Analytical method validation on simultaneous

estimation of Ozenoxacin and Benzoic acid in

pharmaceutical formulation

Acta Chromatographica AMARNATH REDDY RAMIREDDY1 and

DOI:
10.1556/1326.2022.01064
© 2022 The Author(s)
DILIP KUMAR BEHARA2p
1
Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University Anantapur
(JNTUA), Ananthapuramu, Andhra Pradesh, 515002, India
2
Chemical Engineering, JNTUA College of Engineering (Autonomous), Jawaharlal Nehru
Technological University Anantapur (JNTUA), Ananthapuramu, Andhra Pradesh, 515002, India

Received: May 17, 2022 • Accepted: August 11, 2022

ORIGINAL RESEARCH
PAPER
ABSTRACT
In this study, an accurate, simple, economical and precise Reversed-Phase High Pressure Liquid
Chromatography (RP-HPLC) method was developed for the simultaneous estimation of Ozenoxacin
and Benzoic Acid in a pharmaceutical cream formulation, according to the International Conference on
Harmonisation (ICH) guidelines. Chromatographic separation was achieved by gradient elution, on
RP-HPLC Instrument, equipped with column C8 (150 mm 3 4.6 mm, 5 μm particle size) using Ultra
Violet (UV) detector at 235 nm wavelength, by using Mobile Phase A: triethylamine, trifloroacetic acid
and water (1:1:1000) and Mobile Phase B: methanol and Diluent: water, acetonitrile and triethylamine
(500:500:1), at flow rate 0.8 mL min
1; injection volume of 20 μL; column oven temperature 45 8C and
sample temperature: 25 8C; Run time: 15 min. All the validation parameters were within the acceptance
criteria, as per ICH requirements, for Ozenoxacin and Benzoic acid. Consequently, this method has
found to be validated, simple, rapid and successfully applicable, to the simultaneous estimation of
Ozenoxacin and Benzoic acid by RP-HPLC, for routine analytical testing in quality control, with a run
time of 15 min and for future research studies. Forced degradation of Ozenoxacin cream 1% w/w
formulation was performed and found that validated method has stability indicating potential that
needs to be further studied.

KEYWORDS
Ozenoxacin, Benzoic acid, impetigo, RP-HPLC, stability indicating

INTRODUCTION
Ozenoxacin is a novel topical, non-fluorinated quinolone antimicrobial [1]. Ozenoxacin Cream
1% w/w was approved by United States Food and Drug Administration (FDA) in December
2017 to treat Impetigo caused by Staphylococcus aureus or Streptococcus pyogenes in pediatric
patients with two months of age and older as well as adults. Twice-daily application of 0.5 g of
Ozenoxacin Cream 1% w/w for five days is the recommended dosing regimen [2, 3].
Impetigo is the third most common skin disease in children, after dermatitis and viral
warts, with a peak incidence at 2–6 years of age [4, 5]. Lesions are highly contagious and can
p
spread rapidly by direct contact, through a family, nursery, or class. [6].
Corresponding author.
Ozenoxacin belongs to the quinolone group of antimicrobials and is the first non-fluo-
E-mail: dileepbh.chemengg@jntua.ac.
in rinated quinolone, which has a pyridinyl group at C7 [7].
Ozenoxacin appears as a white to pale - yellow crystalline solid material [8]. Ozenoxacin
chemical name is “1 - cyclopropyl - 8 - methyl - 7 - (5 - methyl - 6 - methylamino - pyridine - 3
- yl) - 4 - oxo 1,4 - dihydro - quinolone - 3 - carboxylic acid”, molecular formula is C21 H21 N3
O3 and molecular weight is 363.41 g mol
1. Ozenoxacin structure [9] is as shown in Fig. 1 (a).

Unauthenticated | Downloaded 09/10/22 02:56 AM UTC

2 Acta Chromatographica
Innovator formulation XEPITM existing in a cream form
and each gram of the cream contains 10 mg of Ozenoxacin
and the subsequent inactive excipients: Stearyl alcohol,
peglicol 5 oleate (Labrafil M1944CS: Oleoyl macrogol-6
glycerides), pegoxol 7 stearate (Tefose 63: PEG-6 Stearate
and Ethylene Glycol Palmito Stearate and PEG-32 Stearate),
octyl dodecanol, propylene glycol, Benzoic Acid and purified
water [2, 10, 11].
Benzoic Acid is used in Ozenoxacin Cream 1% w/w, as
an anti-microbial preservative. Benzoic Acid appears as a
white crystalline solid, has a molecular formula of C7H6O2,
has a molecular weight of 122.12 g mol
1 and structure [12]
is as shown in Fig. 1 (b).
Anti-Microbial Preservatives are the ingredients that are
commonly included in pharmaceutical, cosmetic, food and
beverages to inhibit initial microbial loads, quash their
propagation, or preclude new microbial contamination in a
preparation of multiple doses and extend the shelf life of
the product [13, 14]. Semisolid dosage forms generally
comprise of water or water-based solvents inside them.
Chances of microbial growth and contamination increases
with the presence of water. Therefore, Anti-Microbial Pre-
servatives are required to inhibit the microbial growth in
semisolid dosage forms due to any of these reasons [15].
Sufficient quantity of Benzoic Acid is required to inhibit
microbial growth in formulation. For this purpose, in the
same run, Ozenoxacin which is an Active Pharmaceutical
Ingredients and Benzoic Acid which is an anti-microbial
preservative are to be analyzed, for the estimation, in
finished product & stability stage of formulation by using
same analytical testing procedure.
Literature review showed that there are limited reports
available for the determination of Ozenoxacin alone that too
using complicated combination methods [16] liquid chro-
matography, mass spectrometry and electrospray ionization.
Literature review exhibited that there were numerous
publications for the estimation of benzoic acid separately
and along with other ingredients in food and pharmaceu-
tical formulation includes pharmacopeial (USP, BP,
Ph. Eur., IP), non-pharmacopeial and spectrophotometric
methods [17–24].
Fig. 1. Structure of (a) ozenoxacin and (b) benzoic acid
The literature survey disclosed that there is not one
analytical method available for the concurrent measurement
of Ozenoxacin and Benzoic Acid. Thus, it was certainly
important to develop a single run method of Reversed Phase
High Pressure Liquid Chromatography (RP-HPLC), which
will decrease cost and save time as compared to the pre-
vailing individual estimation methods. This research paper
reports a simple, selective RP-HPLC method for concurrent
measurement of Ozenoxacin and Benzoic Acid in cream
formulation, which was established, optimized and reported
for the first time.
MATERIALS AND METHODS
Materials
Ozenoxacin was collected from Optimus Drugs Private
Limited, Hyderabad, India as a gift sample and Benzoic Acid
was purchased from Ganesh Benzoplast, Thane, India. The
pharmaceutical dosage form of their combination OZANEX
cream (Ozenoxacin Cream 1% w/w market sample) was
purchased from Cipher Pharmaceuticals Inc., Canada. All
solvents used in this work were HPLC grade and purchased
from MERCK/RANKEM (India). All other materials used
were of analytical reagent grade.
Instrumentation
The chromatographic partition was executed on a RP-HPLC
system comprising the following: a Waters Alliance 2695
series system with 2489 Photo Diode Array detector,
equipped with quaternary pumps and an autosampler. Data
handling system was performed using Waters Empower
work station.
Development and optimization of chromatographic
conditions
Different aqueous and organic phases were used for best
partitioning of Ozenoxacin and Benzoic Acid to develop a
robust chromatographic method.
Unauthenticated | Downloaded 09/10/22 02:56 AM UTC
Acta Chromatographica 3

Table 1. Gradient Elution Program RESULTS AND DISCUSSION

Time (Minutes) Mobile Phase A Mobile Phase B
0 65 35 RP-HPLC method was developed in agreement with the
2.00 65 35 ICH guideline Q2 (R1) for the simultaneous estimation of
8.00 40 60 Ozenoxacin and Benzoic Acid in a pharmaceutical cream
10.00 40 60 formulation.
10.10 65 35
15.00 65 35 Development and optimization of chromatographic
conditions
Analytical method validation Different aqueous and organic phases, flow rates and
wavelengths were used for best separation of Ozenoxacin
The analytical method used for simultaneous measurement and Benzoic Acid to develop a robust chromatographic
of Ozenoxacin and Benzoic Acid was validated as per the method for the simultaneous measurement of Ozenoxacin
ICH guideline Q2 (R1) and the method validation param- and Benzoic Acid. The improved chromatographic envi-
eters [25] were: specificity, precision (repeatability and in- ronments were attained, when method development was
termediate precision), linearity, accuracy, range and introduced using an aqueous phase (A) comprising trie-
robustness. thylamine: orthophosphoric acid: water at the volume ratio
Forced degradation studies were performed to assess of 1:1:1000 (pH adjustment using orthophosphoric acid to
stability-indicating potential of the validated method. Filter 2.0 ± 0.05) and organic phase (B) comprising Methanol and
validation was also performed. Acetonitrile at the ratio of 700:300 (v/v) with a flow rate of
1.0 mL min
1, UV detector was set at wavelength of 230 nm;
Chromatographic conditions temperature of column oven maintained at 40 8C. Under
The chromatographic partitioning was accomplished using a these situations, elution for Ozenoxacin and Benzoic Acid
Hypersil BDS C8 column (150 mm 3 4.6 mm; 5 μm particle gave sharp peaks but peak shapes were observed irregular.
size). The total run time was 15 min carried out using To resolve this, finally, aqueous phase (A) containing trie-
gradient elution (Table 1) at the flow rate of 0.8 mL min
1. thylamine: trifluoroacetic acid: water in the volume ratio of
The column oven temperature was maintained at 45 8C. The 1:1:1000 and organic phase (B) containing methanol,
Ultraviolet/Visible (UV) detector wavelength was estab- measured at wavelength of 235 nm using UV detector; col-
lished at 235 nm and the injection volume was 20 μL. All the umn oven temperature 45 8C; flow rate of 0.8 mL min
1
determinations were performed at room temperature were used. These changes allowed very good separation of
(∼25 8C). Ozenoxacin and Benzoic Acid and resolution was found to
Preparation of Mobile Phase A: Triethylamine, tri- be greater than 4.0, sharp regular peak shapes were observed.
fluoroacetic acid and purified water were added at the vol- The effectiveness of a separation can be quantified by
ume ratio of 1:1:1000 respectively and mixed well. measuring the resolution. Resolution is a measure of the
Preparation of Mobile Phase B: Methanol. degree of separation between two adjacent signals, detector
Preparation of Diluent: Purified water, acetonitrile and responses, or, in the case of optical instruments, objects [26].
triethylamine were added at the volume ratio of 500:500:1 All of the system suitability parameters (theoretical plates,
respectively and mixed well. tailing factor, resolution and relative SD) were contented the
Preparation of Needle Wash: Purified water and FDA requirements [27]. The Typical chromatogram of
methanol were added at the ratio of 20:80 (v/v) respectively Ozenoxacin and Benzoic Acid Standard solution is shown
and mixed well. in Fig. 2.

Fig. 2. Typical chromatogram of ozenoxacin and benzoic acid standard solution

Unauthenticated | Downloaded 09/10/22 02:56 AM UTC

4 Acta Chromatographica

Analytical method validation to be 0.1% and 0.1% respectively (Acceptance Criteria: less
than 2.0%).
System suitability. To establish the system suitability, Specificity. Solutions of individual impurities, Ozenoxacin
standard solution of Ozenoxacin and Benzoic Acid was sample, Ozenoxacin Placebo and Spiked Solution were
prepared and injected five times. Results were given in prepared and analyzed. Results were summarized in Table 2.
Supplemental file (Table S1 & Table S2). Peak purity complies as per the Ozenoxacin and Benzoic
Theoretical plates of Ozenoxacin and Benzoic Acid peaks Acid peak requirement obtained from the chromatogram
in standard solution were found to be 3,308 and 7,921 of spiked sample solution. Benzoic Acid was well separated
respectively (Acceptance Criteria: more than 2,000). The from Ozenoxacin obtained from the chromatogram of
tailing factor was found to be 1.22 and 1.22 respectively spiked sample solution and all process related impurities
(Acceptance Criteria: not more than 2.0) for Ozenoxacin were well separated from Ozenoxacin and Benzoic Acid.
and Benzoic acid peaks in standard solution. The % Relative A typical chromatogram of the spiked sample is shown
SD (RSD) of the Ozenoxacin and Benzoic acid in five in Fig. 3.
replicate injections from standard solution was found to be Absolute difference between % Assay during specificity
0.1% and 0.1% respectively (Acceptance Criteria: less than was given in Supplemental file (Table S4). Specificity
2.0%). Resolution between Ozenoxacin and Benzoic acid Chromatograms were given in Supplemental file (Figure S1
peaks was found to be 4.23 (Acceptance Criteria: more than to Figure S11).
2.0). All of the system suitability parameters were contented
the FDA requirements [27]. Repeatability (Method precision). A sample solution of
Ozenoxacin Cream 1% w/w at working concentration for six
System precision. The standard solution was prepared and times (six individual sample preparations) was analyzed to
injected six times as per the method of analysis to establish determine the method precision. Results were given in
the system precision. Results were given in Supplemental file Supplemental file (Table S8).
(Table S3).
%RSD of the Ozenoxacin and Benzoic Acid peak areas of Ruggedness (Intermediate precision). A sample solution of
six replicate injections from standard preparation was found Ozenoxacin Cream 1% w/w at working concentration for six
times (six individual sample preparations) was analyzed to
Table 2. Specificity Results determine the ruggedness of the method using different
Compound Retention Time Purity Purity Peak analysts, different instruments, different reagents and
Name (Minutes) Angle Threshold Purity different columns on different days. Details of variations,
System Suitability Parameters & Areas for ruggedness and
Ozenoxacin 5.57 0.130 0.268 Pass
results of Method Precision & Intermediate Precision were
Benzoic Acid 6.98 0.357 1.114 Pass
Quinoline Ester 11.16 Not Applicable
given in Supplemental file (Table S5 to Table S8).
Impurity The % assay values obtained from six sample prepara-
Peak-1 tions (method precision/intermediate precision) for Oze-
Quinoline Ester 11.60 noxacin and Benzoic Acid were within specification
Impurity (95%–105%). The %RSD for % assay values obtained from
Peak-2 six sample preparations (method precision/intermediate
Acid Impurity 12.38 precision) for Ozenoxacin and Benzoic Acid was within
OZE Stage 1 12.92 specification (less than 2%). The overall %RSD for % assay
Impurity values obtained from replicate sample preparations
Blank No interference was observed at Ozenoxacin & (12 results) of method precision and intermediate precision
Placebo Solution Benzoic Acid retention time.
was within specification (less than 3.0%).

Fig. 3. Typical Chromatogram of Spiked sample

Unauthenticated | Downloaded 09/10/22 02:56 AM UTC

Acta Chromatographica 5

Table 3. Linearity data of Ozenoxacin and Benzoic Acid

Linearity of Ozenoxacin Linearity of Benzoic Acid
Linearity Level Concentration (μ/mL) Average Area (mAU) Concentration (μg mL
1) Average Area (mAU)
80% 32.1792 3,452,159 3.2140 375,574
90% 36.2016 3,886,263 3.6157 422,891
100% 40.2240 4,320,794 4.0174 471,607
110% 44.2464 4,795,229 4.4192 516,586
120% 48.2688 5,170,025 4.8209 568,692
Correlation coefficient 0.9995 0.9998
Coefficient of determination 0.9990 0.9996
Intercept
19,804
8,878
Slope 108,013 119,466
Regression equation (y 5 mx þ c) y 5 108,013 3 –19,804 y 5 119,466 3 –8,878
Y-Bias (%)
0.46
1.88
Residual Sum of Squares 1901692651.6 9395234.1

Linearity. Ozenoxacin and Benzoic Acid composite solu-
tions, with five concentrations of 80%, 90%, 100%, 110%,
120%, were prepared from the standard stock solution and
the experiments were performed in triplicate for each con-
centration level to determine the linearity. 80%, 90%, 100%,
110%, 120% are concentration levels relative to the label
claim/declared content. The linearity curves were acquired
between concentrations and peaks areas (mAU: milli
Ampere Units) of five different solutions of Ozenoxacin
and Benzoic Acid at concentration 32–48 μg mL
1 and
3.2–4.8 μg mL
1 respectively. Ozenoxacin and Benzoic Acid
linearity results were shown in Table 3.
Calibration curves of Ozenoxacin & Benzoic acid were
given in Supplemental file (Figure S12 & Figure S13).
Correlation coefficient values were not less than 0.999
and 0.999 for Ozenoxacin and Benzoic Acid respectively.
Y-Bias was found to be within ±2.0%. The relationships
between peak areas and concentrations were satisfactory
considering the calibration curve regression data.
Accuracy. Ozenoxacin and Benzoic Acid composite solu-
tions containing 80%, 100% and 120% of were analyzed to
determine the method’s accuracy. Each solution was pre-
pared individually in triplicate and analyzed to determine
recovery values by component recovery method, which
involves calculating the amount added (spiked) and the
amount measured (recovered). Results are given in Table 4.
The individual % of recovery was between 97.0% and
103.0%. The mean % of recovery was between 98.0% and
102.0%. The %RSD was less than 2% at each level.
Range. This method validation test was performed from the
lower concentration (80%) to upper concentration (120%)
of both Ozenoxacin and Benzoic Acid, which were used in
precision, accuracy and linearity. As per ICH requirements,
the results demonstrated that all system suitability and method
validation parameters were within the acceptance criteria [25].
Robustness. Flow rate and column oven temperature were
varied to establish robustness of the method. Analysis of

Table 4. Accuracy or Recovery data of Ozenoxacin and Benzoic Acid

Analyte Spike Level Amount added in μg mL
1 Amount found in μg mL
1 % Recovery Mean %RSD
OZENOXACIN 80% Level 32.11 32.44 101.0 101.0 0.0
32.11 32.43 101.0
32.11 32.42 101.0
100% Level 40.13 40.63 101.2 101.2 0.3
40.13 40.54 101.0
40.13 40.72 101.5
120% Level 48.16 48.50 100.7 100.7 0.0
48.16 48.50 100.7
48.16 48.51 100.7
BENZOIC ACID 80% Level 3.22 3.24 100.6 100.6 0.0
3.22 3.24 100.6
3.22 3.24 100.6
100% Level 4.03 4.07 101.0 101.2 0.3
4.03 4.08 101.2
4.03 4.09 101.5
120% Level 4.83 4.90 101.4 101.4 0.0
4.83 4.90 101.4
4.83 4.90 101.4

Unauthenticated | Downloaded 09/10/22 02:56 AM UTC

6 Acta Chromatographica

Table 5. Robustness data for Ozenoxacin and Benzoic Acid

Retention Time (Minutes) Assay (%w/w)
S. No. Condition Ozenoxacin Benzoic Acid Ozenoxacin Benzoic Acid
1 Actual 5.72 7.28 99.78 100.83
2 Low Column Temp. 5.91 7.44 100.16 100.64
3 High Column Temp. 5.54 7.12 99.99 100.75
4 Low Flow Rate 6.42 8.07 100.05 100.68
5 High Flow Rate 5.16 6.62 100.31 101.20

standard solution and sample solution was performed in
each varied condition. One parameter was changed while
keeping the other one unchanged from the actual parameter.
Retention time & Assay results are given in Table 5.
Parameters varied, System Suitability Parameters, Areas
observed during Robustness are given in Supplemental file
(Table S9 to Table S15).
The assay values of Ozenoxacin and Benzoic Acid were
within specification (95%–105%).
Bench top solution stability of standard solution and sam-
ple solutions. Standard solution and sample solution con-
taining Ozenoxacin and Benzoic Acid were kept at room
temperature. The solution stability was monitored by
analyzing standard solution and sample solution in the
initial and 48 h intervals.
It was observed that the standard solution and sample
solution were stable for 48 h. The difference in % assay be-
tween initial and solution stability samples was found to be
not more than ±2.0%. The similarity factor for standard was
found to be in the range of 0.98–1.02.
Summary results were given in Supplemental file
(Table S16).
Bench top stability of mobile phase. Mobile phase was kept
at room temperature and monitored by analyzing the
standard solution in the initial and 48 h intervals.
The Resolution between Ozenoxacin and benzoic acid
peaks was 4.41. No Haziness and particles were observed in
Mobile Phase up to 2 days.
Summary results were given in Supplemental file
(Table S17).
Filter validation. Two different filters namely, 0.45 μm
Nylon filter and 0.45 μm Polyvinylidene fluoride filter were
used for filter validation of the method. Sample solution was
prepared using the homogenous solution. Different portions
of sample solution were centrifuged, filtered and injected
into the HPLC system.
The difference between filtered samples observed to be
not more than 2%. The assay values of Ozenoxacin and
Benzoic Acid were within specification (95%–105%). Any
significant interference was not found to be between nylon
filter and Polyvinylidene fluoride filter.
Filter Details & Assay Results are given in Supplemental
file (Table S18 & Table S19).
Forced degradation study. To assess the stability-indicating
nature of the Assay by HPLC method in Ozenoxacin Cream
1% w/w, the sample has been stressed by acid, base,
hydrogen peroxide, aqueous hydrolysis, heat, humidity,
white fluorescent light, and UV light. The degraded samples
were injected into the chromatographic system and
analyzed. Results were summarized in Table 6 & Table 7.
Ozenoxacin in the cream formulation was found to be
stable during exposure to humidity, white fluorescent light,
UV light conditions, water hydrolysis, thermal condition,
acid hydrolysis and base hydrolysis. Drastic degradation
was observed in oxidation conditions. The Ozenoxacin peak
was pure and spectrally homogenous in all the above
degradation conditions, proving that degrading peaks were
not co-eluting with Ozenoxacin peak.
Benzoic Acid in the cream formulation was found to be
stable during exposure to humidity, white fluorescent light,
UV light conditions, water hydrolysis, thermal condition,

Table 6. Ozenoxacin Parameters observed during Forced degradation Study

S. No. Condition Assay (% w/w) Purity Angle Purity Threshold Peak Purity
1 Control Sample 99.50 0.148 0.253 Pass
2 Acid Sample (1N HCl, 24 h at 60 8C) 98.88 0.142 0.252 Pass
3 Base Sample (1N NaOH, 24 h at 60 8C) 96.78 0.162 0.249 Pass
4 Water Sample (24 h at 60 8C) 99.76 0.143 0.257 Pass
5 Peroxide Sample (1% H2O2, 15 min at 60 8C) 74.18 0.413 0.752 Pass
6 Thermal Degradation Sample (60 8C for 48 h) 98.71 0.149 0.259 Pass
7 White Fluorescent Exposure 99.84 0.161 0.260 Pass
Sample (1.2 Million Lux Hours)
8 UV light Exposure Sample (200-W hours/square meter) 98.13 0.156 0.257 Pass
9 90% RH Humidity Exposure Sample 100.00 0.159 0.260 Pass

Unauthenticated | Downloaded 09/10/22 02:56 AM UTC

Acta Chromatographica 7

Table 7. Benzoic Acid Parameters observed during forced degradation study

S. No. Condition Assay (% w/w) Purity Angle Purity Threshold Peak Purity
1 Control Sample 99.81 0.333 0.866 Pass
2 Acid Sample (1N HCl, 24 h at 60 8C) 99.18 0.370 0.855 Pass
3 Base Sample (1N NaOH, 24 h at 60 8C) 99.17 0.293 0.546 Pass
4 Water Sample (24 h at 60 8C) 100.05 0.357 0.977 Pass
5 Peroxide Sample (1% H2O2, 15 min at 60 8C) 99.15 2.515 6.693 Pass
6 Thermal Degradation Sample (60 8C for 48 h) 98.84 0.378 0.993 Pass
7 White Fluorescent Exposure Sample (1.2 Million Lux Hours) 100.28 0.384 0.993 Pass
8 UV light Exposure Sample (200-W hours/square meter) 98.02 0.401 0.944 Pass
9 90% RH Humidity Exposure Sample 99.40 0.371 0.968 Pass

acid hydrolysis, base hydrolysis and oxidation conditions.
The Benzoic Acid peak was pure and spectrally homogenous
in all the above degradation conditions, proving that
degrading peaks were not co-eluting with Benzoic Acid peak.
CONCLUSION
The results obtained in this study demonstrated that the
concurrent measurement of Ozenoxacin and Benzoic Acid,
in pharmaceutical cream formulation using RP-HPLC by
gradient elution was specific, accurate, precise, linear and
rugged. The method was found robust at column oven
temperature variation and flow rate variation. Solution
stability established and found standard solution and
sample solutions are stable up to 48 h at room temperature.
Mobile phase stability established and found stable up to
48 h at room temperature. Filter validation established and
found standard solution and sample solutions are suitable in
0.45 μm Nylon filter and Polyvinylidene fluoride filter.
Forced degradation of Ozenoxacin cream 1% w/w formu-
lation was performed and found that validated method has
stability indicating potential that needs to be further studied.
The method was validated according to the ICH guide-
line and presented equivalent outcomes that demonstrate
that this method will be valuable for concurrent measure-
ment of Ozenoxacin and Benzoic Acid by RP-HPLC, which
was established, optimized and reported first time. This
method can be used for routine analysis of Ozenoxacin and
Benzoic Acid in any form of pharmaceutical and throughout
the stability study with a run time of just 15 min. This
unique method can play a dynamic role for the development
of other studies (e.g., In Vitro Release Testing and In Vitro
Permeation Testing of Ozenoxacin) for the quantitative
analysis for future use.
DECLARATIONS
Funding: The authors declare that no funds, grants, or other
support were received during the preparation of this
manuscript.
Competing interests: The authors have no relevant financial
or non-financial interests to disclose.
Conflict of interest section: The authors declare that no po-
tential conflicts of interest for this article’s research,
authorship, and/or publication.
Author contributions: Amarnath Reddy Ramireddy: The
acquisition, analysis, interpretation of data for the work and
drafting of the work. Dilip Kumar Behara: The conception,
design of the work and final approval of the version to be
published.
Ethical approval: This article does not contain any studies
with human participants or animals performed by any of the
authors.
ACKNOWLEDGEMENTS
Authors are thankful to Optimus Pharma Private Limited,
Hyderabad for providing facilities to perform this research.
One of the authors Amarnath Reddy Ramireddy is thankful
to the Jawaharlal Nehru Technological University, Ananta-
pur, for enrolling as a research scholar.
SUPPLEMENTARY MATERIALS
Supplementary data to this article can be found online at
https://doi.org/10.1556/1326.2020.01064.
REFERENCES
1. Wren, C.; Bell, E.; Eiland, L. S. Ozenoxacin: a novel topical qui-
nolone for impetigo. Ann. Pharmacother. 2018, 52(12), 1233–7.
https://doi.org/10.1177/1060028018786510.
2. https://www.xepicream.com/resources/full-prescribing-information
(accessed Mar 13, 2022).
3. Rosen, T.; Albareda, N.; Rosenberg, N.; García Alonso, F.; Roth, S.;
Zsolt, I. Efficacy and safety of Ozenoxacin cream for treatment of
adult and pediatric patients with impetigo: a randomized clinical
trial. JAMA Dermatol. 2018, 154(7), 806–13. https://doi.org/10.
1001/jamadermatol.2018.1103.
4. Dagan, R. Impetigo in childhood: changing epidemiology and new
treatments. Pediatr. Ann. 1993, 22, 235–40. https://doi.org/10.3928/
0090-4481-19930401-07.

Unauthenticated | Downloaded 09/10/22 02:56 AM UTC

8 Acta Chromatographica
5. Bruijnzeels, M. A.; van Suijlekom-Smit, L. W. A.; van der Velden, J.;
van der Wouden, J. C. The Child in General Practice. Dutch Na-
tional Survey of Morbidity and Interventions in General Practice;
Erasmus University Rotterdam: Rotterdam, 1993.
6. Hlady, W. G.; Middaugh, J. P. An epidemic of bullous impetigo in a
newborn nursery due to Staphylococcus aureus: epidemiology and
control measures. Alaska Med. 1986, 28, 99–103.
7. López, Y.; Tato, M.; Espinal, P.; Garcia-Alonso, F.; Gargallo-Viola, D.;
Cantón, R.; Vila, J. In vitro activity of Ozenoxacin against quinolone-
susceptible and quinolone-resistant gram-positive bacteria. Anti-
microb. Agents Chemother. 2013, 57(12), 6389–92. https://doi.org/10.
1128/AAC.01509-13.
8. https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/
208945lbl.pdf (accessed Apr 21, 2022).
9. https://pubchem.ncbi.nlm.nih.gov/compound/Ozenoxacin
(accessed Mar 13, 2022).
10. https://www.gattefosse.com/pharmaceuticals-products/labrafil-m-
1944-cs (accessed Apr 21, 2022).
11. https://www.gattefosse.com/pharmaceuticals-products/tefose-63
(accessed Apr 21, 2022).
12. https://pubchem.ncbi.nlm.nih.gov/compound/Benzoic-acid
(accessed Apr 21, 2022).
13. Sam, T.; Ernest, T. B.; Walsh, J.; Williams, J. L. European Paediatric
Formulation Initiative (EuPFI). A benefit/risk approach towards
selecting appropriate pharmaceutical dosage forms - an application
for paediatric dosage form selection. Int. J. Pharm. 2012, 435(2),
115–23. https://doi.org:10.1016/j.ijpharm.2012.05.024 (Epub 2012
May 22. PMID: 22626885).
14. JibenRoy (2011). The stability of medicines. An introduction to
pharmaceutical sciences - production, chemistry, techniques and
technology. p. 170.
15. Kar, M.; Chourasiya, Y.; Maheshwari, R.; Tekade, R. K. Current
developments in excipient science: implication of quantitative
selection of each excipient in product development. In Basic
Fundamentals of Drug Delivery. A Volume in Advances in Phar-
maceutical Product Development and Research; 2019, pp 29–83.
16. Santos, B.; Ortiz, J.; Gropper, S. In vitro percutaneous absorption
and metabolism of Ozenoxacin in excised human skin. Future
Microbiol. 2014, 9(8s), S3–9. https://doi.org/10.2217/fmb.14.81.
17. Gupta, V. D. Quantitative determination of benzoic acid and salicylic
acid in ointments by high-pressure liquid chromatography. J. Pharm.
Sci. 1977, 66(1), 110–1. https://doi.org/10.1002/jps.2600660128.
Open Access. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://
creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the
original author and source are credited, a link to the CC License is provided, and changes – if any – are indicated.
18. Cheng, Z.; Qin, R.; Liu, J.; Deng, X.; Qiu, H.; Jia, Z.; Su, X. Rapid
determination for benzoic acid, sorbic acid, phenyllactic acid,
phenylalanine, and saccharin sodium in vinegar by high-perfor-
mance liquid chromatography–UV. Food Anal. Method. 2020, 13,
1673–80. https://doi.org/10.1007/s12161-020-01784-6.
19. Kubota, K.; Horai, Y.; Kushida, K.; Ishizaki, T. Determination
of benzoic acid and hippuric acid in human plasma and urine
by high-performance liquid chromatography. J. Chromatogr.
1988, 425(1), 67–75. https://doi.org:10.1016/0378-4347(88)
80007-0.
20. Heinanen, M.; Barbas, C. Validation of an HPLC method for the
quantification of ambroxol hydrochloride and benzoic acid in a
syrup as pharmaceutical form stress test for stability evaluation.
J. Pharm. Biomed. Anal. 2001, 24(5–6), 1005–10. https://doi.org/
10.1016/s0731-7085(00)00533-1.
21. Kulikov, A. U.; Verushkin, A. G. Simultaneous determination of
paracetamol, caffeine, guaifenesin and preservatives in syrups by
micellar LC. Chromatographia 2008, 67, 347–55. https://doi.org/10.
1365/s10337-007-0510-5.
22. Mansour, A. M.; Ibrahiem, M. M. Simultaneous determination of
azelaic and benzoic acids in topical preparations by liquid chro-
matography. Chromatographia 2002, 55, 435–7. https://doi.org/10.
1007/BF02492273.
23. Kokya, T. A.; Farhadi, K.; Kalhori, A. A. Optimized dispersive
liquid–liquid microextraction and determination of sorbic acid and
benzoic acid in beverage samples by gas chromatography. Food
Anal. Methods 2012, 5, 351–8. https://doi.org/10.1007/s12161-011-
9245-x.
24. Zhang, X.; Xu, S.; Sun, Y.; Wang, Y.; Wang, C. Simultaneous
determination of benzoic acid and sorbic acid in food products by
CE after on-line preconcentration by dynamic pH junction. Chro-
matographia 2011, 73, 1217–21. https://doi.org/10.1007/s10337-
011-2009-3.
25. Validation of analytical procedures: text and methodology Q2(R1).
https://www.ich.org/page/quality-guidelines (accessed Mar 13, 2022).
26. https://www.chromatographyonline.com/view/chromatography-
fundamentals-part-viii-meaning-and-significance-chromatographic-
resolution (accessed Aug 09, 2022).
27. Reviewer Guidance Validation of chromatographic methods.
https://www.fda.gov/regulatory-information/search-fda-guidance-
documents/reviewer-guidance-validation-chromatographic-
methods (accessed Mar 13, 2022).
Unauthenticated | Downloaded 09/10/22 02:56 AM UTC