Bulletin of Faculty of Pharmacy, Cairo University (2013) 51, 175–184
Cairo University
Bulletin of Faculty of Pharmacy, Cairo University
www.elsevier.com/locate/bfopcuwww.sciencedirect.com
ORIGINAL ARTICLE
Determination of ofloxacin and dexamethasone
in Dexaflox eye drops through different ratio
spectra manipulating methods
M. Nebsena,b, Ghada M. Elsayed
a,*, M. AbdelKawya, S.Z. ELkhateeb
a
a Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr-El-Aini, 11562 Cairo, Egyptb Pharmaceutical Chemistry Department, Faculty of Pharmacy and Drug Technology, Heliopolis University,3 Cairo-Belbeis Desert Road, 2834 El-Horria, Cairo, Egypt
Received 17 February 2013; accepted 23 April 2013Available online 4 June 2013
* Corresponding author. Tel.
0580.E-mail address: ghada_mosta
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KEYWORDS
Ofloxacin;
Dexamethasone;
Derivative ratio;
Ratio subtraction;
Mean centering
Abstract Different sensitive and selective spectrophotometric methods for the determination of
ofloxacin and dexamethasone in their binary mixture were presented. Ofloxacin was determined
simply by zero order at its kmax 293.4 nm in a linear range of 1.5–12 lg mL�1 with mean percentage
recovery of 100.07 ± 0.66% without any interference of dexamethasone even in low or high con-
centrations. Dexamethasone was determined by first derivative of ratio spectra 1DD at 266.5 nm,
ratio subtraction and mean centering at 243 nm with methods in a linear range of 2.5–27.5 lg mL�1
with mean percentage recoveries of 100.09 ± 0.70%, 100.00 ± 0.72% and 99.92 ± 0.62, respec-
tively. These methods were applied to the analysis of pharmaceutical dosage form and bulk powder
where good recoveries were obtained. The proposed methods were validated according to USP
guidelines.ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
Open access under CC BY-NC-ND license.
1. Introduction
Ofloxacin (Oflox., Fig. 1) (±)-9-fluoro-2,3-dihydro-3-methyl-
10-(4-methyl-L-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid)1 is a fluoroquinolone
0 548 6038; fax: +20 202 491
hoo.com (G.M. Elsayed).
aculty of Pharmacy, Cairo
g by Elsevier
ng by Elsevier B.V. on behalf of F
://dx.doi.org/10.1016/j.bfopcu.201
antibacterial agent, which is highly active against bothGram-positive and Gram-negative bacteria. It is also activeagainst mycoplasma, chlamydia and legionella.2
The mechanism of the bactericidal effect of Oflox. is basedon the inhibition of the DNA gyraze of the bacteria, theenzyme that produces a negative supercoil in DNA and thuspermits transcription and replication.3
Several methods are available for analysis of Oflox. such ashigh-performance liquid chromatography,4–8 capillary electro-phoresis,9 chemiluminescence10 and chemometry.11
Dexamethasone (Dexa. Fig 2) [9-Fluoro-11b,17,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione]1, a potentsynthetic corticosteroid with anti-inflammatory and
immunosuppressive properties, is frequently used as an
aculty of Pharmacy, Cairo University.
3.04.004
Figure 2 Structural formula for dexamethasone (MW= 392.47).
Figure 1 Structural formula for ofloxacin (MW= 361.38).
176 M. Nebsen et al.
anti-inflammatory agent.12 It has been widely used to treatinflammation, allergy and diseases related to adrenal cortex
insufficiency.Several methods had been reported in the literature describ-
ing the analysis of Dexa. including LC,13–17 spectrophotome-try,18 TLC chromatography19 and voltametry.20
Dexa. was determined with Oflox. in their binary mixtureby RF HPLC21 or in their ternary mixture with ephedrine bymicroemulsion LC.22
The aim of this study was to develop and validate spectro-photometric methods for the determination of Oflox. andDexa. The methods were validated by using the guidelines of
USP.1
2. Experimental
2.1. Instrumentation
Spectrophotometric measurements were carried out using adouble beam UV visible spectrophotometer, Schimadzu Japan,model 1601 PC, connected to IBM compatible computer and
HP 800 ink Jet printer, the bundle software was a UV PC per-sonal spectroscopy software version 3.7. The spectral bandwidth was 0.2 nm and wavelength scanning speed was
2800 nm min�1.
2.2. Chemicals and reagents
Oflox. standard material was kindly supplied by EL Nile com-pany, Egypt. Its purity was checked in our laboratory accord-
ing to the USP HPLC method1 and was found to be99.98 ± 0.73%. Dexa. standard material was kindly suppliedby EIPICO Company, Egypt. Its purity was checked in our
laboratory according to USP HPLC method1 and was foundto be 100.30 ± 1.38%. Market sample of Dexaflox eye dropswas manufactured by Gamgom pharma, Saudi Arabia, each1 ml claimed to contain 3 mg Oflox. and 1 mg Dexa., batch
number KC 082 was purchased from the local market.All reagents and solvents used were of analytical grade. 3%
of 0.1 N HCl in methanol was used as a reagent for the prep-
aration of standard solutions.
2.3. Standard stock and working solutions
Oflox. standard stock solution: 0.25 mg mL�1 in 3% of 0.1 NHCl/methanol reagent.
Oflox. standard working solution: 0.1 mg mL�1 in 3% of0.1 N HCl/methanol reagent.
Dexa. standard stock solution: 0.25 mg mL�1 in 3% of0.1 N HCl/methanol reagent.
Dexa. standard working solution: 0.1 mg mL�1 in 3% of0.1 N HCl/methanol reagent.
2.4. Procedures
2.4.1. Spectral characteristics of ofloxacin and dexamethasoneThe zero order absorption spectra of 9, 12 lg of Oflox. and 2.5,27.5 lg of Dexa. solutions were recorded against 3% of 0.1 NHCl/methanol reagent as a blank over the range of 200–
400 nm.
2.4.2. Construction of calibration curvesAliquots equivalent to 15–120 lg mL�1 Oflox. and 25–275 lg mL�1 Dexa. are accurately transferred from their stan-dard working solutions (0.1 mg/mL) into two separate series of
10-ml volumetric flasks then completed to volume with 3% of0.1 N HCl/methanol reagent. The spectra of the prepared stan-dard solutions are scanned from 200 to 400 nm and stored in
the computer.
2.4.2.1. For zero order method. The content of Oflox. was
determined from the measurements of its absorbance of zeroorder spectra at kmax 293.4 nm and plotted against their corre-sponding concentrations. The same procedure was used todetermine the content of Oflox. in the laboratory prepared
mixtures and in pharmaceutical formulations.
2.4.2.2. For first derivative of ratio spectrophotometric method.
The computer stored spectra of Dexa. were divided by the pre-viously stored spectrum of Oflox. of concentration 12 lg ml�1
and the first derivative of the ratio spectra were obtained with
Dk = 4 nm and a scaling factor of ten. The content of Dexa.was calculated from the measurements of the amplitude ofthe first derivative peak of (Dexa/Oflox) at 266.5 nm and plot-
ted against their corresponding concentrations. The same pro-cedure was used to determine the content of Dexa. in the
Determination of ofloxacin and dexamethasone in Dexaflox eye drops through different ratio 177
laboratory prepared mixtures and in pharmaceutical
formulations.
2.4.2.3. For ratio subtraction method. The content of Dexa. wascalculated from the measurements at kmax 240 nm and plotted
against their corresponding concentrations then the regressionequation was computed.
For the determination of Dexa. in laboratory prepared mix-
tures, different ratios of Dexa. and Oflox. were prepared thenthe prepared standard solutions were scanned from 200 to400 nm and stored in the computer. The same procedure was
used to determine the content of Dexa. in the pharmaceuticalformulation.
2.4.2.4. For mean centering method. The computer stored spec-tra of Dexa. were divided by the previously stored spectrum ofOflox. of concentration 12 lg mL�1 then the obtained ratiospectra were mean centered. The content of Dexa. was calcu-
lated from the measurements of the mean centered peak of(Dexa/Oflox) at 243 nm and plotted against their correspond-ing concentrations. The same procedure was used to determine
the content of Dexa. in the laboratory prepared mixtures andin pharmaceutical formulations.
2.4.3. Analysis of Oflox. and Dexa. laboratory preparedmixturesInto a series of 10-ml volumetric flasks aliquots of Oflox. and
Dexa. were accurately transferred from their correspondingworking solutions (0.1 mg mL�1) each. Then the volumes werecompleted with blank reagents each one separately to prepare
mixtures containing different ratios of the two drugs. The zeroorder spectrum of each laboratory prepared mixture was re-corded against blank reagents then stored in the computer.
2.4.3.1. For Oflox. The absorbance of the zero order spectra at293.4 nm was recorded, then the concentrations of the intact
(d)
(c)
Abs.
Wav
Figure 3 Zero order absorption spectra of Oflox. (a) 12, (b) 9 lg ml�
in methanol reagent as blank.
drug were calculated from their corresponding regression
equation.
2.4.3.2. For Dexa. (1DD). The obtained spectra were then di-vided by the spectrum of 12 lg ml�1 of Oflox. The peak ampli-
tudes of the first derivative of ratio spectra 1DD at 266.5 weremeasured then the concentrations of the intact drug were cal-culated from their corresponding regression equation.
2.4.3.3. For Dexa. (Ratio subtraction). The obtained spectrawere then divided by the spectrum of 9 lg ml�1 of Oflox. to
obtain new division spectra. The absorbance in the plateau re-gion at 300–320 nm giving a constant value was subtractedfrom the division spectra, the obtained curves were multiplied
by the spectrum of 9 lg/ml Oflox. The obtained curves can beused for direct determination of Dexa. at 240 nm and the con-centrations were calculated from the corresponding regressionequation.
2.4.3.4. For Dexa. (mean cetering). The obtained spectra werethen divided by the spectrum of 12 lg ml�1 of Oflox. The mean
centered values of ratio spectra at 243 nm were measured Thenthe concentrations of the intact drug were calculated fromtheir corresponding regression equation.
2.4.4. Analysis of Oflox. and Dexa. in Dexaflox eye dropsFromDexaflox eye drops bottle (each 1 ml contains 1 mgDexa.
and 3 mg Oflox.), a volume 1 ml was transferred quantitativelyinto 10-ml volumetric flask, the volume was then completed tomark with a blank reagent. Aliquots were transferred into 10-
ml volumetric flasks, the volume was then completed to markwith a blank reagent then analyzed as described under analysisof laboratory prepared mixtures. The concentrations were cal-culated from the corresponding regression equations.
Moreover, standard addition technique was applied to as-sess the validity of the method.
(b)
(a)
e length (nm)
1 (__) and Dexa. (c) 27.5, (d) 2.5 lg ml�1 (—) using 3% 0.1 N HCl
(a)
(c)
Abs.
Wave length (nm)
(b)
Figure 4 Zero order spectra of laboratory prepared mixtures of Oflox.: Dexa., respectively: (a) 12 lg ml�1 : 4 lg ml�1, (b) 9 lg ml�1 :
15 lg ml�1 , (c) 7.5 lg ml�1 : 7.5 lg ml�1.
Abs.
Wave length (nm)
Figure 5 Ratio spectra of Dexa. 2.5–27.5 lg ml�1 using Oflox. 12 lg ml�1 as a divisor and 3% 0.1 N HCl in methanol reagent as blank.
178 M. Nebsen et al.
3. Results and discussion
The primary goal of this study was to develop and validate spec-trophotometric methods that could assay Dexa. and Oflox. intheir binary mixture with sufficient selectivity and sensitivityeither in bulk powder or in pharmaceutical preparation.
3.1. Zero order spectrophotometric method
Spectral characteristics of Oflox. and Dexa. are shown in
Fig. 3. Oflox. can be directly determined by zero order spectro-photometry but direct analysis of Dexa. in the presence ofOflox. cannot be performed.
266.5nm
Abs.
Wave length (nm)
Figure 6 First derivative of ratio spectra of Dexa. 2.5–27.5 lg ml�1 using Oflox. 12 lg ml�1 as a divisor and 3% 0.1 N HCl in methanol
reagent as blank.
Determination of ofloxacin and dexamethasone in Dexaflox eye drops through different ratio 179
A linear relationship was constructed between the absor-
bance and the concentration of Oflox. at its kmax 293.4 nmand regression equation was calculated and found to be:
Abs: ¼ 0:0963 Cþ 0:0078 r ¼ 1
Direct determination of Oflox in laboratory prepared mix-tures was applied (Fig. 4).
3.2. First derivative ratio spectrophotometric method (1DD)
Derivative ratio spectrophotometric technique was widely usedfor resolving either binary23,24 or tertiary25,26 mixtures. Themain advantage of the ratio spectra derivative spectrophotom-
etry is the chance of doing easy measurements in correspon-dence of peaks so it permits the use of the wavelength ofhighest value of analytical signals (a maximum or a minimum).
It is necessary to study and to optimize the following parame-ters: concentration of the standard spectrum used as divisor,Dk to obtain the first derivative, smoothing function andzero-crossing wavelengths.26
Dexa. was quantified in the presence of Oflox. by 1DD. Thismethod resulted in a suitable regression equation for Dexa. Acorrect choice of standard divisor and working wavelength are
of capital importance.Hence, the method was tested with various divisor concen-
trations. In measurements the spectrum of 12 lg ml�1 of
Oflox. was used as a standard divisor for the determinationof Dexa. (Fig. 5). Even the presence of more than one peakin the obtained derivatized spectrum but only that atk = 266.5 nm gave reproducible results (Fig. 6). This assured
the best compromise in terms of sensitivity, repeatabilityand signal to noise ratio. A linear relationship was foundbetween the peak amplitudes and the concentration of
Dexa. at 266.5 nm in the range of 2.5–27.5 lg ml�1 from
which the linear regression equation was calculated and found
to be:
Peak amplitude ¼ 0:0626 Cþ 0:0004 r ¼ 0:99995
3.3. Ratio subtraction method
The ratio subtraction technique27,28 starts by scanning zero or-der spectra of the prepared standard solutions of Dexa., thenthe linearity is checked between absorbance at the selected
wavelength of 240 nm and the corresponding concentrationof Dexa.
The method is successfully used for the determination of
Dexa. in its binary mixture where the spectrum of Oflox. ismore extended Fig. 3, the zero order absorption spectra ofthe laboratory-prepared mixtures (Oflox. and Dexa.) were
scanned (Fig. 4), dividing them by a carefully chosen concen-tration of standard Oflox. (9 lg/mL) as a divisor producingnew ratio spectra which represent Dexa./Oflox. + constantas shown in Fig. 7, then subtraction of the values of these con-
stants Dexa./Oflox. in the plateau region (300–320 nm) asshown in Fig. 8, followed by multiplication of the obtainedspectra by the divisor Oflox. (9 lg/mL) as shown in Fig. 9. Fi-
nally, the original spectra of Dexa. could be obtained (Fig. 9)which are used for the direct determination of Dexa. at 240 nmand calculation of the concentration from the corresponding
regression equation obtained by plotting the absorbance valuesof the zero order curves of Dexa. at 240 nm against the corre-sponding concentrations.
The computed regression equation was found to be:
Abs: ¼ 0:0395 Cþ 0:0028 r ¼ 0:99995
Oflox. can be determined directly from its zero order spec-tra as mentioned before in 3.1.
Abs.
Wave length (nm)
Figure 8 Ratio spectra of laboratory prepared mixtures of Dexa. and Oflox. using 9 lg ml�1 Oflox. as a divisor. After subtraction of the
constant.
constant
Abs.
Wave length (nm)
Figure 7 Ratio spectra of laboratory prepared mixtures of Dexa. and Oflox. using 9 lg ml�1 Oflox. as a divisor.
180 M. Nebsen et al.
3.4. Mean centering method
For further improvement of the selectivity to resolve the over-
lap present between Oflox. and Dexa., a simple method is ap-plied which is based on the mean centering of ratio spectra. Iteliminates the derivatization step and therefore the signal-to-
noise ratio is enhanced.29,30
As the appropriate divisor was chosen, then the obtainedratio spectra is mean centered, so upon division; the Oflox. be-comes constant then the mean center of constant equals zero
so the Dexa. concentration is obtained without any interferingof Oflox.28
Mean centering method is applied to quantitatively
determineDexa. in its laboratory preparedmixtures and in phar-
320310300290280270260250240230220-2
-1
0
1
2
3
Wave length (nm)
MC
val
ues
243nm
Figure 10 Mean centered ratio spectra of Dexa. (2.5–27.5 lg ml�1).
(a)
(b)
(c)
Abs.
Wave length (nm)
Figure 9 The zero order absorption spectra of Dexa. (a) 15 lg ml�1, (b) 7.5 lg ml�1, (c) 4 lg ml�1 obtained by the proposed ratio
subtraction method for the analysis of laboratory prepared mixtures after multiplication by the divisor 9 lg ml�1 Oflox.
Determination of ofloxacin and dexamethasone in Dexaflox eye drops through different ratio 181
maceutical preparations. The absorption spectra of Dexa. aredivided by the absorption spectrum of 12 lg/ml Oflox. (Fig. 5).
The obtained ratio spectra aremean centered and the concentra-tions ofDexa. determined bymeasuring the amplitude at 243 nm(Fig. 10). The linear regression equation is found to be:
Mean centering value ¼ 0:0823 Cþ 0:0097 r ¼ 0:99995
The choice of divisor is different from one method to an-other, which attributed to optimization between increasingthe sensitivity of the method by choosing smaller divisor and
the produced noise which affects the reproducibility of theobtained results.
4. Method validation
Validation was done according to USP guidelines.1
4.1. Linearity and range
The linearity of the methods was evaluated by analyzing eightconcentrations of Oflox. and six concentrations of Dexa., Each
concentration was repeated three times. The assay was per-formed according to the experimental conditions previouslymentioned. The range and linear equations are summarized
in Table 1.
Table 2 Specificity of the proposed spectrophotometric methods for determination of Oflox. and Dexa. in laboratory prepared
mixtures.
Ratio Concentration (lg ml�1) Oflox Dexa
Oflox. Dexa. Zero order
method at 293.4 nm
Derivative ratio
method at 266.5 nm
Ratio subtraction
method at 240 nm
Mean centering
method at 243 nm
Recovery% Recovery% Recovery% Recovery%
3:1 12 4 100.58 100.50 100.50
1:1 7.5 7.5 101.32 99.07 99.07 103.01
3:5 9 15 99.67 99.87 99.87 98.72
1:5 3 15 101.30 100.73 100.73 100.65
1:2 7.5 15 101.99 101.00 101.00 99.99
Mean 100.97 100.23 100.23 100.59
SD 0.88 0.77 0.77 1.80
RSD% 0.87 0.77 0.77 1.80
Table 1 Assay validation parameters of the proposed spectrophotometric methods for the determination of pure Oflox. and Dexa.
Parameters Oflox Dexa
Zero order First derivative ratio Ratio subtraction Mean centering
Linearity range
(lg ml�1) 1.5–12 2.5–27.5 2.5–27.5 2.5–27.5
Accuracy
Mean + SD 100.07 ± 0.66 100.09 ± 0.70 100.00 ± 0.72 99.92 ± 0.62
Precision
Repeatability a 100.44 ± 1.24 99.74 ± 0.805 99.60 ± 0.74 100.07 ± 1.23
Reproducibility b 99.97 ± 1.43 100.12 ± 1.152 100.08 ± 0.94 100.59 ± 0.94
Regression
Slope 0.0963 0.0626 0.0395 0.0823
SE of slope 0.0002 0.0003 0.0020 0.0003
Intercept 0.0078 0.0004 0.0028 0.0097
SE of intercept 0.0017 0.0063 0.0034 0.0055
Correlation coefficient 1 0.99995 0.99995 0.99995
a Repeatability (n= 3), average of three concentrations (4.5, 6, and 7.5 lg ml�1) for Oflox. and (12.5, 17.5, and 22.5 lg ml�1) for Dexa.
repeated three times within the day.b Reproducibility (n= 3), average of three concentrations (4.5, 6, and 7.5 lg ml�1) for Oflox. and (12.5, 17.5, and 22.5 lg ml�1) for Dexa.
repeated three times in three successive days.
182 M. Nebsen et al.
4.2. Accuracy
The accuracy of the results was checked by applying the pro-
posed methods for the determination of different samples ofOflox. and Dexa. The concentrations were obtained from thecorresponding regression equations. From which the percent-age recoveries were calculated with mean percentage recovery
shown in Table 1.Accuracy of the methods was further assured by the use of
the standard addition technique, it was performed by the addi-
tion of known amounts of pure Oflox. andDexa. to known con-centrations of the pharmaceutical preparation, the resultingmixtures were assayed, and the results obtained were compared
with the expected results Table 2. The good recoveries of stan-dard addition technique suggested good accuracy of the pro-posed methods.
4.3. Precision
4.3.1. RepeatabilityThree concentrations of Oflox. (4.5, 6, 7.5 lg mL�1) and Dexa.(12.5, 17.5, 22.5 lg mL�1) were analyzed three times intra-daily
using the proposed methods. The relative standard deviationswere calculated Table 1.
4.3.2. ReproducibilityThe previous procedures were repeated inter-daily on three dif-ferent days for the analysis of the three chosen concentrations.The relative standard deviations were calculated Table 1.
4.4. Specificity
Specificity of the methods was achieved by the analysis of dif-ferent laboratory prepared mixtures of Oflox. and Dexa. with-in the linearity range. Satisfactory results are shown in Table 2.
4.5. Robustness
The linearity concentrations of both Oflox and Dexa were pre-pared using 2%, 3%, 5% 0.1 N HCl/methanol and analyzedusing the proposed methods. The relative standard deviationswere found to be below 2.0% and the methods proved to be
robust.
Table 4 Statistical comparison between the results obtained by applying the proposed spectrophotometric methods and USP HPLC
method (1) for the determination of pure sample of Oflox., USP HPLC method (1) for determination of pure sample of Dexa.
Values Ofloxacin Dexamethasone
Zero order
method
Official HPLC
methodaDerivative ratio
method
Ratio subtraction
method
Mean centering
method
Official HPLC
methodb
Mean 100.07 99.98 100.09 100.00 99.92 100.30
SD 0.66 0.73 0.70 0.77 0.62 1.37
RSD% 0.66 0.73 0.70 0.77 0.62 1.37
Variance 0.44 0.53 0.49 0.59 0.38 1.88
n 8 5 6 6 6 6
t-testc 0.23 (2.201)* 0.33 (2.228)* 0.47 (2.228)* 0.62 (2.228)*
F-value c 1.22 (4.12) 3.83 (5.05)* 3.17 (5.05)* 4.88 (5.05)*
a Perfectsil� Target ODS-3 column using a mobile phase consisting of sodium dodecyl sulphate0.24%: acetonitrile: glacial acetic acid (58:40:2
by volume).b Perfectsil� Target ODS-3 column using a mobile phase consisting of water: acetonitril (60:40 v/v).c The values in the parenthesis are the corresponding theoretical values of t and F at (P = 0.05).
Table 3 Quantitative determination of Oflox. and Dexa. in Dexaflox eye drops (each 1 mL claimed to contain 1 mg Dexa and 3 mg
Oflox) by the proposed spectrophotometric methods and the application of standard addition technique.
Pharmaceutical formulation Recovery % a Standard addition
Dexaflox eye drops B.N KC082 Taken (lg mL�1) Added (lg mL�1) Recovery %
Oflox.
Zero order
97.89% 4.5 3.00 101.00
4.50 101.11
6.00 98.33
Mean 100.15
SD 1.57
Dexa.1DD
105.50% 4 2.50 100.40
4.00 99.85
7.50 99.46
Mean 99.90
SD 0.47
Dexa.
Ratio subtraction
105.00% 4 2.50 100.00
4.00 99.00
7.50 100.67
Mean 99.89
SD 0.84
Dexa.
Mean centering
104.99% 4 4.00 99.42
7.50 101.34
Mean 100.38
SD 1.35
a Average of at least four determinations.
Determination of ofloxacin and dexamethasone in Dexaflox eye drops through different ratio 183
5. Analytical application
The proposed methods were applied for the determinationof Oflox. and Dexa.in their combined pharmaceuticalformulation in Dexaflox eye drops that indicates high
accuracy for the determination of the studied drugs as shownin Table 3.
6. Statistical analysis
Results obtained by the proposed procedures for the
determination of pure samples of Oflox. and Dexa. are statis-tically compared to those obtained by the official methods.1
The results showed no significant differences between themTable 4.
These proposed methods have the advantages of time sav-ing, simple software program used, no need of expensive sol-vents compared with their official HPLC methods.
7. Conclusion
The binary Oflox. and Dexa. mixture is easily determined bythe proposed simple, accurate and reproducible spectrophoto-metric methods where Oflox. is determined in its zero order
without any interference of Dexa., the interference of Oflox.with the spectrum of Dexa. was overcome by applying firstderivative of ratio spectra 1DD, ratio subtraction and meancentering methods. These reproducible quantitative analysis
methods can be used for the determination of Oflox. and Dexa.in pure bulk powder and in pharmaceutical eye drops and are
184 M. Nebsen et al.
also suitable for quality control laboratories where economy
and time are essential.
8. Conflict of interest
None.
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