Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066

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Spectrochimica Acta Part A: Molecular and

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

Simultaneous determination of ofloxacin and bromfenac in combined

dosage form using four different spectrophotometric methods
Ashraf M. Taha a, Ragab A.M. Said b,c, Ibrahim S. Mousa d, Tarek M. Elsayed d,⇑
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Sinai University, Al-Arish, Egypt
b
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo 11751, Egypt
c
Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, Egypt
d
Pharmaceutics Department, Faculty of Pharmacy, Sinai University, Al-Arish, 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

Four simple, accurate, and precise

spectrophotometric methods were
used for simultaneous determination
of ofloxacin and bromfenac.

The methods were successfully
applied for ofloxacin and bromfenac
determination in laboratory prepared
mixtures and combined formulation.

The results obtained showing
satisfactory recoveries, precision and
repeatability.

Methods validation was performed
according to ICH guidelines.

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

Article history: Four simple, precise, accurate and validated spectrophotometric methods have been developed for the
Received 12 October 2021 simultaneous determination of ofloxacin (OFL) and bromfenac sodium (BROM). Firstly, first and second
Received in revised form 12 February 2022 derivative spectrophotometric methods (1D & 2D) using a zero-crossing technique utilizing 309.3 and
Accepted 20 February 2022
257.5 nm for OFL and 290.7 and 246.5 nm for BROM as optimum working wavelengths in a binary mix-
Available online 23 February 2022
ture, respectively. Secondly, the first derivative ratio spectrophotometric method (1DD) in which peak
amplitudes at 297.3 nm and 260.7 nm were chosen to simultaneously estimate OFL and BROM, respec-
Keywords:
tively. Thirdly, dual wavelength (DW) method based on two selected wavelengths for each drug in such a
UV VIS spectrophotometry
Ratio derivative
way that the difference in absorbance is zero for the second one. At wavelengths 296.4, 348.4 nm BROM
Derivative spectrophotometry has equal absorbance values, therefore, these two wavelengths have been used to determine OFL.
Ofloxacin Similarly, 271.7 nm and 313.1 nm were selected to determine BROM in the combined formulation.
Bromfenac Finally, the fourth method depends on ratio difference spectrophotometry (RDSM), in which the differ-
ence between amplitudes at 305.6 nm and 326.5 nm on the ratio spectrum of the mixture was directly
proportional to the concentration of OFL; independent of the interfering components. Similarly, the dif-
ference between amplitudes at 265.1 nm and 275.4 nm on the ratio spectrum was used for the determi-
nation of BROM.
The linearity was confirmed in the range of 4 – 18 mg/ml for OFL and BROM for the four methods. The
proposed methods were used to determine both drugs in their laboratory prepared mixture and com-
bined formulation with mean percentage recoveries of 99.41 ± 1.35% for OFL and 99.98 ± 1.30 % for
BROM in method (A). In method (B), the mean percentage recoveries were 101.70 ± 1.61% for OFL and
101.90 ± 1.45% for BROM. In method (C) OFL was 99.57 ± 1.61% and 100.90 ± 1.62% for BROM. Finally,
in method (D) the mean percentage recoveries were 99.37 ± 1.67% for OFL and 100.70 ± 1.59% for BROM.

⇑ Corresponding author.
E-mail address: totarek7@gmail.com (T.M. Elsayed).

https://doi.org/10.1016/j.saa.2022.121066
1386-1425/Ó 2022 Elsevier B.V. All rights reserved.
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al.
The developed methods were successfully employed for determination of OFL and BROM in laboratory
prepared mixtures and combined formulation showing satisfactory recoveries. Methods validation was
performed according to the International Conference on Harmonization (ICH) guidelines. The obtained
results conformed to the accepted ranges of recovery, precision and repeatability.
1. Introduction
Ofloxacin (OFL) is a quinolone antibiotic with rapid bactericidal
activity against a wide variety of bacteria, including the most com-
mon causes of postoperative endophthalmitis, Staphylococcus epi-
dermidis and Staphylococcus aureus. Compared to other
fluoroquinolones (ciprofloxacin and norfloxacin), topical ofloxacin
achieves significantly higher aqueous concentrations in nonin-
flamed eyes. It penetrates the cornea and achieves therapeutic
levels in the aqueous humor [1].
Bromfenac (BROM) is a brominated nonsteroidal anti-
inflammatory drug (NSAID) with strong in vitro anti-
inflammatory effect. It is indicated for treatment of pain and
inflammation associated with inflammatory eye disorders and
postoperative inflammation following cataract surgery. It can pen-
etrate all eye tissues and reach conjunctiva, cornea, lens, iris, ciliary
body, aqueous humor, choroid, retina and sclera. Therapeutically
effective aqueous humor levels can be maintained for up to 12 h
after application of a single drop of 0.1% bromfenac solution in
the eye [2]. A new sustained release ophthalmic dosage form com-
bining both drugs provide an advantage for the patient for postsur-
gical prophylaxis as it can increase patient compliance and reduce
the burden of repeated dosing.
A validated analytical method is required for determination of
drug content in dosage forms. No method was reported for simulta-
neous determination of OFL and BROM in a pharmaceutical dosage
form. Various methods can be developed including high-
performance liquid chromatography (HPLC) and spectrophotomet-
ric determination. Spectrophotometric methods are faster and less
costly compared to HPLC. Derivative spectrophotometry methods
do not require separation of studied compounds. In such methods,
the overlapped spectra of substances in a mixture are resolved by
the application of several derivative techniques, e.g., first and second
derivative spectrophotometry [3-8]. Salinas et al. developed a new
spectrophotometric method, which is based on the use of the first
derivative of the ratio spectra for resolving binary mixtures [9-11].
Also, different spectrophotometric methods were developed based
on two wavelength techniques using the absorbance or amplitude
at two wavelengths and recording the difference of the values at
these wavelengths to permit either resolution of the overlapped
spectra in order to help in the analysis of complex mixtures, or mea-
surement of the individual components in the mixtures with satis-
factory degree of accuracy and precision. Several methods could
be applied such as dual wavelength [12–14], ratio difference [9,15-
19], constant center [18], bivariate [20], ratio subtraction [21],
absorbance subtraction and amplitude modulation method [22,23].
The aim of this study is to develop a spectrophotometric method
for simultaneous determination of ofloxacin and bromfenac in ocu-
lar insert containing a combination of both drugs. The use of differ-
ent derivative spectrophotometry techniques was explored.
2. Experimental
2.1. Apparatus
Shimadzu UV–Visible spectrophotometer (1700 UV – Visible)
with UV Win PC software and equipped with 1-cm quartz cuvettes
2
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066
Ó 2022 Elsevier B.V. All rights reserved.
was used for all the absorbance measurements and treatment of
data.
2.2. Chemicals and reagents
Ofloxacin and bromfenac sodium were received as gifts from El
Qahera for Pharmaceutical & Chemical Industries and Orchidia
pharma, Egypt, respectively. Analytical grade methanol was pur-
chased from El-Gomahria for Chemical Industries, Egypt.
2.3. Preparation of standard and sample solutions
2.3.1. Preparation of stock standard solutions
Standard stock solutions of OFL or BROM at 1 mg/ml concentra-
tion were prepared by dissolving 10 mg of each drug in 10 ml of
methanol/water (1:1) in a volumetric flask. With the appropriate
dilution of these stock solutions with the same solvent, working
standard solutions with concentrations 4, 6, 8, 10, 14 and 18 lg/
ml were prepared. Stock solutions and working standard solutions
were freshly prepared on each day.
2.3.2. Preparation of sample solutions
For the determination of recovery, variable amounts of binary
mixtures of BROM and OFL (5, 10 or 15 mg, each) were weighed
and mixed with a 90 mg of placebo in 10 ml volumetric flasks
for preparation of low, medium and high concentration sample
solutions. About 7 ml of methanol:water (1:1) was added. The mix-
ture was then sonicated for three minutes, and the volume was
completed to the mark with the same diluent after the flask was
let to cool down. It was then filtered with a 0.45 lm syringe filter.
2 ml of the filtrate was diluted to 10 ml with the same solvent.
Then 400 lL was diluted to 10 ml with the same solvent. The pre-
pared sample solutions have nominal concentrations of 4, 8 and
16 lg/ml. The placebo is a mixture of equal amounts of car-
boxymethyl cellulose, hydroxypropyl methyl cellulose and
polyvinylpyrrolidone.
2.4. Procedures
2.4.1. Construction of the calibration curves
Different aliquots of both OFL and BROM standard solution
(1 mg/ml) were transferred to 10-ml volumetric flasks and com-
pleted to volume with methanol/water solvent (1:1) to prepare
solutions with concentrations ranging from (4 – 18 mg/ml). The
absorption spectra (from 200 to 400 nm) of these solutions were
recorded using methanol/water as blank and stored in the
computer.
2.4.2. First and second derivative spectrophotometric method (1D and
2
D)
The first and second derivative corresponding to each spectrum
of studied drugs (D k = 4 nm) was recorded. The amplitude values
were measured at 309.3 and 257.5 nm for OFL and at 290.7 and
246.5 nm for BROM, respectively.
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al.
2.4.3. First derivative ratio spectrophotometric method (1DD)
The previously scanned spectra of OFL in the range of 4 – 18 mg/
ml were divided by a standard spectrum of 18 mg/ml BROM. Simi-
larly, the zero-order spectra of the prepared solutions of BROM in
the range of 4 – 18 mg/ml were divided by a standard spectrum
of 18 mg/ml OFL and the first derivative of the ratio spectra (1DD)
were then obtained with D k = 4 nm and scaling factor = 1. Calibra-
tion curves were constructed by plotting the peak amplitude at
297.3 and 260.7 nm versus the corresponding concentrations of
OFL and BROM, respectively, and the regression parameters were
calculated.
2.4.4. Dual wavelength method (DW)
The quantitative determination of OFL in the binary mixture
was done by calculation the absorbance difference between
296.4 and 348.4 nm. The difference in absorbance values of BROM
at these wavelengths shows zero value. Calibration curve was con-
structed by plotting the absorbance difference between 296.4 and
348.4 nm versus the corresponding concentrations of OFL and the
regression parameters were calculated. The quantitative determi-
nation of BROM in the binary mixture was done similarly by calcu-
lation of the absorbance difference between 271.7 and 313.1 nm.
The difference in absorbance values of OFL at these wavelengths
shows zero value. Calibration curve was constructed by plotting
the absorbance at zero-order versus the corresponding concentra-
tions of BROM and the regression parameters were calculated.
2.4.5. Ratio difference spectrophotometric method (RD)
From the ratio spectra obtained in section 2.4.3 (First derivative
ratio spectrophotometric method) the difference in peak ampli-
tudes (DP) was measured at 305.6 and 326.5 nm for OFL and at
265.1 and 275.4 for BROM, then, calibration curves were con-
structed by the difference between two different peak amplitudes
versus drug concentrations, and the regression parameters were
calculated.
2.4.6. Laboratory prepared mixtures
Mixtures containing different ratios of OFL and BROM were pre-
pared, and the absorption spectra of the laboratory prepared mix-
tures were recorded. Then the procedures were completed as
described in section 2.4.1 (Construction of the calibration curves).
3. Results and discussion
3.1. Derivative spectrophotometry (1D and 2D methods)
It is obvious that the simultaneous determination of OFL and
BROM is not possible by direct measurement of absorbance signals
since there is a marked overlap between the absorption spectra of
the two drugs, as shown by Fig. 1. The first and second derivative
spectrophotometry has been extensively used in the determination
of drugs in multicomponent systems having overlapping spectra.
This eliminates interference from matrix formulation by using
the zero-crossing techniques [8]. Hence, these two methods were
investigated for the possibility of simultaneous determination of
OFL and BROM in modified release ocular insert.
3.1.1. Selection of Dk and scaling factor (SF)
Initially, derivative spectra were obtained using different values
of Dk and scaling factor (SF). The obtained values shown in Table 1
indicate that in 1D, Dk = 4 nm and SF = 1 for both drugs and in 2D,
Dk = 4 nm and SF = 20 for both drugs, resulted in high peak ampli-
tude and highly smooth spectra with maximum sensitivity [21].
3
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066
3.1.2. Selection of suitable wavelengths
The first and second derivative spectra of several concentrations
of OFL and BROM were overlapped to determine their zero crossing
wavelengths. For the first derivative spectra, the zero crossing
points were 249.5 nm and 309.3 nm for OFL and 255.3 nm and
290.7 nm for BROM. While for the second derivative spectra, they
were 228.6 nm, 241.3 nm, 257.5 nm and 289.0 nm for OFL and
246.5 and 249.9 nm for BROM (Figs. 2, 3, 4 and 5). Out of these zero
crossing points, 309.3 nm and 257.5 nm for OFL and 290.7 and
246.5 nm for BROM were chosen as optimum working wave-
lengths for the simultaneous determination of OFL and BROM in
a binary mixture by first and second derivative methods. At these
wavelengths, the absolute value of the total derivative showed the
best linear response to the analyte concentration, not affected by
the concentration of any other component, and gives a near-zero
intercept on the ordinary axis of the calibration curve.
At these wavelengths, the values of mean recoveries and the
corresponding relative standard deviations and coefficients of vari-
ation of each drug from their prepared mixtures are shown in
Table 2. Following ICH guidelines [24], the limit of detection
(LOD) and limit of quantification (LOQ) were determined by 3.3
r/s and 10 r/s criteria, respectively; where r is the standard devi-
ation of the analytical signal and s is the slope of the corresponding
calibration curve. Beer’s law was obeyed in concentration ranges 4
– 18 mg/ml for OFL and 4 – 18 mg/ml for BROM.
3.2. First derivative of ratio spectra method (1DD)
Derivation of the ratio spectra for resolving binary mixtures
allows the use of the wavelength of highest value of analytical sig-
nals with several maxima and minima, which permits the determi-
nation of drugs in presence of other compounds which may
interfere in the analysis [19]. This method permits the use of the
wavelength of greatest sensitivity (a maximum or a minimum)
as the signal of measurement. Moreover, the presence of a several
of maxima and minima is another advantage as these wavelengths
give an opportunity for the determination of active compounds in
the presence of other possibly interfering compounds [15]. Hence,
ratio-spectra derivative spectrophotometric method is another
useful technique for the estimation of drugs in their mixtures.
Investigation of the main instrumental parameters affecting the
shape of derivative spectra was performed. These parameters
include divisors concentration, smoothing factor and working
wavelength. They must be optimized in order to obtain suitable
signal-to-noise ratio, good selectivity and high sensitivity.
3.2.1. Effect of divisor concentration
To obtain the most suitable concentration used as a divisor, the
spectra of the working standard solutions of OFL (4 – 18 mg/ml)
were divided by the stored spectrum of working standard solutions
of BROM with different concentrations (6, 10 and 18 mg/ml) as
lower, medium and high concentrations of divisor. Similarly, the
spectra of the working standard solutions of BROM (4 – 18 mg/
ml) were divided by the stored spectrum of working standard solu-
tions of OFL with different concentrations (6, 10 and 18 mg/ml). The
concentration of 18 mg/ml was chosen for both OFL and BROM as a
convenient divisor concentration regarding average recovery per-
cent [25], where it showed a good signal-to-noise ratio, good slope
and intercept values and more repeatable results.
3.2.2. Selection of wavelength
The spectra of solutions containing OFL and BROM binary mix-
tures were divided by the spectrum of 18 mg/ml BROM standard
solution in order to obtain the OFL ratio spectra (Fig. 6a). The first
derivatives of the obtained ratio spectra of both drugs were applied
with Dk = 4.0 nm and SF = 1.0. For the 1st derivative of OFL ratio
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al.
Fig. 1. Zero order absorption spectra for 18 mg/ml of OFL (a.) and 18 mg/ml of BROM (b) in methanol–water.
Table 1
Optimum Dk and SF Values for 1st and 2nd Derivative Spectra
Derivative Dk
1
D 4
2
D 4
spectra, there was one minimum at 312.9 nm and one maximum at
297.3 nm (Fig. 6b). Similarly, the spectra of solutions containing
OFL and BROM mixtures were divided by the spectrum of 18 mg/
ml OFL standard solution in order to obtain the BROM ratio spectra
(Fig. 7a), 1st derivative of BROM ratio spectra was obtained, there
were two minima at 244.7 and 272.2 nm and two maxima at 236.2
and 260.7 nm (Fig. 7b). It was observed that from 320 to 400 nm,
Fig. 2. First derivative (1D) spectra for OFL (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) and 18 mg/ml of BROM (dotted line) in methanol–water.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066
the noise increases and affects the amplitude of selected analytical
wavelengths, consequently, the wavelength scale of (Figs. 7a and
SF 7b) was cut after 320 nm to make the analytical wavelengths
1
amplitude clearer. The wavelengths 297.3 nm for OFL and
20 260.7 nm for BROM were chosen as the optimum working wave-
lengths for the simultaneous determination of OFL and BROM in
their binary mixture, where they have the most suitable values
of the regression equations’ parameters involving better linearity
range, best slope and intercept values with respect to sensitivity.
(Table 2).
3.3. Dual wavelength method
With the application of the dual wavelength method, it can be
made possible to determine each of OFL and BROM separately in
4
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066

Fig. 3. First derivative (1D) spectra for BROM (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) and 18 mg/ml of OFL (dotted line) in methanol–water.

Fig. 4. Second derivative (2D) spectra for OFL (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) and 18 mg/ml of BROM (dotted line) in methanol–water.

Fig. 5. Second derivative (2D) spectra for BROM (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) and 18 mg/ml of OFL (dotted line) in methanol–water.

their binary mixture, even with the overlap of their UV-absorption
spectra. In this method, two wavelengths are selected, where the
interfering component shows the same absorbance value while
the component of interest shows significant difference in absor-
bance with concentration. At these two points on the spectra, the
absorbance difference is directly proportional to the component
of interest, independent of the interfering component [14].
3.3.1. Selection of wavelength
The wavelengths evaluated were 255.1, 277.3 nm and 296.4,
348.4 nm for OFL and 236.7, 273.8 nm and 271.7, 313.1 nm for
BROM. For determination of OFL, the absorbance values at 296.4
and 348.4 nm (where different BROM concentrations has the same
absorbance) gave the best selectivity. While for BROM, the absor-
bance values at 271.7, 313.1 nm gave the best results.

5
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066

Table 2
Statistical parameters of the calibration graphs for the determination of OFL and BROM by derivative and ratio derivative spectrophotometry.

Parameters OFL BROM

First derivative Second derivative Ratio derivative First derivative Second derivative Ratio derivative
1 2 1 1 2 1
D 309.3 D 257.5 DD297.3 D 290.7 D 246.5 DD260.7
Linearity range (mg/ml) 4–18 4–18 4–18 4–18 4–18 4–18
Slope (b)a 0.00138 0.00529 0.05121 0.00149 0.0024 0.0019
Intercepta 0.00104 5.71 E-4 0.01128  8.67 E-4 5.06 E-4 9.47 E-4
Correlation coefficient (R2) 0.9995 0.9991 0.9992 0.9997 0.9991 0.9992
LOD (mg/ml)b 0.65 0.88 0.86 0.5 0.90 0.57
LOQ (mg/ml)c 1.98 2.66 2.62 1.51 2.74 1.73
Accuracy (Mean R % ± RSD%) 99.41 ± 1.35 99.10 ± 1.69 101.70 ± 1.61 99.98 ± 1.30 100.53 ± 1.72 101.90 ± 1.45

Fig. 6a. Ratio spectra of different concentrations of OFL (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) divided by zero-order absorption spectrum of BROM (18 mg/ml) (dotted line).

Fig. 6b. First derivative ratio spectra of OFL (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) using BROM (18 mg/ml) (dotted line) as a divisor.

Fig. 7a. Ratio spectra of different concentrations of BROM (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) divided by zero-order absorption spectrum of OFL (18 mg/ml) (dotted line).

6
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al.
Fig. 7b. First derivative ratio spectra of BROM (4, 6, 8, 10, 14 and 18 mg/ml) (solid lines) using OFL (18 mg/ml) (dotted line) as a divisor.
3.4. Ratio difference (RD) method
In the ratio difference spectrophotometric method, the ampli-
tude difference between two points on the ratio spectra of a mix-
ture is directly proportional to the concentration of the
component of interest; independent of the interfering component.
For a mixture of the two drugs (X) and (Y), X can be determined by
dividing the spectrum of the mixture by a known concentration of
Y as a divisor (Y-). The division will give a new curve that
represents
ðX þ YÞ=Y ¼ X=Y þ Y=Y ¼ X=Y þ constant
By selecting two wavelengths (l1 and l2) on the obtained ratio
spectrum and subtracting the amplitudes at these two points, the
constant Y/Y- will be cancelled along with any other instrumental
error or any interference from the sample matrix. Suppose the
amplitudes at two selected wavelengths are P1 and P2 at l1 and
l2, respectively; by subtracting the two amplitudes, the interfering
substance Y show no interference; then,
    lytical methods.
P1  P2 ¼ ðX= Y Þ1 þ constant  ðX=Y Þ2 þ constant
¼ ðX= Y Þ1  ðX=Y Þ2
The concentration of X is calculated by using the regression
equation representing the linear relationship between the differ-
ences of ratio spectra amplitudes at the two selected wavelengths
versus the corresponding concentration of drug (X). Similarly, Y
could be determined by the same procedure using a known con-
centration of X as a divisor X- [13].
3.4.1. Selection of wavelength
This is a two-step method where divisors are chosen as dis-
cussed in method 2 (Fig. 6a), followed by the selection of the wave-
lengths at which measurements are recorded (analytical
wavelengths). They are two wavelengths that show dissimilar
amplitudes in the ratio spectrum and excellent linearity at each
of them independently. The wavelength pairs explored for OFL
are 305.6–326.5, 305.6–338.1, and 305.6–349.2. The pair 305.6–
326.5 nm gave the best results (Fig. 6a). While for BROM, the pairs
265.1–275.4 nm, 265.1–264.2 nm and 265.1–229.2 nm were
applied. The pair 265.1–275.4 nm gave the best results (Fig. 7a).
4. Validation of the proposed methods
4.1. Linearity and range
Calibration graphs were constructed by plotting the amplitudes
of the first and second derivative spectrophotometric (1D & 2D), the
7
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066
first derivative ratio spectrophotometric (1DD) and the ratio differ-
ence spectrophotometric methods or the difference of absorbance
of the dual wavelength method versus drug concentrations in mg/
ml. Linear relationships were obtained over the concentration
ranges stated in Tables 2 and 3 and the adherence of the calibration
curves to Beer’s law was verified by the high regression coeffi-
cients. The values of correlation coefficients were close to unity
indicating good linearity, the characteristic parameters for the con-
structed equations are summarized in Tables 2 and 3.
4.2. Accuracy/Recovery
The accuracy of the proposed methods was determined by
investigating the percentage recoveries at three concentration
levels (5, 10 and 15 mg/ml), for both OFL and BROM, each in tripli-
cate. The mean recoveries and the relative standard deviations
were calculated (Tables 2 and 3). The results showed that the per-
cent recovery values were within the range of 100 ± 2% with low
standard deviations indicating high accuracy of the proposed ana-
4.3. Precision
The repeatability of the analytical methods was evaluated by
assaying freshly prepared solutions in triplicates within one day
in the concentration range of 5 – 15 mg/ml for both OFL and BROM.
Inter-day precision was evaluated by assaying triplicates of freshly
prepared solutions for three successive days using the same con-
centration range for both drugs. The percentage relative standard
deviations (R.S.D.) did not exceed 1.34% (Tables 2 and 3) indicating
a high reproducibility of the results and the precision of the pro-
posed methods.
4.4. Selectivity
Method selectivity was evaluated by preparing different mix-
tures of the studied drugs at various concentrations within the lin-
earity range. The synthetic mixtures in presence of amounts of
carboxymethyl cellulose, hydroxypropyl methyl cellulose and
polyvinylpyrrolidone as excipients were analyzed according to
the procedures described under the proposed methods. Tables 2
and 3 indicate the high selectivity of the proposed methods for
determining the studied drugs in their mixture.
4.5. Limits of detection and quantitation
The limits of detection (LOD) and quantitation (LOQ) were cal-
culated as follows: LOQ = 10  r/s and LOD = 3.3  r/s, where r is
A.M. Taha, Ragab A.M. Said, I.S. Mousa et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 273 (2022) 121066

Table 3
Statistical parameters of the calibration graphs for the determination of OFL and BROM by Dual wavelength and Ratio Difference ratio spectrophotometry.

Parameters OFL BROM

Dual wavelength Difference ratio Dual wavelength Difference ratio
Linearity range (mg/ml) 4–18 4–18 4–18 4–18
Difference in absorbance Difference in amplitude Difference in absorbance Difference in amplitude
between 296.4 and 348.4 nm between 305.6 and 326.5 nm between 271.7 and 313.1 nm between 265.1 and 275.4 nm
Slope (b)a 0.05241 0.39593 0.02847 0.02354
Intercepta 2.408 E-4 0.0413 0.00506 0.00379
Correlation coefficient (R2) 0.9993 0.9991 0.9994 0.9993
LOD (mg/ml)b 0.77 0.90 0.75 0.77
LOQ (mg/ml)c 2.34 2.73 2.28 2.33
Accuracy (Mean R % ± RSD%) 99.57 ± 1.61 99.37 ± 1.67 100.90 ± 1.62 100.70 ± 1.59
a
Regression equation: y = a + bc, where y is the peak amplitude, a is the intercept, b is the slope and c is the concentration.
b
LOD, limit of detection.
c
LOQ, limit of quantification.

the standard deviation of response and s is the slope of the calibra-
tion curve. The values of LOD and LOQ shown in Tables 2 and 3
reveal methods sensitivity.
4.6. Robustness
The robustness of a method is its ability to remain unaffected by
small changes in parameters. Variation of temperature by ± 0.5 did
not have a significant effect on the absorbance or amplitudes of the
mentioned methods. Testing for robustness involved performing
the spectral measurements at ± 0.2 nm. The robustness was calcu-
lated as percentage recoveries and relative standard deviation as
shown in Tables 2 and 3. The results revealed that the methods
gave robust results (RSD < 2).
5. Conclusion
The developed derivative spectrophotometry, first derivative of
ratio spectra method, dual wavelength and ratio difference meth-
ods have been successfully applied for simultaneous determination
of OFL and BROM in a mixture. They were found to be rapid, sim-
ple, sensitive and accurate. Once the equations were analyzed, they
require only measuring the absorbance values of the sample solu-
tion at the selected wavelengths followed by a few simple calcula-
tions. The suggested methods were completely validated showing
satisfactory data for all the method validation parameters tested.
Recovery studies indicated that practically there was no interfer-
ence from dosage form excipients, so these methods can be easily
and conveniently adopted for routine content determination of OFL
and BROM.
CRediT authorship contribution statement
Ashraf M. Taha: Methodology, Investigation, Data curation,
Writing – original draft. Ragab A.M. Said: Visualization, Methodol-
ogy, Writing – review & editing. Ibrahim S. Mousa: Conceptualiza-
tion. Tarek M. Elsayed: Conceptualization, Methodology, Writing –
review & editing, Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
8
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