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Novel spectrophotometric methods for simultaneous determination
of timolol and dorzolamide in their binary mixture

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Biomolecular Spectroscopy

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

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.

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

equation, in addition to a comparative study was done between the

Timolol maleate (TIM), (S)-1-[(1,1-dimethyl)amino]-3-[[4-(4-mor- two novel methods (AS and AM) and six recently well established

pholinyl9-1,2,5-thiadiazol-3-yl]oxy]-2-propanol is a nonspeciﬁc-adren- spectrophotometric methods (SRS, RD, RS, EXRS, CM and MCR) in

ergic blocker (Fig. 1a). It was the ﬁrst-blocker to be used as an terms of speciﬁcity and validation. These two novel methods are con-

antiglaucoma agent [1]. None of the newer-blockers were found to be sidered as new approach of the isosbestic point method either in zero

more effective than timolol [13]. Dorzolamide hydrochloride (DOR), order absorption spectrum i.e. isoabsorptive point [27,31–37] or in

(4S,6S)-4-ethylamino-5,6-dihydro-6-methyl-4H-thieno (2,3-b)thiopy- the ratio spectrum [38] at which the total concentration of both com-

ran-2-sulfonamide-7,7-dioxide monohydrochloride (Fig. 1b), is a human ponents in the mixture was calculated, by using smart mathematical

carbonic anhydrase-II inhibitor used in eye drops to treat increased pres- techniques utilizing the absorption factor [39,40] in zero order

sure in the eye caused by open-angle glaucoma and to treat a condition absorption spectra or constants in the ratio spectra which could be

called ocular hypertension [1]. Both drugs have been co-formulated adapted to isosbestic point analysis for separate quantitative estima-

and widely used to reduce intraocular pressure in open-angle glaucoma tion of each drug in their mixture using uniﬁed regression equation.

and for treating ocular hypertension. The proposed methods are very simple, accurate, precise and do not

Only few methods have been reported for the determination of require any sophisticated apparatus or computer programs.

DOR based on HPLC assay with ultraviolet detection in human ser-

um and urine [2–4] and capillary electrophoresis [5]. Several ana- Theoretical background

lytical procedures have been reported for determination of TIM,

including GC [6], HPLC in plasma [7,8], in pharmaceuticals [9–11] Absorbance subtraction method (AS)

and HPTLC [12].

To the best of my knowledge, very few spectrophotometric This method is based on the same principles as the absorption

methods [13,14], capillary electrophoretic method [15] and few factor method [39,40]. The method could be applied for the analy-

HPLC methods [16,17] were described for the simultaneous deter- sis of a mixture of two drugs X and Y having overlapped spectra

mination of both drugs in eye drops. intersect at isoabsorptive point and Y is extended over X, while X

The main problem of spectrophotometric multicomponent anal- does not show any contribution at another wavelength (k2). In this

ysis is the simultaneous determination of two or more compounds method the isoabsorptive point kiso could be used for separate

in the same mixtures without preliminary separation. Several uni- quantitative estimation of each X and Y in their mixture (X + Y).

variate spectrophotometric methods have been used for resolving The determination can be done using mathematically calculated

mixtures with overlapping spectra such as derivative spectropho- factor of one of these components. By simple manipulation step,

tometry [18], H-point standard addition method for binary [19] we can get the absorbance value corresponding to X and Y, sepa-

and ternary [20] mixtures, the ratio derivative spectrophotometry rately. So, the concentration of each component could be obtained

for binary [21] and derivative ratio spectra-zero crossing for ternary via the isoabsorptive point regression equation without any need

mixtures [22–27], the double divisor-ratio spectra derivative meth- for a complementary method.

od for determination of ternary mixtures [18,26,28]. Multivariate The absorbance values corresponding to X and Y at kiso were cal-

spectrophotometric methods also were reported as partial least culated by using absorbance factor which is a constant for pure Y

squares regression [29], principal component regression [28] and representing the average of the ratio between the absorbance val-

multiple linear regression analysis [30]. ues of different concentrations of pure Y at k1 (Aiso) to those at k2

The aim of this work is to develop and conduct two novel methods (A2) i.e {Aiso/A2}

namely; absorbance subtraction (AS) and amplitude modulation

(AM) methods for resolving those binary mixtures with spectral abs1

interfering problems, either drugs in mixture or drug and its degrada- Absorbance of Y in the mixture at kiso ¼ absk2 ðX þ YÞ

abs2

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

Fig. 1a. Chemical structure of timolol maleate.

concentrations X or Y respectively).

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

Fig. 1b. Chemical structure of dorzolamide hydrochloride.

H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207 199

Am, AX, AY are the absorbance values of the mixture, component X where (Y) is extended, then a new curve is obtained after subtrac-

and component Y, respectively at isoabsorptive point. tion of the constant. This can be summarized as the following:

½Am ¼ ½aX C X þ ½aY C Y XþY X Y X

¼ 0 þ 0 ¼ 0 þ constant ð1Þ

Dividing the previous equation with spectrum of Y0 as a divisor Y0 Y Y Y

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-

X X

þ constant constant ¼ 0 ð2Þ

lowing equation was obtained: Y0 Y

Simultaneous ratio subtraction (SRS)

Pm ¼ PX þ PY The concentration of X is calculated using the regression equa-

tion representing correlation between the amplitudes of ratio spec-

where Pm, PX and PY are the amplitudes of the mixture, component X tra YX0 and the corresponding concentration of X in lg/mL.

and component Y, respectively at that isosbestic point. The concentration of the second component (Y) is calculated via

The amplitude of constant (PY) [aYCY]/[aYCY0 ] can be measured measured constant value (CV) using the regression equation repre-

directly from the spectrum in the region where Y is extended since senting correlation between the amplitudes of ratio spectra YY0 and

the constant is a straight line parallel to the wavelength axis. the corresponding concentration of Y in lg/mL [39].

Upon using the normalized spectrum of Y0 , the following equa-

tion was obtained:

Simultaneous ratio subtraction coupled with constant multiplication

Pm ¼ PX þ PY (SRS–CM)

The concentration of X is determined as previously detailed in

Pm ¼ ½aX C X =½aY þ ½aY C Y =½aY SRS, while the concentration of Y can be determined using constant

multiplication method (CM) by multiplying the previously

Pm ¼ ½aXC X =½aY þ constant 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

PY ¼ ½aY C Y =½aY representing correlation between the absorbance values of the zero

order spectra of Y at its kmax against the corresponding

PY ¼ ½C Y concentrations.

The recorded amplitude of the constant is modulated to concen- Y

tration, so it represents the concentration of Y [CY]. (CRecorded of Y). Y¼ Y0; ð3Þ

Y0

For determination of X, the measured value of the constant is

subtracted from the ratio spectrum then the amplitude at kiso

was recorded: Ratio subtraction (RS) coupled with extended ratio subtraction (EXRS)

The mixture of X and Y could be analyzed using well established

PX ¼ Pm PY ratio subtraction method [43] where zero order spectrum of com-

ponent X could be obtained by multiplying the obtained ratio spec-

PX ¼ ½aX C X =½aY þ constant constant trum in Eq. (2) by the divisor Y0

PX ¼ ½aX C X =½aY X

Y0 ¼ X ð4Þ

Y0

At that isosbestic point aX = aY

Another extension of the already developed method has been

PX ¼ ½C X established as a new approach namely extended ratio subtraction

This obtained amplitude of ratio spectrum is modulated to con- [39,42] to get the zero order spectrum of second component (Y),

centration and it represents concentration of X [CX]. (CRecorded of X). by dividing the obtained D0 spectrum of X in Eq. (3) by a known

To eliminate any error, this recorded concentration of X and Y concentration of X as a divisor (X0 ) to get the constant XX0 for each

could be corrected to the actual concentration by using the follow- concentration in the mixtures then follow the same procedure of

ing uniﬁed regression equation at that isosbestic point: the ratio subtraction by dividing each mixture using a known con-

centration of X as a divisor then subtracting the corresponding con-

C Recorded ¼ slope C intercept stant XX0 and multiplying by (X0 )

The slope is found to be around unity and intercept around zero.

Y

Where CRecorded represents the recorded amplitude correspond- X0 ¼ Y ð5Þ

X0

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- The concentrations of X or Y in the mixture were calculated

sents the corresponding concentration of X or Y. from the corresponding regression equations (obtained by plotting

the absorbance values of the zero order spectra of each drug at its

Simultaneous ratio subtraction, simultaneous ratio subtraction kmax against its corresponding concentration).

coupled with constant multiplication and ratio subtraction coupled

with extended ratio subtraction methods Ratio difference spectrophotometric method (RD)

These methods are applied for the analysis of a mixture of two Ratio difference spectrophotometric method [39,44] was re-

drugs X and Y having overlapped spectra and one of them is ex- cently developed for analyzing a mixture of two drugs X and Y hav-

tended, one can determine X by dividing the spectrum of the mix- ing overlapped spectra depend on the amplitude difference

ture by a known concentration of Y as a divisor (Y0 ). The division between two wavelengths k1 and k2 in the ratio spectra where

will give a new curve that represents YX0 þ constant. If we measure the interfering substance should be contributed at these chosen

this constant which is parallel to the wavelength axis in the region 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 spectra of the resulting solution were measured in the range of

instrumental error. This can be summarized as the following: 200–400 nm and stored in the computer.

X X

P1 P2 ¼ 1 2

Y0 Y0 Absorbance Subtraction method (AS)

The scanned spectra of 5–40 lg/mL TIM were measured at

where P1 is the peak amplitude of the ratio spectrum at k1, P2 is the 272.8 nm and 315 nm. The absorbance factor was calculated which

peak amplitude of the ratio spectrum at k2. is the ratio of the absorbance at these two wavelengths. Calibration

The concentration of X is calculated using the regression equa- curve was constructed relating the absorbance of the zero order

tion representing the linear correlation between the differences of spectra of TIM at 272.8 nm versus the corresponding concentra-

ratio spectra amplitudes at the two selected wavelengths using Y tions of TIM and the regression equation was computed.

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 ). Amplitude modulation method (AM)

The stored spectra of 5–40 lg/mL TIM were divided by the nor-

Mean centering of ratio spectra spectrophotometric method (MCR) malized spectrum of TIM. The obtained ratio spectra of TIM were

measured at 272.8 nm and 315 nm. Calibration curve was con-

This is a well established spectrophotometric method in which structed relating the recorded concentrations of TIM at 272.8 nm

binary mixtures of X and Y could be determined without previous versus the corresponding concentrations of TIM and the regression

separation. In this method the ratio spectra are obtained after equation was computed.

which the constant is removed by mean centering of the ratio spec-

tra [44–47]. Simultaneous ratio subtraction methods (SRS)

The stored spectra were divided by the absorption spectrum of

Experimental standard solution of TIM (30 lg/mL), where the obtained ratio

spectra were recorded. Calibration curves relating the amplitudes

Chemicals and reagents of the ratio spectra of TIM representing constant value at plateau

region 315–325 nm versus the corresponding concentrations of

Timolol maleate (TIM) was kindly supplied by EIPICO, Cairo, TIM, the absorbance of zero order absorption spectra at 297.5 nm

Egypt. BN: TMM-021341. Its purity was found to be 99.96 versus the corresponding concentrations of TIM and the ampli-

± 0.51% according to the reported method [14]. tudes of the ratio spectra at 250 nm versus the corresponding con-

Dorzolamide hydrochloride (DOR) was kindly supplied by centrations of DOR were constructed and the regression equations

Merck Sharp & Dohm Company, USA. Batch number HC-B12-02- were computed.

001148102291. Its purity was found to be 100.25 ± 0.46% accord-

ing to the reported method [14].

Ratio difference spectrophotometric method (RD)

CosoptÒ eye drops bottles were manufactured by Global Napi

The scanned spectra of TIM and DOR solutions were divided by

Pharmaceuticals-Egypt. BN:21182/2001, each 1 mL is claimed to

the absorption spectra of standard solution of DOR (40 lg/mL) and

contain 20 mg dorzolamide and 5 mg timolol.

TIM (30 lg/mL), respectively. The obtained ratio spectra were re-

Methanol was E. Merck, Darmstadt, Germany.

corded. Calibration curves for TIM and DOR were constructed by

plotting the difference between the amplitudes of the obtained ra-

Instruments and software tio spectra at 250–290 nm and 250–280 nm versus the corre-

sponding concentrations of TIM and DOR, respectively; and the

Spectrophotometric measurements were carried out on Shima- regression equations were computed.

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. Ratio subtraction methods coupled with extended ratio subtraction

For MCR computations, MatlabÒ 7 was used along with PLS_ methods (RS–EXRS)

toolbox. 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-

Spectral characteristics

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

The absorption spectra of 40 lg/mL TIM and DOR, separately,

their corresponding concentrations were constructed and the

and that of a laboratory mixture containing equal concentrations

regression equation was computed.

of both drugs (each, 20 lg/mL) in methanol were scanned over

the range 200–400 nm as shown in (Fig. 3)

Mean centering of ratio spectra method (MCR)

Solutions and calibrations The scanned spectra were exported to Matlab for subsequent

calculation, then the spectra of DOR were divided by the absorp-

Stock solutions of TIM and DOR (each, 1 mg/mL), calculated tion spectrum of standard solution of TIM (30 lg/mL), the obtained

equivalent to their base, were prepared by dissolving the com- ratio spectrum was then mean centered. Also the spectra of TIM

pounds in methanol then completing in 100-mL volumetric ﬂasks. were divided by the absorption spectrum of standard solution of

Aliquots of the prepared stock solutions were further diluted with DOR (40 lg/mL) and the obtained ratio spectra were mean cen-

methanol to a ﬁnal volume of 100-mL. The diluted solutions were tered. The calibration curves for TIM and DOR were constructed,

used as the working solutions for TIM and DOR (each, 100 lg/mL). each by plotting the mean centered values at 250 nm and

Standard solutions containing 5–60 lg/mL of TIM and 5–40 lg/mL 249.9 nm, respectively versus their corresponding concentrations

of DOR were prepared separately in methanol. The absorption and the regression equations were computed.

H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207 201

For preparation of laboratory mixtures, into a series of 10-mL CosoptÒ eye drops bottle each 1 mL is claimed to contain

volumetric ﬂasks, aliquots equivalent to 50–600 lg of TIM and 22.26 mg dorzolamide hydrochloride equivalent to 20 mg dorzola-

50–400 lg of DOR were accurately transferred from their working mide and 6.83 mg timolol maleate equivalent to 5 mg timolol.

solutions (each, 100 lg/mL) with different ratios of the two drugs For AS and AM methods, from eye drops bottle 1 mL was trans-

and the volume was completed with methanol. The spectra of ferred into 50-mL volumetric ﬂask then the volume was completed

the prepared mixtures were scanned from 200 to 400 nm and with methanol to obtain a ﬁnal concentration of 100 lg/mL of TIM

stored in the computer. 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-

Absorbance subtraction method (AS) tration 5 lg/mL of TIM and 20 lg/mL of DOR.

The absorbance of TIM in the mixture was calculated using the For SRS, RD, RS, EXRS, CM and MCR methods, Aliquots equiva-

absorbance factor equation, and then the absorbance of DOR was lent to 100 lg of TIM and 400 lg of DOR were transferred into

calculated after subtraction of TIM interference. The concentra- 10-mL volumetric ﬂasks. The volume was completed with metha-

tions of TIM and DOR were calculated from the computed uniﬁed nol to obtain ﬁnal concentration 10 lg/mL of TIM and 40 lg/mL of

regression equation at 272.8 nm. DOR.

The proposed methods were applied for the analysis of the

studied drugs in their pharmaceutical formulation using the proce-

Amplitude modulation method (AM) dures mentioned under analysis of laboratory prepared mixtures

The absorption spectra of different laboratory prepared mix- for each method and the concentrations of the cited drugs were

tures were divided by the normalized spectrum of TIM. The ratio calculated from the corresponding regression equations.

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. Results and discussion

Simultaneous Ratio Subtraction Method (SRS), Ratio Subtraction tive and accurate analytical methods for simultaneous determina-

coupled with extended ratio subtraction (RS–EXRS) tion of TIM and DOR in their bulk powders and pharmaceutical

The stored zero order absorption spectra of the laboratory pre- dosage form with satisfactory precision and accuracy. As well, to

pared mixtures were divided by the absorption spectrum of stan- construct a statistical comparison between the ability of the pro-

dard TIM0 (30 lg/mL), then the amplitudes in the plateau region posed methods to determine both drugs in their pure form, labora-

at k 315–325 nm (the constant) was recorded and subtracted from tory prepared mixture and in their pharmaceutical formulations.

the obtained ratio spectra respectively. The amplitude of the ob- By scanning the absorption spectra of TIM and DOR in metha-

tained spectra were recorded at 250 nm and used for the determi- nol, severely overlapped spectral bands were observed in the

nation of DOR using its corresponding regression equation of SRS wavelength region of 200–300 nm which hinders their direct

or via ratio subtraction method by multiplying the obtained ratio determination (Figs. 2a and 2b), so different methods were applied

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.

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.

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- Fig. 2a. Zero-order spectra of 20 lg/mL of DOR (—) and 5 lg/mL of TIM (. . .),

ceed as under the proposed method. The concentration of each separately in methanol, in presence of 5 lg/mL of benzalkonium chloride (h h) as

drug was calculated using the speciﬁed regression equation. preservative.

202 H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207

Fig. 3. Ratio spectra of 40 lg/mL of DOR (—) and TIM (. . .), separately in methanol,

Fig. 2b. Zero-order spectra of 40 lg/mL of DOR (—) and TIM (. . .), separately in and binary of a mixture of DOR and TIM, 20 lg/mL of each (- - -) using the

methanol, and binary of a mixture of DOR and TIM, 20 lg/mL of each (- - -) in normalized spectrum of TIM as a divisor.

presence of 5 lg/mL of benzalkonium chloride (h h) preservative.

Absorption of DOR at kiso 272:8 nm

abs1

¼ abs272:8 ðTIM þ DORÞ abs315

for achieving best resolution and quantitative determination of abs2

each drug without any interference from the other. where abs272.8 and abs315 are absorption of TIM in the mixture

All spectral measurements were done without interference of at 272.8 nm (isoabsorptive point) nm and 315 nm (no interference)

benzalkonium chloride (preservative present in CosoptÒ eye drops) and abs1 is the absorbance factor is absorption ratio of pure TIM at

abs2

which did not show any absorption and its contribution to the 272.8 nm/315 nm. The concentrations of TIM and DOR were calcu-

absorption of the mixture above 220 nm was considered to be neg- lated using the corresponding uniﬁed regression equation (ob-

ligible at a concentration up to 100 lg/mL, so the ternary mixture tained by plotting the absorbance of the zero order spectra of

in range (220–400 nm) acts as a binary mixture of TIM and DOR TIM or DOR at kiso 272.8 nm against the corresponding

(Fig. 2a). concentrations).

The absorbance subtraction method has advantage that the two

Absorbance subtraction method (AS) drugs in the mixture can be determined using uniﬁed regression

equation at kiso in contrary to the previously established

TIM and DOR present in their dosage form in ratio (1:4); in isoabsorptive point method [27,31–38] which determines total

which their absorption spectra in methanol are severely concentration of two drugs while one of the drugs is measured

overlapped in the wavelength region of 200–300 nm (Fig. 2a) and by using conventional spectrophotometric method as a comple-

intersect at isoabsorptive point 272.8 nm. This could be conﬁrmed mentary method.

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

Amplitude modulation method (AM)

(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

As shown in (Fig. 2b), the absorption spectra of timolol (TIM)

permit one to conclude that the mixture of drugs act as a single

and dorzolamide (DOR) in methanol shows isoabsorptive point at

component and give the same absorbance value as pure drug at

272.8 nm (aTIM = aDOR) which is the same place in the ratio spec-

their isoabsorptive point.

trum of TIM using the normalized spectrum of TIM as a divisor ob-

The absorption spectra of the standard solutions of TIM with

tained by dividing the whole spectrum of any concentration by its

different concentrations were recorded in the wavelength range

corresponding concentration to give a new spectrum represents

of 200–400 nm. The value of absorbance factor of pure TIM repre-

the absorptivity of the analyte of interest versus all the measured

senting the average of the ratio between absorbance at two wave-

wavelengths (Fig. 3). The amplitude modulation method was ap-

lengths, one of them showing interference from DOR (kiso) while

plied to solve the mixture of TIM and DOR of overlapping spectra

DOR has no contribution at the other wavelength (k2) was found

by dividing the spectrum of the mixture by 1 lg/mL TIM as a divi-

to be 0.833. Quantitative estimation of DOR in mixture (DOR + -

sor. The division will give a new curve that represents

TIM) was carried out by subtracting the absorbance due to TIM DOR

TIM0

þ constant (Fig. 4). The constant value will be measured in pla-

at kiso using the following equations:

teau (315–325 nm), after subtraction of the measured value the

abs1 constant; the obtained ratio spectrum TIM DOR

0 is used for calculation

Absorption of TIM at kiso 272:8 nm ¼ abs315

abs2 the concentration of DOR in the mixture.

H.M. Lotfy et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 197–207 203

Fig. 4. Ratio spectra of Timolol (TIM) 5 lg/mL and Dorzolamide (DOR) 20 lg/mL Fig. 5. Division spectra of laboratory prepared mixture of Dorzolamide (DOR) 15,

(. . .) and their laboratory prepared mixture with the same ratio (1:4) (- - -) using the 20, 30, 40 lg/mL and Timolol (TIM) 10 lg/mL using spectrum of TIM (30 lg/mL) as

spectrum of TIM (—) as a divisor. a divisor.

The concentration of TIM and DOR in the mixture could be ob- tudes values of the ratio spectra of DOR at 250 nm against the cor-

tained from the corresponding regression equation (obtained by responding concentrations).

plotting the absorbance values of the isosbestic point of ratio spec- The concentration of TIM in the mixture could be obtained

tra of TIM or DOR at 272.8 nm against the corresponding either via constant value step by recording the constant value

concentrations). and substituting in equation representing the constant values

The advantages of amplitude modulation method over other against the corresponding concentration of TIM or via constant

mathematical techniques utilizing the constants that it reduces multiplication step by multiplying the obtained constant of the

the manipulation steps and only one divisor is needed in order to laboratory mixture by TIM (the divisor), to get the original zero or-

determine each component in the mixture separately. By using der spectra of TIM (Y) in the mixture, which is used for direct deter-

the normalized spectrum of the divisor which represent the mination of TIM at 297.5 nm (Fig. 6) and calculation of the

absorption spectrum, therefore the results will not affected by concentration from the corresponding regression equation (ob-

the use of different concentrations of divisor and it has advantage tained by plotting the absorbance values of the zero order curves

over the isoabsorptive point at zero order that measure the total of TIM at 297.5 nm against the corresponding concentrations).

concentration therefore there is always a need of other conven- The advantages of simultaneous ratio subtraction method [39]

tional method as complementary one to measure one of the com- that only one divisor was needed and minimum manipulation

ponents in the mixture (Fig. 2b). steps in order to determine both components in the mixture

This method has advantages over the newly developed absor- therefore, it is very simple, rapid, accurate and the two drugs in

bance subtraction using uniﬁed regression equation at isoabsorp- the mixture could be determined in contrary to the previously

tive point that the obtained values at the ratio spectrum will established ratio subtraction method [43] which determines one

represent the concentration of each component and it will cancel drug only.

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- Ratio difference spectrophotometric method (RD)

duced by eliminating of the absorbance factor calculation step

since the value of the constant is the same at the two selected In the RD spectrophotometry, the absorption spectrum of the

wavelengths. 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

Simultaneous ratio subtraction method (SRS) difference spectrophotometric method (RD) was applied to solve

the problem of the overlapped absorption spectra of the cited

TIM

TIM

The ratio subtraction method was applied to solve the mixture of drugs DOR 1 DOR 2 or DOR

TIM

1 DOR

TIM

2, where the interfering sub-

TIM and DOR of overlapping spectra by dividing the spectrum of the stance was cancelled and subsequently shows no interference.

mixture by a known concentration of TIM (30 lg/mL) as a divisor. The overlapped spectra of the two drugs suggested that a RD

DOR

The division will give a new curve that represents TIM 0 þ constant method was a suitable method for simultaneous determination

(Fig. 5). The constant will be measured at plateau (315–325 nm). of TIM and DOR, respectively.

DOR

If we subtract this constant, the obtained ratio spectrum TIM 0 is used The two main steps affecting the ratio difference method is the

for calculation the concentration of DOR in the mixture from the choice of the divisors and the two selected wavelengths. The se-

corresponding regression equation (obtained by plotting the ampli- 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).

(EXRS)

maximum sensitivity while, the requirements of the two chosen

wavelengths are that the contribution of the interfering substance

The ratio subtraction method was applied to solve the mixture

at them so, the amplitude at 250 and 290 nm were selected for

of TIM and DOR of overlapping spectra by dividing the spectrum of

determination of TIM in the mixture using DOR (40 lg/mL) as a

the mixture by a known concentration of TIM (30 lg/mL) as a divi-

divisor (Fig. 7). Similarly, the two selected wavelengths for the esti-

sor. The division will give a new curve that represents

mation of DOR using TIM (30 lg/mL) as a divisor were 250 and DOR

TIM0

þ constant in plateau (315–325 nm) (Fig. 5). If we subtract this

280 nm (Fig. 8).

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

DOR in the mixture (Fig. 9). 6

The determination of TIM could be done by the extended ratio

MCN values

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

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.

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.

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

They were determined using three concentrations of each of

The accuracy of the results was checked by applying the pro- TIM and DOR (5, 10, 20 lg/mL) which were analyzed three times

posed methods for determination of different blind samples of intra-daily and inter-daily on three different days using the pro-

TIM and DOR. The concentrations were obtained from the corre- posed methods. The relative standard deviations were calculated

sponding regression equations. From which the percentage recov- (Table 1).

eries suggested good accuracy of the proposed methods (Table 1).

Stability

trophotometric changes up to 4 weeks when stored at 4 °C.

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- Application of the methods in assay of eye drops

tical preparations to give accurate precise and linear results (Table

1). 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-

Selectivity pared with that of the reported method [14]. The good percentage

recoveries conﬁrm the suitability of the proposed methods for the

Selectivity of the methods was achieved by the analysis of dif- routine determination of these components in their combined

ferent laboratory prepared mixtures of TIM and DOR within the formulation.

linearity range. Satisfactory results were shown in (Table 2).

Statistical analysis

Robustness

Tables 3 and 4 showed statistical comparison of the results ob-

Three concentrations of each of TIM and DOR (5, 10, 20 lg/mL) tained by the proposed methods and reported spectrophotometric

were prepared using 80%, 75%, 70% methanol and analyzed three method [14]. The calculated t and F values were less than the the-

times using the proposed methods. The relative standard devia- 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.

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.

Table 4 [4] M.L. Satuf, J.C. Robles, H.C. Goicoechea, A.C. Oliveri, Anal. Lett. 32 (1999) 2019–

One way ANOVA testing for the different proposed and the reported methods used for 2033.

determination of Timolol (TIM) and Dorzolamide (DOR) in pure form. [5] R.C. Tim, R.A. Kautz, B.L. Karger, Electrophoresis 21 (2000) 220–226.

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Source DF Sum of squares Mean square F value F crit Pharm. Sci. 69 (1980) 1111–1115.

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Between exp. 7 1.518 0.217 0.206 2.233 [8] K. Kubota, H. Nakamura, E. Koyama, T. Yamada, K. Kikuchi, T. Ishizaki, J.

Chromatogr. B 533 (1990) 255–263.

Within exp. 72 75.768 1.052

[9] B.R. Patel, J.J. Krischbaum, R.B. Poet, J. Pharm. Sci. 70 (1981) 336–338.

DOR [10] D.J. Mazzo, P.A. Snyder, J. Chromatogr. 438 (1988) 85–92.

Between exp. 6 0.160 0.027 0.059 2.443 [11] D.J. Mazzo, J. Chromatogr. 299 (1984) 503–507.

Within exp. 44 19.966 0.454 [12] S.P. Kulkarni, P.D. Amin, J. Pharm. Biomed. Anal. 23 (2000) 983–987.

[13] N. Erk, J. Pharm. Biomed. Anal. 28 (2002) 391–397.

At the 0.05 level. [14] L.I. Bebawy, J. Pharm. Biomed. Anal. 27 (2002) 737–746.

The population means are not signiﬁcantly different. [15] I.M. Palabiyik, M.G. Caglayan, F. Onur, Chromatographia 73 (2011) 541–548.

[16] N. Sharma, S.S. Raol, A.M. Reddy, J. Chromatogr. Sci. 50 (2012) 745–755.

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[24] F. Onur, C. Yucesoy, S. Dermis, M. Katral, G. Kukdil, Talanta 51 (2000) 269–279.

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(2001) 203–210.

[26] E. Dinc, I.M. Palabyik, O. ustundag, F. Yustsever, F. Onur, J. Pharm. Biomed.

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[27] K.A. Connors, Text Book of Pharmaceutical Analysis, USA, 1967.

[28] E. Dinc, C. Yucesoy, F. Onur, J. Pharm. Biomed. Anal. 28 (2002) 1091–1100.

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[29] S. Wold, M. Sjostrom, L. Eriksson, Chemom. Intell. Lab. Syst. 58 (2001) 109–

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and could be used for more complex binary and ternary mixtures. [30] M. Blanco, J. Gene, H. Iturriaga, S. Maspoch, J. Riba, Talanta 34 (1987) 987–993.

The proposed methods were very simple with minimum manipu- [31] V.S. Erram, H.P. Tipnis, Indian Drugs 30 (9) (1993) 462–467.

[32] V.S. Erram, H.P. Tipnis, Indian Drugs 30 (7) (1993) 319–323.

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apparatus or a special program and could be easily applied in qual- [34] V.S. Erram, H.P. Tipnis, Indian Drugs 30 (11) (1993) 555–560.

ity control laboratories as they are having equal accuracy and pre- [35] V.S. Erram, H.P. Tipnis, Indian Drugs 31 (2) (1993) 65–68.

[36] N.Y. Hassan, S.M. Elgizawy, H.M. Lotfy, S.S. Saleh, Int. Res. J. Pure Appl. Chem. 3

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[42] H.M. Lotfy, S.S. Saleh, N.Y. Hassan, S.M. Elgizawy, TACL 3 (2) (2013) 70–84.

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