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

Peer review under responsibi

University.

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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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