Development of stability indicating HPLC method for the separation and validation of enantiomers of miconazole

Article abstract of DOI:10.1002/chir.22858

A selective and sensitive stability indicting HPLC method was developed for the analysis of enantiomers of miconazole. For this purpose, six different polysaccharide-based chiral columns were evaluated. Optimization was performed using several polar organic and alcohol-hydrocarbon mobile phases. As a result of optimization studies, the analysis was carried out using Lux Cellulose-3, methanol as a mobile phase at a flow rate of 1?mL·min^?1, and the detection wavelength was arranged to 230?nm. Developed method has been fully validated according to International Council on Harmonization guidelines. Method was found linear in the concentration range of 1 to 200?μg·mL^?1. Coefficient of determination (R²) was calculated as 0.9996, intraday precision of the method was found with the RSD% of 0.56, and the recovery of the method was calculated close to 100%. Furthermore, some other validation parameters like specificity, selectivity, LOD, and LOQ were also investigated. Stability indicating capability of this method was shown by forced degradation studies, and the run time for each analysis was less than 6?minutes. As a result, simple, fast, reliable HPLC method was developed for the separation and determination of the enantiomers of miconazole. Applicability of the developed method was shown with the application of marketed pharmaceutical preparations.

Full text of DOI:10.1002/chir.22858

Received: 29 January 2018
DOI: 10.1002/chir.22858
Revised: 25 February 2018
Accepted: 26 February 2018
R E G U L A R A R T I C L E
Development of stability indicating HPLC method for the
separation and validation of enantiomers of miconazole
1
1
2
1
Nadia Bounoua | Khaled Sekkoum | Mehmet Gumustas
| Nasser Belboukhari |
3
Sibel A. Ozkan
1
Bioactive Molecules and Chiral
Abstract
Separation Laboratory, Faculty of Science
and Technology, University of Béchar,
Béchar, Algeria
A selective and sensitive stability indicting HPLC method was developed for
the analysis of enantiomers of miconazole. For this purpose, six different poly-
saccharide‐based chiral columns were evaluated. Optimization was performed
using several polar organic and alcohol‐hydrocarbon mobile phases. As a
result of optimization studies, the analysis was carried out using Lux Cellu-
2
Department of Forensic Toxicology,
Institute of Forensic Sciences, Ankara
University, Ankara, Turkey
3
Department of Analytical Chemistry,
−
1
Faculty of Pharmacy, Ankara University,
Ankara, Turkey
lose‐3, methanol as a mobile phase at a flow rate of 1 mL·min , and the
detection wavelength was arranged to 230 nm. Developed method has been
fully validated according to International Council on Harmonization guide-
lines. Method was found linear in the concentration range of 1 to
Correspondence
Mehmet Gumustas, Department of
Forensic Toxicology, Institute of Forensic
Sciences, Ankara University, Ankara,
Turkey
−
1
2
2
00 μg·mL . Coefficient of determination (R ) was calculated as 0.9996, intra-
day precision of the method was found with the RSD% of 0.56, and the recov-
ery of the method was calculated close to 100%. Furthermore, some other
validation parameters like specificity, selectivity, LOD, and LOQ were also
investigated. Stability indicating capability of this method was shown by
forced degradation studies, and the run time for each analysis was less than
Email: mgumustas@hotmail.com;
mgumustas@ankara.edu.tr
Sibel A. Ozkan, Department of Analytical
Chemistry, Faculty of Pharmacy, Ankara
University, Ankara, Turkey.
Email: ozkan@pharmacy.ankara.edu.tr
6
minutes. As a result, simple, fast, reliable HPLC method was developed
for the separation and determination of the enantiomers of miconazole. Appli-
cability of the developed method was shown with the application of marketed
pharmaceutical preparations.
KEYWORDS
chiral separation, enantiomer, HPLC, miconazole, polysaccharide‐based chiral column, validation
1
1
| INTRODUCTION
cell membrane. On the other hand, enantiomer of (R)
miconazole and the racemic mixture have similar antifun-
1
Miconazole (MIC), 1‐[(2RS)‐2‐[(2,4‐dichlorobenzyl)oxy]‐
‐(2,4‐dichlorophenyl)ethyl]‐1H‐imidazole (Figure 1), is
gal properties.
2
Miconazole has a number of known impurities, which
are close in structure to the main compound. The high‐
performance liquid chromatography (HPLC) method for
the determination of the related substances is described
in the European Pharmacopeia (Ph.Eur), a set of texts
concerning the qualitative and quantitative composition
of pharmaceutical preparations and the tests to be carried
an imidazole antifungal agent. It is commonly applied
topically to fungal infections as it has strong activity
against dermatophytes and Candida alibicans. The mech-
anism of MIC acted by inhibiting the synthesis of ergos-
terol, a critical component of fungal cell membranes,
and creating a sterol, which is toxic to the yeast species
Chirality. 2018;1–9.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
BOUNOUA ET AL.
>
In these modalities, chiral separation can be
achieved by either changing the stationary phases and/
or adding the chiral selectors into the mobile phase.
The former approach has only historical value because
of high cost due to the chiral selectors that were wasted.
On the other hand, CSPs have been used widely all over
the world and about 99% chiral separations were carried
out by this way. In the literature, there are some
information about chiral selectors that have been used
to prepare CSPs and the most important are
FIGURE 1 Structural formula of enantiomers of miconazole
5
out on medicines, on the raw materials used in the pro-
duction of pharmaceutical preparations, and on the inter-
polysaccharides.
The goal of the present study was to develop a simple,
sensitive, selective, fast liquid chromatographic method
for the determination of enantiomers of miconazole not
only from its bulk form but also from pharmaceutical
preparations. For this reason, step‐by‐step optimization
strategy was also followed in order to find efficient
separation.
2
mediates of synthesis.
The specialized literature indicates a series of
methods for miconazole determination from pharmaceu-
tical preparations or biological fluids such as reverse‐
phase liquid chromatography, gas chromatography, and
3
others with the application of bulk and cream,
4-6
pharmaceutical dosage forms, biological samples such
7-9
9
as plasma, and saliva. In addition, there are papers
discussing the use of other analytical techniques such
2
| MATERIALS AND METHODS
.1 | Apparatus
10,11
as spectrophotometry.
These methods did not report
anything about simultaneous determination of the
enantiomers of the MIC. Furthermore, on the basis of
the literature survey, enantiomers of miconazole are
separated by different analytical techniques such as
capillary electrophoresis (CE) using dual cyclodextrin
2
HPLC experiments were performed with an Agilent 1100
HPLC instrument (Agilent Technologies, Waldbronn,
Germany) equipped with a G1313A ALS autosampler, a
G1316A temperature controller, G1311A quaternary
pump, and 1315B DAD variable wavelength detector.
Chemstation B.04.02‐SR2 software was used for instru-
ment control, data acquisition, and data handling. As a
stationary phase, six polysaccharide‐based chiral columns
12
system with long retention time (>16 min). Imran and
13
Hassan separated the enantiomers of MIC by HPLC
using cellulose tris(3,5‐dichlorophenylcarbamate) chiral
stationary phase where they found the good resolution
(
Rs = 2.40) but long analysis time.
(
Lux Cellulose‐1, Lux Cellulose‐2, Lux Cellulose‐3, Lux
Chirality on the molecular level was discovered by
14
Cellulose‐4, Lux Amylose‐1, and Lux Amylose‐2) with
the dimensions of 250 × 4.6 mm packed with 3 μm parti-
cles were provided by Phenomenex Inc (Torrance, CA,
USA). The structures of the chiral selectors used in this
study are shown in Figure 2.
Pasteur in 1848 when he manually separated the two
enantiomers of sodium ammonium tartrate crystals by
using a magnifying glass. Nowadays, it is well recognized
that chirality has significant impact on various disciplines
like toxicology, biology, medicine, pharmacology, and
many branches of chemistry. As a result of all these
efforts in 20th century, it became established that drug
metabolism and action of chiral drugs are mostly
2
.2 | Chemicals and reagents
3
enantioselective.
Because of the importance of chirality in human life,
optically pure active pharmaceutical ingredients have
been manufactured by either stereoselective synthesis or
instrumental chiral separation techniques. For this rea-
son, several separation strategies and methods have been
developed like HPLC, capillary electrophoresis, and
supercritical fluid chromatography. Among these separa-
tion techniques, HPLC is the most universally used one
because of ease of operation, the amount of available chi-
ral stationary phases (CSPs), and the flexibility of the
Miconazole was kindly provided from Fako Drug Inc
(Istanbul, Turkey). The pharmaceutical preparations of
®
the MIC, Miko‐Penotran 1200 mg vaginal ovule, and
®
Persmant gel 30 g were kindly supplied by Embil
Pharm and Orva ilac (Istanbul, Turkey), respectively.
HPLC grade methanol, acetonitrile, isopropanol, and
reagent grade ammonium acetate, formic acid, and
diethylamine were purchased from Merck (Darmstadt,
Germany). All other chemicals were analytical reagent
grade. Doubly distilled water was used while the water
is necessary.
4
mobile phases.

BOUNOUA ET AL.
3
FIGURE 2 Structure of chiral selectors used in this study
−
1
2.3 | Chromatographic conditions
concentration of 0.6 mg·mL , sonicated for 20 minutes,
and then completed to the volume with the same solvent.
The optimization was performed using several polar
organic and alcohol‐hydrocarbon mobile phases with the
addition of various amount of water and ammonium ace-
tate separately. The separation was carried out using Lux
Cellulose‐3 chiral stationary phase that has a chiral selec-
tor, namely, cellulose tris(4‐methylbenzoate) with metha-
0
.45 μm membrane syringe filter was used for filtration.
Also, Persmant gel formulation was accurately weighed
and then sonicated and filtrated by using same procedure
with the ovule formulation. Appropriate dilutions were
made with mobile phase to obtain final concentrations.
−
1
nol as a mobile phase at a flow rate of 1 mL·min and an
injected volume of 5 μL. UV detection performed at
2.5 | Validation
2
30 nm, and column oven temperature was maintained
at 45°C. The mobile phase was prepared daily and soni-
cated before use.
The analytical method development and validation were
performed according to the International Council on Har-
15
monization (ICH) guidelines. The methods were vali-
dated to quantify the enantiomers of miconazole from
its raw formulation and marketed preparations with the
following parameters: specificity, linearity, range, preci-
sion, accuracy, limit of detection, and quantification. On
the other hand, before the validation studies, it must be
shown that the separation conditions were suitable for
the next steps. For this reason, system suitability test
results should be checked; a system suitability test can
be defined as a test to ensure that the method can gener-
ate results of acceptable accuracy and precision. In gen-
eral, at least two of these criteria are required to
demonstrate suitability for the performed method.
2
2
.4 | Preparation of solutions
.4.1 | Preparation of stock solutions and
calibration curves
Stock solutions of MIC were prepared by weighting 5 mg
solid MIC and dissolving in 10 mL methanol. Final con-
centration of this solution is 0.5 mg·mL . Standard solu-
tions for HPLC were prepared with mobile phase by
varying the concentration of MIC in the range of 1 to
−
1
−
1
2
50 μg·mL . Calibration curve was constructed by plot-
ting the peak area of the drug to the concentration of
the pharmaceutical active ingredient.
2
.5.1 | Specificity
2
.4.2 | Preparation of pharmaceutical
The specificity of the method can be described based on
the ability to verify the analyte in the presence of excipi-
ents, matrix, and impurities.
chromatograms, the pharmaceutical preparation and its
placebo (mixture of the API and excipients) showed
almost no interfering peaks within the retention time
formulations
15,16
The studied pharmaceutical preparations included one
ovule and one gel formulation. Ovule (Miko‐Penotran)
was containing 1200 mg of MIC transferred to a 100 mL
calibrated flask and diluted with methanol to the final
According to the final

4
BOUNOUA ET AL.
ranges. On the other hand, forced degradation tests were
realized to support these data.
3 | RESULTS AND DISCUSSION
.1 | Optimization of the chromatographic
3
system
2
.5.2 | Linearity and range
The six chiral selectors were used in the present study.
The columns have chiral selectors that are physically
adsorbed onto the stationary phases. Four out of six sta-
tionary phases contain cellulose derivatives, and two of
them have amylose backbone. All columns except Lux
Cellulose‐3 has polysaccharide carbamate–based chiral
selector while Lux Cellulose‐3 has benzoate–based chiral
selector.
The concentrations were varied in the range of 1 to
−
1
5
00 μg·mL , and the linearity was obtained within the
−
1
range of 1 to 250 μg·mL . The calibration graph for this
method was constructed by plotting the peak area of the
drug against the active pharmaceutical ingredient (API)
concentration using repeated analysis. The linear regres-
sion was demonstrated and tabulated by the necessary
parameters.
The nature, backbone, and the moieties of these chiral
selectors have a significant effect on the separation of the
enantiomers of MIC. On the other hand, several mobile
phase variations were also tried to find the optimum con-
ditions such as polar organic (methanol, acetonitrile, eth-
anol, and isopropanol), reverse phase, and normal phase
(alcohol‐hydrocarbon mixtures). With these six columns
at least six different mobile phase mixtures and different
temperature and flow rates, more than 100 different con-
ditions were tested for the optimization step. According to
the results, the optimum separation with the shortest
analysis time was achieved by Lux Cellulose‐3 column
to further investigate the influence of the mobile phase
such as polar organic (methanol, acetonitrile,
isopropanol, and ethanol), normal‐phase mode (hexane/
isopropanol), and reverse‐phase mode (organic with
water and/or buffer solution). From the results, it can be
concluded that better separation was appeared for metha-
nol from the viewpoint of analysis time and sensitivity.
However, baseline separation was achieved by using n‐
2
.5.3 | Limit of detection and
quantification
The limit of detection (LOD) is the lowest amount of ana-
lyte in a sample that can be detected but not necessarily
quantities as an exact value under the stated experimental
conditions. The quantification limit (LOQ) of an individ-
ual analytical procedure is the lowest amount of analyte
in a sample that can be quantitatively determined with
suitable precision and accuracy.
LOD and LOQ were calculated from the equations in
which the standard deviation of response and the slope
of the calibration curve were used.
17
2.5.4 | Precision and accuracy
In order to determine the precision of the method, repeat-
ability and intermediate precision were applied. The
intermediate precision was assessed by carrying out the
analysis of at least three working solutions in three con-
secutive days that is called as interday. The repeatability
of analytical responses is expressed in terms of relative
standard deviation in (RSD%).
hexane/isopropanol
mixture,
acetonitrile,
and
isopropanol as mobile phases. The effect of varied compo-
sitions of water into the methanol and acetonitrile (3‐10%,
v/v) was evaluated, and an increased amount of water
caused to longer analysis time with higher resolution.
Ammonium acetate buffer was used instead of water,
and at the end of these investigations, methanol and
water mixture proved to be better than the mixture of
buffer and/or acetonitrile. The peaks for the enantiomers
were better defined and resolved each other and almost
free from tailing (Table 1).
It can be seen from the table that increasing amount of
water extends the analysis time. Same achievements were
reached using the buffer as an additive. Because of that
reason, methanol was chosen as a mobile phase without
any additive. For further optimization, the effect of flow
rate and temperature on enantioresolution and analysis
time was also investigated, respectively. Flow rate was
Accuracy is the main requirement of any chromato-
graphic methods especially working on pharmaceuticals.
Because of the possible interferences from the placebo of
the pharmaceutical preparations, potential effects from
matrix components must be investigated. For this reason,
recovery experiments need to be performed in the pres-
18
ence of the matrix. The recovery of the procedure was
carried out by spiking the already analyzed samples of
marketed formulations with the known amounts of stan-
dard solutions of pharmaceutical active compounds. If
the obtained results were close to 100%, it was shown that
the proposed method was free from the interferences of
the excipients used in the formulations.
−
1
altered between 0.5 and 1.25 mL·min , and the results
were demonstrated in Table 2.

BOUNOUA ET AL.
5
TABLE 1 The effect of water as an additive
MeOH/
O,
Lux Cellulose‐3
H
2
v/v
t
1
t
2
k
1
k
2
Rs
α
60 C
1
9
9
9
9
00/0
9/1
4.88
5.40
5.96
6.39
8.78
6.18
0.54
0.69
0.87
1.0
0.95
1.25
1.56
1.86
3.41
5.23
6.21
7.01
7.37
8.76
1.27
1.33
1.37
1.41
1.61
45 C
7.16
8.17
9.13
35 C
2
5 C
7/3
15 C
5/5
5
C
0/10
14.13
1.74
0
0
2
4
6
8
10
12
FIGURE 3 Effect of the temperature
TABLE 2 The effect of flow rate
Lux Cellulose‐3
Flow rate,
mL·min
−1
3.2 | Investigation of thermodynamic
parameters
t
1
t
2
k
1
k
2
Rs
α
0
0
1
1
.5
9.76
6.48
4.92
3.82
12.00
7.95
6.05
4.65
0.56
0.56
0.57
0.52
0.92
0.91
0.93
0.86
7.89
7.10
6.46
5.79
1.23
1.23
1.23
1.22
The enantioseparation on CSP is based on formation of
complex between analyte and CSP; separation can be
based on free and complexed states energy balanced,
and it would be studied from thermodynamic consider-
.75
.25
ations. The equilibrium‐binding constant (k ) that mea-
i
sures the binding strength may based on standard Gibbs
free energy change (ΔG):
Because of the high background pressure, the flow
rate kept constant at 1 mL·min .
−
1
As a last step, the temperature of the column oven
was changed in the range from 5°C to 60°C. Table 3
°
ΔG ¼ −RTInk :
i
and Figure
3
illustrated that the resolution
The Gibbs free energy is composed of enthalpic and
entropic contributions (ΔH° and ΔS°), the enantiomeric
strong binding driven by intermolecular interaction as
measured by the enthalpy change ΔH. The process of
complexation is usually paid off by an entropic cost ΔS,
shown as following Gibbs‐Helmholtz equation:
increases from 5°C to 45°C. However, as expected, the
enantioresolution started to decrease after high tempera-
tures such as 60°C.
The correlation between lnk1, lnk2, and lnα with 1/T
for MIC enantiomers was calculated as follows:
°
°
°
ΔG = ΔH − TΔS .
2
lnk1 ¼ 1562:9x−5:4693; R ¼ 0:9997:
This gives Van't Hoff equation, which gives the clear
way for straightforward determination of thermodynamic
parameter of enantioseparation, such as the standard
enthalpy (ΔH) and entropy (ΔS) of transfer of the solute
2
lnk2 ¼ 1605:3x−5:1171; R ¼ 0:9973:
2
lnα ¼ 203:49x−0:434; R ¼ 0:9504:
17
from the mobile phase to the CSP.
′
°
°
InK ¼ −ΔH =RT þ ΔS ;
TABLE 3 The effect of column oven temperature
Cellulose‐3
Temperature,
C
°
°
Inα ¼ −ΔΔH =RT þ ΔΔS ;
°
t
1
t
2
k
1
k
2
Rs
α
5
6.95
6.30
5.72
5.30
4.92
4.50
6.95
9.29
7.38
6.71
6.05
5.33
1.16
0.96
0.80
0.68
0.57
0.46
1.89
1.59
1.32
1.13
0.93
0.73
4.31
5.39
6.18
6.73
6.46
5.82
1.33
1.32
1.29
1.27
1.23
1.18
where k represents the retention factor, R is the universal
gas constant (8.3144 J/[mol·K]), T is the absolute temper-
ature, ΔH and ΔS are the molar enthalpy and molar
entropy of the adsorption, ΔΔH and ΔΔS are the differ-
ences ΔH2 − ΔH1 and ΔS2 − S1, and Φ is the column
phase ratio. The slope and intercept are −ΔH/R and ΔS/
R + lnΦ (ΔS*). For the linear plot of lnα versus 1/T, the
1
2
3
4
6
5
5
5
5
0

6
BOUNOUA ET AL.
slope and intercept are −ΔΔH/R and ΔΔS/R, respectively.
The logarithm of retention factor (Ink) was plotted versus
inverted temperature in Kelvin.
3.3 | Validation of the method
19
System suitability is the approval of suitability of any
separation system for analysis. In present work,
because of the system suitability test parameter, our
optimized method deemed as suitable as demonstrated in
Table 4.
The resultant chromatogram is illustrated in Figure 4.
Selectivity is another essential parameter for the anal-
ysis of pharmaceuticals. In present work, selectivity was
demonstrated by forced degradation studies. For this
It is observed that with the increasing temperature, k
values decreased. The same trend was observed also for
α value. After this investigation, 45°C was selected as opti-
mum condition due to the decreased analysis time and
better resolution. The relation between chromatographic
retention and column temperature can be described by
the Van't Hoff plots. In this work, linear plots of lnk ver-
sus 1/T were obtained having the regression coefficient
2
R = 0.999 and 0.997 for both the enantiomer as shown in
TABLE 4 System suitability test parameters results
Figure 3. The plots of lnα versus 1/T and resolution versus
column temperature were also linear having the regres-
sion coefficient R = 0.995. The corresponding Van't Hoff
Parameters
2
Compounds
(−) MIC
(+) MIC
plots were represented.
Retention time
4.88
0.57
6.19
0.93
1.65
6.46
1.27
12116
Capacity factor (k)
Selectivity factor (α)
Resolution factor (Rs)
Tailing
′
°
°
Ink ¼ −ΔH =RT þ ΔS =R þ InΦ:
1.31
ΔG ¼ −ΔHT:ΔS:
Theoretical plate numbers
plate/column)
12824
(
ΔG ¼ −RT lnðKÞ:
RSD% of retention time
0.046
0.079
ΔG1 ¼ −RT lnK1:
8
0
0
0
0
0
ΔG ¼ −RT lnK :
4.88
2
2
6
4
2
6
.19
(-)
lnðk1=k2Þ ¼ −ΔH=R ð1=T2−1=T1Þ:
(+)
On the basis of the calculations, ΔH = −32.95 KJ/mol,
ΔG = −1.2952 KJ, and ΔS = −4.064 J/K.
0
2
4
6
8
10
With variation of temperature, almost baseline separa-
tions could be achieved for two enantiomers of MIC. The
selectivity factor and for the enantiomers of all investi-
gated compound decreased with increasing temperature.
The resolution factor was observed with higher tempera-
tures. The natural logarithms of the retention factors
FIGURE 4 Chromatogram related with optimized conditions of
enantiomers of miconazole
TABLE 5 The degradation amounts calculated with the compar-
ison of the peak areas of untreated MIC enantiomers after 24 h
(lnk) of the investigated MIC depended linearly on the
inverse of temperature (1/T). Van't Hoff plots afforded
thermodynamic parameters, such as the apparent change
in enthalpy (ΔH°), the apparent change in entropy (ΔS°),
and the apparent change in Gibbs free energy (ΔG°) for
the transfer of analyte from the mobile to the stationary
phase. The thermodynamic constants (ΔH°, ΔS°, and
ΔG°) were calculated in order to promote an understand-
ing of the thermodynamic driving forces for retention in
this chromatographic system.
(
−) MIC
(+) MIC
Conditions
M NaOH
0.1 HCl
Degradation Amount, %
1
9.19
0.59
4.78
0.97
1.36
11.30
0.24
7.55
0.79
1.78
3
% H
0°C
2 2
O
8
Photolytic degradation 254 nm

BOUNOUA ET AL.
7
reason, bulk MIC was treated under acidic and alkali
hydrolysis conditions by using 0.1M HCl and NaOH sepa-
rately, oxidation by 3% H O , heat in the oven in sold
form and heat in the water bath in solution, and photo-
lytic degradation was applied under UV light.
The results of the degradation amounts calculated
with the comparison of the peak areas of untreated MIC
enantiomers (Table 5), and related chromatograms are
illustrated in Figure 5.
2
2
As a result of forced degradation studies, all peaks
®
were evaluated by using Chemstation software and possi-
ble interferences were purely separated from the main
peaks. It shows that developed method is selective enough
for the further validation steps.
8
0
Untreated sample
60
40
20
0
Linearity studies were performed by analyzing varied
−
1
concentrations (1 to 250 μg·mL ) that are explained in
the above sections. Least square regression was applied
to the results of the series of mixture analyses across the
ranges. Linearity was evaluated by a coefficient of deter-
0
2
4
6
8
10
12
14
16
18
2
1
M NaOH, 2 hours
mination values (R > 0.999) that shows the developed
80
60
40
20
0
method is linear in the stated ranges for both enantio-
mers. All of the calculated LOD and LOQ values obtained
from the calibration curve for each enantiomer were
−1
below 1 μg·mL . Thus, the method can be considered
to be sufficiently sensitive for the quantification of enan-
tiomers of MIC from pharmaceutical formulations. All
necessary parameters for the calibration equation were
tabulated in Table 6.
Precision of the method demonstrated the closeness of
agreement between a series of analysis of a sample. Intra-
day and interday precision was evaluated by injecting
three different levels of calibration solutions (n = 5) on
the same day and three consecutive days respectively.
Results are expressed by the percentage of relative stan-
dard deviation (RSD%) and illustrated in Table 6. The
mean of RSD% values was found acceptable and lower
than 2%.
0
2
4
6
8
10
12
14
16
18
80
60
40
20
0
0.1 M HCl, 2 hours
0
2
4
6
8
10
12
14
16
18
80
60
40
20
0
2 2
3% H O , 2 hours
Accuracy or trueness of the method is examined by
the standard addition technique. Results are expressed
in terms of both recovery and bias from spiking the three
0
2
4
6
8
10
12
14
16
18
o
8
0 C in water bath 2 hours
80
60
40
20
0
TABLE 6 Characteristics of the linear regression analysis of
miconazole
(−) MIC
(+) MIC
−1
Linearity range (μg·mL
Slope
)
1‐250
3.014
−4.176
0.999
1.019
0.0028
0.47
1‐250
3.018
−4.104
0.999
0.992
0.0027
0.14
0
2
4
6
8
10
12
14
16
18
Intercept
Correlation coefficient
SE of slope
1
00
Photolytic degradation
54 nm, 24 hours
2
8
6
4
2
0
0
0
0
0
SE of intercept
−1
Limit of detection (μg·mL
)
−
1
Limit of quantification (μg·mL
)
0.91
0.43
a
0
2
4
6
8
10
12
14
Within‐day precision (RSD%)
0.56
0.35
a
Between‐day precision (RSD%)
0.77
0.66
FIGURE 5 Chromatograms related with stress degradation
studies
a
Mean of five repeated analysis.

8
BOUNOUA ET AL.
TABLE 7 Assay results and mean recovery studies of MIC ovule and MIC gel in pharmaceutical dosage forms
(
−) MIC
(+) MIC
Ovule
Labeled claim, mg
200
198.86
0.689
0.57
200
199.77
0.270
0.115
50
a
Amount found, mg
a
RSD, %
Bias, %
Added, mg
25
50
100
99.40
99.40
0.51
25
100
98.54
98.54
0.31
a
Found, mg
28.28
103.7
0.33
50.67
102.4
0.47
26.09
104.36
0.19
50.52
101.2
0.252
−1.20
Recovery, %
a
RSD% of recovery
Bias, %
−3.77
−2.43
0.60
−4.36
1.46
Gel
Labeled claim, mg
100
96.56
0.15
100
96.18
0.51
a
Amount found, mg
a
RSD, %
Bias, %
3.44
3.81
Added, mg
25
50
100
25
50
100
a
Found, mg
25.40
101.23
0.89
51.03
102.06
0.43
100.43
100.43
0.44
25.21
100.84
1.13
50.32
100.64
0.29
100.01
100.01
0.25
Recovery, %
a
RSD% of recovery
Bias, %
−1.23
−2.06
−0.43
−0.84
−0.64
−0.01
a
Mean of 5 repeated analysis.
different levels followed by five repetitive analyses. On the
basis of the recovery results of the commercial products
4 | CONCLUSION
(
100.2‐103.77%), the method was free from the interfer-
A novel selective, specific, precise, accurate, and stability
indicating chiral HPLC method was developed using
polysaccharide‐based chiral column namely, Lux Cellu-
lose‐3. For this reason, the effect of chiral stationary
phases, mobile phases (normal phase, polar organic,
and reverse phase), flow rate, and the column oven tem-
perature on the enantioseparation of the enantiomers of
MIC was investigated. Evaluation of chromatographic
data revealed that chiral stationary phase and the
mobile phase have significant effects on the analysis
time as well as enantioresolution of the enantiomers.
As a result of the optimization steps, the enantiomers
of MIC were separated about 6 minutes. On the other
hand, stability indicating capability of the proposed
method was evaluated by stress degradation studies.
The optimized method was fully validated by evaluation
of the validation parameters. The method was demon-
strated to be highly feasible and reproducible. High per-
centage recovery suggested that the method was free
from the interferences of the commonly used excipients
and additives in the formulations. Finally, the developed
method further underlines the ease of use of the polar
ences of the commonly used excipients and additives in
the pharmaceutical preparation (Table 7 and Figure 6).
(
-)
(+)
a
0
0
1
2
3
4
5
6
7
b
(+)
(
-)
0
0
1
2
3
4
5
6
7
®
FIGURE 6 The HPLC chromatogram of the (a) Miko‐Penotran
ovule and (b) Persmant gel formulations (15 μg·mL
®
−1
)

BOUNOUA ET AL.
9
organic solvents such as methanol in chiral separation of
the enantiomers of MIC.
11. Khashaba P, El‐Shabouri S, Emara K, Mohamed A. Analysis
of come antifungal drugs by spectrophotometric and spectroflu-
orometric methods in different pharmaceutical dosage forms.
J. Pharm. Biomed. Anal. 2000;222:363‐376.
ACKNOWLEDGMENTS
1
1
1
2. Xiuli L, Wanguo H, Chenfu Z. Enantiomer separation of micon-
azole by capillary electrophoresis with dual cyclodextrin
systems. Anal Sci. 2003;19:1509‐1512.
The authors would like to thank University of Tahri
Mohamed Béchar for supplying internship. The authors
also grateful to Professor Dr Bezhan Chankvetadze for
his kind support and his guidance.
3. Imran A, Hassan YA. Enantioseparation of some clinically used
drugs by HPLC using cellulose tris (3.5‐dichlorophenylcarbamate)
chiral stationary phase. Biomed Chromatogr. 2003;17:113‐117.
4. Pasteur L. On the asymmetry of naturally occurring organic
compounds. In: Richardson GM, ed. The Foundations of Stereo
Chemistry: Memoirs by Pasteur, Van't Hoff, Le Bel, and
Wislicenus. New York: American Book Co; 1901:1‐33.
ORCID
Mehmet Gumustas http://orcid.org/0000-0003-2793-7154
Sibel A. Ozkan http://orcid.org/0000-0001-7494-3077
1
5. ICH guidelines, Q1A (R2): stability testing of new drug sub-
stances and products (revision 2), International Conference on
Harmonization. Available from: 〈http://www.fda.gov/down-
loads/RegulatoryInformation/Guidances/ucm128204.pdf〉
http://www.fda.gov/downloads/RegulatoryInformation/Guid-
ances/ucm128204.pdf〉, 2003.
6. ICH guideline (Q1AR) stability testing of new drug substances
and products. International Conference on harmonization
IFPMA, Geneva, 2000.
REFERENCES
1
. Mangas‐Sanchez J, Busto E, Gotor‐Fernandez V, Malpartida F,
Gotor V. Asymmetric chemoenzymatic synthesis of miconazole
and econazole enantiomers. The importance of chirality in their
biological evaluation. J Org Chem. 2011;76(7):2115‐2122.
1
2
3
4
. Rao D, Satanarayana N, Malleswara Reddy A, Sait S, Chakole D,
Mukkanti K J. Pharmaceut Biomed Anal. 2010; 51:736–742.
1
7. Gumustas M, Kurbanoglu S, Uslu B, Ozkan SA. UPLC versus
HPLC on drug analysis: advantageous, applications and their
validation parameters. Chromatographia. 2013;76:21‐22.
. Reddy IK, Mehvar R. Chirality in Drug Design and Development.
New York: CRC Press; 2004.
. Chankvetadze B. Recent developments on polysaccharide‐based
chiral stationary phases for liquid‐phase separation of enantio-
mers. J Chromatogr A. 2012;1269:26‐51.
18. Singh S, Bakshi M. Guidance on conduct of stress testing to
determine inherent stability of drugs. Pharm Technol. 2000;1‐14.
1
9. Dattatraya V, Wani Vipul P, Rane SNM. Liquid chromatographic
separation and thermodynamic investigation of lorcaserin
hydrochloride enantiomers on immobilized amylose based chi-
ral stationary phase. Chirality. 2017;1‐9.
5
. Krstulovic AM. Chiral Separations by HPLC. UK: Ellis Horwood
Limited; 1989.
6
. Cavrini V, Di Pietra A, Raggi M. High‐pressure liquid chromato-
graphic (HPLC) analysis of imidazole antifungals in commercial
dosage forms. Int J Pharmaceut. 1982;102:119‐124.
How to cite this article: Bounoua N, Sekkoum K,
Gumustas M, Belboukhari N, Ozkan SA.
Development of stability indicating HPLC method
for the separation and validation of enantiomers of
miconazole. Chirality. 2018;1–9. https://doi.org/
7
. Kobylinska M, Kobylinska K, Sobik B. J Chromatography B.
1
996;658:191‐195.
8. Sternson L, Patton T, King T. J Chromatography B. 1982;221:223‐228.
9
. Turner A, Warnock D. Determination of miconazole in human
saliva using high‐performance liquid chromatography.
J. Chromatography B. 1982;2271:229‐232.
10.1002/chir.22858
1
0. Wróbel K, Wróbel K, de la Garza Rodríguez I, López‐de‐Alba P,
López‐Martínez L. J Pharmaceut Biomed Anal. 1999;201‐2:99‐105.