lg/mL and Timolol (TIM) 10 lg/mL.
mixture 20 lg/mL TIM and 40 lg/mL DOR (- - -) using spectrum of DOR (40 lg/mL)
as a divisor showing the two selected wavelengths (250 and 290 nm).

Ratio subtraction (RS) coupled with extended ratio subtraction method

maximum sensitivity while, the requirements of the two chosen
wavelengths are that the contribution of the interfering substance
at them so, the amplitude at 250 and 290 nm were selected for
determination of TIM in the mixture using DOR (40 lg/mL) as a
divisor (Fig. 7). Similarly, the two selected wavelengths for the esti-
mation of DOR using TIM (30 lg/mL) as a divisor were 250 and
280 nm (Fig. 8).
(EXRS)
The ratio subtraction method was applied to solve the mixture
of TIM and DOR of overlapping spectra by dividing the spectrum of
the mixture by a known concentration of TIM (30 lg/mL) as a divi-
sor. The division will give a new curve that represents
DOR
TIM0
þ constant in plateau (315–325 nm) (Fig. 5). If we subtract this
constant, then multiply the new curve obtained by TIM0 (the divi-
 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207 205

80

60

40

MCN values
20

-20

-40
200 210 220 230 240 250 260 270 280 290 300
Wavelength (nm)

Fig. 11. Mean centered ratio spectra of TIM (5–60 lg/mL) using 40 lg/mL of DOR as
a divisor.

Fig. 10. Division spectra of laboratory prepared mixture of Dorzolamide (DOR) 20, 10
30, 40 lg/mL and Timolol (TIM) 10 lg/mL using spectrum of DOR (40 lg/mL) as a
divisor.
8

sor), therefore we can obtain the original zero order spectrum of

DOR in the mixture (Fig. 9). 6
The determination of TIM could be done by the extended ratio
MCN values

subtraction by dividing this obtained spectrum of DOR by carefully

4
chosen concentration of standard DOR (40 lg/mL) producing ratio
spectrum represent the constant DOR/DOR0 in plateau (250–
255 nm). The previously scanned zero order absorption spectrum 2
of the laboratory-prepared mixture (TIM and DOR), dividing by
the standard DOR0 as a divisor producing a new ratio spectrum that
represent TIM/DOR0 + constant as shown in (Fig. 10), then subtrac- 0

tion of the obtained constant DOR/DOR0 , followed by multiplication

of the obtained spectrum by the standard DOR0 (the divisor). Final- -2
ly, the original spectrum of TIM could be obtained which is used for 200 210 220 230 240 250 260 270 280 290 300
direct determination of TIM at 297.5 nm (Fig. 6) and calculation of Wavelength (nm)
the concentration TIM in the mixture from the corresponding
Fig. 12. Mean centered ratio spectra of DOR (5–40 lg/mL) using 30 lg/mL of TIM as
regression equation was done (obtained by plotting the absorbance
a divisor.
values of the zero order curves of TIM at 297.5 nm against the cor-
responding concentrations). the amplitude at 250 nm and 249.9 nm corresponding to a maxi-
The extended ratio subtraction method [39,42] has advantage mum wavelength for each drug respectively (Figs. 11 and 12).
that the two drugs in the mixture can be determined at their kmax For determination of the concentration of TIM and DOR in labora-
in contrary to the previously established ratio subtraction method tory-prepared mixtures and dosage form samples of a pharmaceu-
[43] which determines one drug only. This method is valid for the tical preparation, the same procedure was used.
analysis of binary and ternary mixtures with extended spectra. For all the proposed methods, the statistical parameters of the
regression equations and the concentration ranges are shown in
(Table 1), the table shows that the proposed methods were applied
Mean Centring Method (MCR)
for the determination of pure drugs and satisfactory results were
obtained. The proposed method was successfully applied to the
For optimization of MCR method, the effect of divisor
analysis of TIM and DOR in their laboratory prepared mixtures
concentration on analytical parameters, such as slope, intercept,
and in eye drops dosage form (Table 2).
and correlation coefficient of the calibration graphs was tested.
Mean-centering of the ratio spectra was obtained in the wave-
length range of 200–300 nm. It was observed that changing the Method validation
concentration had no significant effect on the linear calibration
range and calculated analytical parameters. So, the absorption Validation was done according to ICH recommendations [48].
spectra of the standard solutions of the TIM and DOR with different
concentrations were recorded in the wavelength range of 200– Linearity
300 nm and divided by the spectrum of 40 lg/mL of standard
DOR and 30 lg/mL of TIM, respectively to obtain the ratio spectra. The linearity of the methods was evaluated by analyzing twelve
The concentration of TIM and DOR was determined by measuring concentrations of TIM and eight concentrations of DOR ranging
206 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207

Table 1
Regression parameters and results of determination of pure samples of TIM and DOR by the proposed methods.

Parameter TIM TIM or DOR DOR

SRS RD EXRS & CM MCR AS AM SRS RD RSM MCR
Linearity (lg/ 5–60 5–60 5–60 5–60 5–40 5–40 5–40 5–40 5–40 5–40
ml)
Slope 0.0156 0.7179 0.029 0.0843 0.013 1.0039 0.2401 0.2345 0.0398 0.2043
Intercept 0.0332 0.36388 0.0158 0.4679 0.0025 0.0845 0.1266 0.0811 0.0184 0.129
Correlation 0.9997 0.9998 0.9999 0.9998 0.9999 0.9999 1 1 1 1
Coefficient (r)
Mean ± SD 99.96 ± 0.919 99.78 ± 0.987 99.75 ± 1.116 100.08 ± 1.286 100.09 ± 0.816 99.98 ± 0.810 100.04 ± 0.535 100.04 ± 0.472 100.15 ± 0.754 100.11 ± 0.736
Accuracy ± SD 100.60 ± 0.252 99.87 ± 0.757 99.68 ± 0.693 100.12 ± 0.693 99.98 ± 0.827 100.18 ± 0.407 100.39 ± 0.368 99.81 ± 0.489 100.40 ± 0.717 99.96 ± 0.616

RSD%a 0.693 0.221 0.383 0.427 0.120 0.405 0.299 0.138 0.077 0.527

RSD%b 0.940 0.472 0.693 0.708 0.315 0.713 0.404 0.271 0.304 0.687

RSD%a &
 
RSD%b: the intra-day and inter-day respectively (n = 3) relative standard deviation of concentrations (5, 10, 20 lg/mL).

Table 2
Determination of the studied drugs in the laboratory prepared mixtures and in eye drops by the proposed methods.

Sample TIM (R%a)

Method AS AM SRS CM RD EXRS MCR
Laboratory-prepared mixtures (n = 5)b 99.645 ± 0.668 100.14 ± 0.604 100.36 ± 0.909 99.87 ± 0.852 100.13 ± 0.596 99.36 ± 0.444 99.90 ± 0.795
CosoptÒ Batch No. 21182/2001 100.32 ± 0.620 100.90 ± 0.197 99.69 ± 0.937 100.26 ± 0.768 100.06 ± 0.462 99.98 ± 0.290 100.16 ± 0.679
DOR (R%a)
AS AM SRS RD RS MCR
Laboratory-prepared mixtures (n = 5)b 100.70 ± 0.746 100.203 ± 0.573 100.28 ± 0.392 100.36 ± 0.338 100.11 ± 0.935 100.25 ± 0.816
CosoptÒ Batch No. 21182/2001 100.48 ± 0.271 100.86 ± 0.042 100.41 ± 0.925 100.04 ± 0.289 99.99 ± 0.567 100.45 ± 0.829
a
Recovery + RSD%.
b
5 sets each of 3 replicates.

from 5–60 lg/mL and 5–40 lg/mL, respectively. Each concentra- tions were found to be below 2.0% and the methods proved to be
tion was repeated three times. The assay was performed according robust.
to the experimental conditions previously mentioned. The linear
equations were summarized in (Table 1). Precision

Accuracy Repeatability and intermediate precision

The accuracy of the results was checked by applying the pro-
posed methods for determination of different blind samples of
TIM and DOR. The concentrations were obtained from the corre-
sponding regression equations. From which the percentage recov-
eries suggested good accuracy of the proposed methods (Table 1).
They were determined using three concentrations of each of
TIM and DOR (5, 10, 20 lg/mL) which were analyzed three times
intra-daily and inter-daily on three different days using the pro-
posed methods. The relative standard deviations were calculated
(Table 1).
Stability

Range TIM and DOR working solutions in methanol showed no spec-

The calibration range was established through considerations of
the practical range necessary according to adherence to Beer’s law
and the concentration of TIM and DOR present in the pharmaceu-
tical preparations to give accurate precise and linear results (Table
1).
Selectivity
Selectivity of the methods was achieved by the analysis of dif-
ferent laboratory prepared mixtures of TIM and DOR within the
linearity range. Satisfactory results were shown in (Table 2).
Robustness
Three concentrations of each of TIM and DOR (5, 10, 20 lg/mL)
were prepared using 80%, 75%, 70% methanol and analyzed three
times using the proposed methods. The relative standard devia-
trophotometric changes up to 4 weeks when stored at 4 °C.
Application of the methods in assay of eye drops
The proposed UV methods were applied for the determination
of TIM and DOR in their combined pharmaceutical formulation
CosoptÒ eye drops and the results are shown in (Table 2) and com-
pared with that of the reported method [14]. The good percentage
recoveries confirm the suitability of the proposed methods for the
routine determination of these components in their combined
formulation.
Statistical analysis
Tables 3 and 4 showed statistical comparison of the results ob-
tained by the proposed methods and reported spectrophotometric
method [14]. The calculated t and F values were less than the the-
oretical ones indicating that there was no significant difference be-
 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207 207

Table 3
Statistical analysis of the proposed methods and the reported method of TIM and DOR in their pure powdered form.

Parameter Timolol (TIM)

AS AM SRS RD EXRS & CM MCR Reported method** [14]
Mean 100.09 99.98 99.96 99.79 99.75 99.89 99.97
SD 0.816 0.810 0.919 0.987 1.116 0.908 0.666
n 8 8 12 12 12 12 4
Variance 0.666 0.656 0.845 0.974 1.245 0.824 0.444
t-Test* 0.034 (2.228)* 0.004 (2.228)* 0.003 (2.145)* 0.049 (2.145)* 0.054 (2.145)* 0.027 (2.145)*
F* 1.500 (8.89)* 1.477 (8.89)* 1.903 (8.79)* 2.194 (8.79)* 2.804 (8.79)* 1.856 (8.79)*
Dorzolamide (DOR)
AS AM SRS RD RS MCR Reported method** [14]
Mean 100.09 99.98 100.04 100.04 100.15 100.11 99.99
SD 0.816 0.810 0.535 0.472 0.754 0.736 0.472
n 8 8 8 8 8 7 4
Variance 0.666 0.656 0.286 0.223 0.569 0.542 0.223
t-Test* 0.040 (2.228)* 0.004 (2.228)* 0.028(2.228)* 0.031(2.228)* 0.068(2.228)* 0.055(2.262)*
F* 2.987 (8.89)* 2.942 (8.89)* 1.283 (8.89)* 1.000 (8.89)* 2.552 (8.89)* 2.430 (8.94)*
*
The figures in parenthesis are the corresponding theoretical values at P = 0.05.
**
The reported method used is the first derivative spectrophotometry at 250.2 nm for DOR and 312.5 nm for TIM, using 0.1 M sodium hydroxide as a blank.

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