Chiral separation of four stereoisomers of ketoconazole drugs using capillary electrophoresis

Article abstract of DOI:10.1002/chir.22416

This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6- tri-O-methyl)-β-cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10mM phosphate buffer at pH2.5 containing 20mM TMβCD, 5mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5-s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively.

Full text of DOI:10.1002/chir.22416

CHIRALITY (2014)
Chiral Separation of Four Stereoisomers of Ketoconazole Drugs
Using Capillary Electrophoresis
WAN AINI WAN IBRAHIM,1,2,3* SITI ROSILAH ARSAD,
1,2,3
1,2,3
MOHD. MARSIN SANAGI,1,2,3
HASMERYA MAAROF,
4
AND HASSAN Y. ABOUL-ENEIN **
1
Separation Science & Technology Group (SepSTec), Department of Chemistry, Faculty of Science, Sukdai, Johor Bahru, Johor, Malaysia
2
Nanotechnology Research Alliance, Universiti Teknologi Malaysia, Sukdai, Johor Bahru, Johor, Malaysia
3
Ibnu Sina Institute for Fundamental Science Studies, Universiti Teknologi Malaysia, Sukdai, Johor Bahru, Johor, Malaysia
4
Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki,
Cairo, Egypt
ABSTRACT
This work aimed to develop a chiral separation method of ketoconazole enantio-
mers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6-
tri-O-methyl)-β-cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively
inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl
sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS con-
centration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral
separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing
2
0 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C
with a 5-s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were
successfully resolved for the first time within 17 min (total analysis time was 28 min including
capillary conditioning). The migration time precision of this method was examined to give re-
peatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively.
Chirality 00:000-000, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: electrokinetic chromatography; chiral separation; azole drugs; ketoconazole;
heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin; sodium dodecyl sulfate
INTRODUCTION
and 2S4S (Fig. 1). The trans diastereomers were found to
be a much weaker inhibitor than the cis pair.
Chirality has been and still remains one of the major issues
Earlier chiral separation of ketoconazole was carried out
using supercritical fluid chromatography (SFC) and high-
performance liquid chromatography (HPLC) on a chiral
stationary phase based on substituted polysaccharides with
different groups such as 3,5-dimethylphenylcarbamates and
(R)-phenyl-ethylcarbamate. The separation of ketocona-
zole using SFC and HPLC only gives two stereoisomers
in drug development and the pharmaceutical industry. The
United States Food and Drug Administration, European Com-
mittee for Proprietary Medicinal Products, and other drug
controlling agencies have issued certain guidelines to phar-
maceutical and agrochemical industries about the marketing
1
of racemates. In the pharmaceutical industry, the separation
of enantiomers is growing in interest because the enantio-
mers of a compound can display quite different activity and
toxicity profiles. For this reason, the U.S. Food and Drug Ad-
ministration has endorsed the development of enantiomer-
with R =0.72–0.83, k =3.7–12.2 min and R =1.53 - 1.70,
s
s
7
k =10.8–17.2 min, respectively. In the year 2000, the chi-
ral separation of ketoconazole using SFC on an amylose-
based column was described. Two stereoisomers of
selective synthesis and analysis methods when a new drug
is going to be developed and marketed.2
ketoconazole were resolved in less than 7 min with high
8
resolution (R =4.29). Recently, the chiral separation of
There is no doubt that enantiomer separation by capillary
electrophoresis (CE) has rapidly attracted attention as a
promising technique due to its high separation efficiency
and flexibility. Among various different CE modes, capillary
zone electrophoresis (CZE) and electrokinetic chromatogra-
phy (EKC), in which only a chiral selector is added to the
usual running buffer solution, are the most widely used for
s
ketoconazole was carried out by using EKC, which fo-
cused on investigating the applicability of a chiral selec-
tor to recognize the enantiomers stereoselectivity. The
most widely used chiral selector in EKC are cyclodex-
trins, due to their low UV absorbance, relative inexpense,
ease of use, and ready availablity.9 Only two stereoiso-
mers of ketoconazole were successfully resolved using
EKC with TMβCD as the chiral selector in a 100 mM
,10
3
,4
enantiomer separations. One of the most attractive advan-
tages of EKC for the separation of enantiomers is an easy
change of separation media in the method development; that
is, one can easily alter the separation solution to find the opti-
mum separation media and one can also use an expensive chi-
*
Correspondence to: Wan Aini Wan Ibrahim, Separation Science & Technol-
ogy Group (SepSTec), Department of Chemistry, Faculty of Science, UTM,
81310 UTM Johor Bahru, Johor, Malaysia. E-mail: wanaini@kimia.fs.utm.my
or waini@utm.my; Hassan Y. Aboul-Enein, Department of Pharmaceutical
and Medicinal Chemistry, National Research Centre, Dokki, Cairo 12311,
Egypt. E-mail: haboulenein@yahoo.com
5
ral selector because of the small amounts required.
Ketoconazole was the first orally active azole introduced in
clinical practice as an effective antifungal agent. Ketoconazole
is chiral and has two stereogenic centers in the molecule.
Their absolute configuration has been determined via synthe-
Received for publication 13 July 2014; Accepted 27 October 2014
DOI: 10.1002/chir.22416
Published online in Wiley Online Library
(wileyonlinelibrary.com).
6
sis by Rotstein et al. as cis-2R,4S and 2S,4R, also trans-2R4R
©
2014 Wiley Periodicals, Inc.

WAN IBRAHIM ET AL.
Fig. 1. Stereoisomers of ketoconazole.
phosphate buffer (pH3.5). Good precision was obtained, where
the RSD was found to be lower than 1.2, 4.9, and 6.1% for migra-
tion time, enantiomeric resolution, and corrected peak areas,
detector window) obtained from Polymicro Technologies (Phoenix, AZ).
Background electrolytes (BGE) were prepared by dissolving anionic sur-
factant, SDS, and TMβCD at an adequate concentration in a solution of
disodium hydrogen phosphate 12-hydrate where phosphoric acid was
used to adjust the preferred pH of the phosphate buffer. Before use, the
new capillary was rinsed with 1.0 M NaOH solution for 40 min, 0.1 M
NaOH solution for 40 min, with deionized water for 40 min, and finally with
the running buffer for 40 min. In between runs, the capillary was condi-
tioned with 0.1 M NaOH for 3 min, followed by deionized water for 3 min
and with BGE for 5 min.
10
respectively. The use of high buffer concentration is not favor-
able in CE analysis, as it will generate high current and lead to
11–13
Joule heating.
To the best of our knowledge, only two stereoisomers of
ketoconazole were successfully resolved using SFC, HPLC,
and CE. Thus, in this study we modified and optimized the
composition of background electrolyte (BGE), which is the
key parameter in CE to separate all four stereoisomers of ke-
toconazole. Our main interest was to apply an anionic surfac-
tant solution, sodium dodecyl sulfate (SDS), in the BGE,
which has been the most popular technique for many years.
Generally, addition of anionic surfactant solution into BGE
at a higher concentration than the critical micelle concentra-
tion (CMC) is effective for the separation of ionic analytes
and also electrically neutral or nonionic analytes. Thus, the
addition of anionic surfactant into BGE is suitable for drug
analyses involving cationic, anionic and neutral drugs.14
SDS functions as an anionic selector and also carrier for the
neutral analyte ketoconazole.
RESULTS AND DISCUSSION
Method Development
The method development for chiral separation was initially
performed by optimizing the experimental parameters such
as concentration of chiral selector, concentration of running
electrolyte, pH of buffer solution, as well as the use of addi-
tives and the presence of an organic modifier. In addition,
the effect of different instrumental conditions such as applied
voltage and separation temperature was also investigated. In
10
a previous article regarding the chiral separation of ketoco-
nazole by EKC, we noted that the use of TMβCD as a chiral
selector with a high concentration of running buffer allowed
a very fast separation with good resolution. However, only
two peaks of stereoisomers were separated, which are cis-
2R4S and cis-2S4R. The linearity of the method was assessed
from 10–100 mg/L for racemic mixture of ketoconazole. The
limit of detection (LOD) of each enantiomer was 0.25 mg/L
but the limit of quantification (LOQ) was not mentioned.
The precision (% RSD) was found to be lower than 1.2, 4.9
and 6.1% for migration, enantiomeric resolution, and
corrected peak areas, respectively. The precision of the
method was found to be 97.5, 98.5, and 96.0% for the tablets,
the syrup, and the gel, respectively. In this current study,
we developed an EKC method for the separation of four enan-
tiomers with addition of an anionic surfactant, SDS.
MATERIALS AND METHODS
Chemicals and Reagents
Ketoconazole was purchased from Dr Ehrenstorfer (Augsburg, Ger-
many) (batch number C14532000, 99.0% purity), heptakis(2,3,6-tri-O-
methyl)-β-cyclodextrin (TMβCD) was purchased from Sigma-Aldrich
À1
(
St. Louis, MO), (batch number H4645-5G, MW: 1429.54 g mol , purity
≥
90%), and sodium dodecyl sulfate (SDS) was obtained from Fisher Scien-
tific (Loughborough, UK). Sodium hydroxide (NaOH) and disodium hy-
drogen phosphate 12-hydrate were purchased from Riedel-de Haen
(Seelze, Germany). All other chemicals and solvents were common
brands of analytical-reagent grade. Water used for dilutions or as buffer
preparation was produced from a Water Purification System from
À1
Millipore (Molsheim, France). The stock solution (1000 mg L ) of the
ketoconazole was prepared by dissolving the drug in HPLC-grade metha-
nol and diluted to 100 mg L as a typical concentration of sample to be
injected. All the separation solutions were filtered through a 0.20-μm ny-
lon syringe filter from Whatman (Clifton, NJ).
À1
Effect of different concentration of TMβCD and buffer solution. In
the first step, the instrumental parameters used were 5 s hy-
drodynamic injection at 50 mbar pressure, 25 °C of separation
temperature, and 25 kV of applied voltage. The concentration
of TMβCD was first varied in the range of 3–30 mM using ini-
tially 40 mM phosphate buffer solution and acidic pH (pH 3.0)
to find the best separation of ketoconazole. We found that
only two of the stereoisomers were successfully separated
at all concentration ranges used. The migration time in-
creased when the concentration of TMβCD increased and
Method
All electropherograms were obtained with the G1600A Agilent capil-
lary electrophoresis system from Agilent Technologies (Waldbronn,
Germany), equipped with temperature control and diode array detection
(DAD). Separations were performed using an untreated fused silica capil-
lary of 64.5 cm × 50 μm i.d. (with an effective length of 56 cm to the
Chirality DOI 10.1002/chir

CHIRAL SEPARATION OF FOUR STEREOISOMERS
improved the resolution of peaks (electropherogram not
shown). We chose 20 mM of TMβCD for further study since
it gave the highest resolution (R =4.24).The influence of
s
phosphate buffer concentrations was investigated in the
range 5–50 mM (electropherogram not shown). Changing
the buffer concentration only affects the migration time and
resolution; still, there were only two enantioseparated peaks
observed. Phosphate buffer concentration at 10 mM was pre-
ferred for further optimization, as it gave good resolution
(
Rs =0.98 À 5.04) with reasonable analysis time (11 À 13 min).
Effect of SDS concentration. The anionic surfactant, SDS, was
added to the buffer solution to enhance the separation of keto-
conazole stereoisomers. Theoretically, the hydrophobic tail of
SDS monomers will be included in the cyclodextrin (CD) cavity
along with the solute. This phenomenon could change the na-
ture of the solute and CD interaction and subsequently change
15
the resolution. A previous study reported that the addition of
an anionic surfactant, SDS, improved enantioseparation of
fenticonazole by increasing the contact time between the chiral
Fig. 2. Electropherograms of enantioseparation of ketoconazole drugs at
different percentages of methanol. Separation conditions: 5 mM SDS, 20 mM
TMβCD in 10 mM phosphate buffer (pH 2.5); capillary, 64.5 cm × 50 μm I.D.
1
6
compound and chiral selector. We optimized the addition of
SDS from 3–20mM to find the best separation of ketoconazole
diastereomers. In general, SDS is the selected anionic surfac-
tant and it is used at concentrations basically higher than the
(effective length, 56 cm); voltage, 25 kV; temperature, 25 °C; detection wave-
length, 200 nm; hydrodynamic injection, 50 mbar for 5 s; analyte concentra-
À1
tion, 100 mg L ).
1
7
CMC. The CMC value for SDS in water without any additive
Effect of methanol. The organic modifiers frequently affect
À3
À1
has been reported as 8.0 ×10 molL . However, when some
the enantiomeric resolution by changing the viscosity of the
18
8,24
additive was added, the value of CMC would be decreased.
In this study, we observed that two smaller peaks appeared at
mM SDS concentration. Increasing the SDS concentration in-
BGE and the stability of the inclusion complex.
In order
to enhance the resolution of enantiomers, methanol as an or-
ganic modifier was added to the running buffer solution.
Addition of methanol into the BGE successfully separated
all four ketoconazole enantiomers, probably due to alter-
5
creased the migration time of the stereoisomers and reduced
the selectivity. The separation with SDS was carried out at
low pH (pH3.0), where the EOF is suppressed. Effective sepa-
ration of ketoconazole enantiomers in the presence of SDS
might be a consequence of the effective ion-pair interactions be-
tween ketoconazole enantiomers and SDS monomers where
micellar solubilization acts over CMC or probably due to direct
electrostatic binding to the negatively charged anionic surfac-
tant, SDS.19 The SDS concentration of 5 mM was selected for
further optimization ( electropherogram not shown).
25
ation of the electrophoretic mobilities of the analytes.
Methanol was added in the range of 0.5 À 2.0% (v/v). The
resolution improved when the percentage of methanol was
increased from 0.5% to 1.0% (v/v). However, poor resolution
was obtained when 2.0% (v/v) of methanol was added.
Therefore, 1.0% (v/v) of methanol was selected as the opti-
mum percentage of organic modifier in running buffer (Fig. 2).
Effect of different voltage and temperature. Further optimization
was carried out by optimizing the separation at different volt-
ages from 20 kV to 28 kV, which basically affects the migra-
tion time and the Joule heating. Generally, increasing the
Effect of buffer pH. The pH of the buffer solution affects both
the charges on the analytes and the magnitude of the electro-
osmotic flow (EOF), hence improving the selectivity in CE.
The pH of the buffer solution has a significant role in
enantioseparation of the drugs, as it may affect the charge
of the analytes, and thus the binding characteristics.1
The effect of running buffer pH on resolution was studied in
the pH range 2.5–9.0 (electropherograms not shown) since
the ketoconazole drug has two different pKa values, 6.51
and 2.94. Increasing the pH decreased the migration time as
well as the resolution. Based on the results, pH 3.0 provided
full resolution of all four diastereomers. However, the effi-
ciency of the peaks was poor since the peak areas are much
smaller. In addition, we observed the two smaller peaks of
trans-2R4R and trans-2S4S that is not stable at pH 3.0. There-
fore, pH 2.5 was selected because the peaks observed have
high peak areas as compared to pH 3.0. A previous study
voltage gives rise to shorter migration times but is
counteracted by the increased Joule heating.2
4,26
An efficient
5,20
resolution with good peak shape and reasonable migration
time was achieved when the separation was performed at
2
5 kV (Fig. 3). Increasing the separation voltage to 28 kV de-
creased the migration time but the resolution become poor,
as the peaks overlapped. The temperature of the capillary
has an effect on the viscosity of the BGE and kinetic complex
formation, which also had a significant effect on the analysis
time.2
4,26
The influence of the temperature (20 À 30 °C) on
the separation of ketoconazole was also studied and the best
enantioseparation was obtained at 25 °C (electropherogram
not shown).
21,22
23
Precision Studies
proved that EKC
and MEKC mode was successfully de-
veloped for imidazole and triazole derivatives using an acidic
phosphate buffer using uncharged CDs. The uncharged CDs
migrate at the same velocity as the EOF, and as a result they
only allow the separation of ionized analytes in a pH buffer be-
In this work, the optimum conditions for separation of the
four ketoconazole enantiomers was obtained by using BGE
consisting of 20 mM TM βCD, 5 mM SDS, 10 mM phosphate
buffer (pH 2.5), 1.0% v/v MeOH at 25 kV separation voltage at
25 °C (Fig. 4). A big difference in peak area between
Chirality DOI 10.1002/chir
2
1
low the pKa of azole derivatives.

WAN IBRAHIM ET AL.
precision.27 In this study, poor reproducibility was observed
for peak areas, probably due to less stability of the complex
formation of ketoconazole drugs with TMβCD. Ketoconazole
is a weak base analyte which is positively charged in acidic so-
lutions and electrostatically attracted to the negatively
charged capillary walls.28 Consequently, the lower precision
is probably due to the irreproducible adsorption, which
caused variations in migration time and peak area. Addition-
ally, impurity peaks may be disguised by the peak tailing or
resolution of enantiomers, in which two analytes migrating
29
closely after each other may result in poor precision.
CONCLUSIONS
The enantioseparation of four stereoisomers of ketocona-
zole was successfully performed using CD-EKC. In the pres-
ent work, the optimum separation of ketoconazole peaks
was accomplished with 20 mM TMβCD, 5 mM SDS in
1
1
0 mM phosphate buffer solution (pH 2.5), and addition of
.0% (v/v) of methanol. A separation time within 17 min was
achieved under the optimum conditions with acceptable Rs
>1.5). It was shown that the presence of SDS below its
Fig. 3. Enantioresolution of ketoconazole at different voltages. Separation
conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5),
(
1
.0% methanol (v/v); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm);
CMC value in the BGE greatly influenced resolution and se-
lectivity of the peaks relevant to the enantiomers of ketocona-
zole when an EKC method was applied using TMβCD in
acidic buffer as a chiral selector. Even though there are
EKC methods that offer shorter analysis, only two enantio-
mers of ketoconazole were observed, and thus is not the
method for an enantiomeric purity check. The precision of
the developed method was measured in terms of repeatability
and reproducibility. Poor reproducibility was obtained for the
enantioseparation of ketoconazole, probably due to the insta-
bility of the drug complexes being formed.
temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection,
À1
5
0 mbar for 5 s; analyte concentration, 100 mg L ).
stereoisomers P3, P4 with stereoisomer P1, P2 as observed in
the electropherogram is probably due to differences in the
percentage of racemic mixture of the compound. Upon opti-
mization of the above-referenced separation conditions, ana-
lytical performance of the methods was also investigated for
chiral separation of ketoconazole. In this study, the parameter
investigated was precision (repeatability and reproducibility).
Precision was assessed as relative standard deviation (RSD)
-
1
for enantioseparation of ketoconazole (100 μg mL ). Repeat-
ability of three consecutive injections obtained for the devel-
oped methods in terms of RSD (n =3) was from 5.42 to
ACKNOWLEDGMENTS
The authors thank the Ministry of Education Malaysia
(MOE) and Universiti Teknologi Malaysia for the Tier 1 Re-
search University Grant (vote no. 04H22). S.R. Arsad also
thanks MOE Malaysia for the MyPhD scholarship.
5
.80% for migration times, and 10.35 to 14.88% for peak areas.
Reproducibility was assessed on 3 different days and on each
day triplicate injections were performed. The RSD (n =9) cal-
culated was from 7.15 to 8.88% for migration times, and 7.6 to
2
5.68% for peak areas.
Even though CE offers a broad range of selectivity in com-
LITERATURE CITED
1
2
. Ali I, Aboul-Enein HY, Gaitonde VD, Singh P, Rawat MSM, Sharma B.
Chiral separations of imidazole antifungal drugs on Amy Coat RP column
in HPLC. Chromatographia 2009;70(1-2):223–227.
. Bernal JL, Toribio L, del Nozal MJ, Nieto EM, Montequi MI. Separation of
antifungal chiral drugs by SFC and HPLC: a comparative study. J
Biochem Biophys Methods 2002;54(1-3):245–254.
bination with high separation efficiency and fast analysis
time, CE still has some major problems, such as poor
3
4
5
6
. Terabe S, Otsuka K. Review. Separation techniques of enantiomers by
capillary electrophoretic. J Chromatogr A 1994;666:295–319.
. Nishi H, Terabe S. Optical resolution of drugs by capillary electrophoretic
techniques. J Chromatogr A 1995;694(1):245–276.
. Nishi H. Enantiomer separation of drugs by electrokinetic chromatogra-
phy. J Chromatogr A 1996;735(1-2):57–76.
. Rotstein DM, Kertesz DJ, Walker KAM, Swinney DC. Stereoisomers
of ketoconazole: preparation and biological activity. J Med Chem
1
992;35(15):2818–2825.
7
8
. Saag MS, Dismukes WE. Minireview azole antifungal agents: emphasis.
Antimicrob Agents Chemother 1988;32(1):1–8
. Bernal JL, del Nozal MJ, Toribio L, Montequi MI, Nieto EM. Separation of
ketoconazole enantiomers by chiral subcritical fluid chromatography. J
Biochem Biophys Methods 2000;43(1-3):241–250.
Fig. 4. Enantioresolution of ketoconazole using optimum CD-EKC condi-
tions. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate
buffer (pH 2.5), 1.0% methanol (v/v); capillary, 64.5 cm × 50 μm I.D. (effective
length, 56 cm); voltage, 25 kV; temperature, 25 °C; detection wavelength,
9
. 9.Gübitz G, Schmid MG. Chiral separation by capillary electromigration
techniques. J Chromatogr A 2008;1204(2):140–156.
2
1
00 nm; hydrodynamic injection, 50 mbar for 5 s; analyte concentration,
10. 10.Castro-Puyana M, Crego AL, Marina ML. Enantiomeric separation of
ketoconazole and terconazole antifungals by electrokinetic
À1
00 mg L ).
Chirality DOI 10.1002/chir

CHIRAL SEPARATION OF FOUR STEREOISOMERS
chromatography: rapid quantitative analysis of ketoconazole in pharma-
ceutical formulations. Electrophoresis 2005;26(20):3960–3968.
of pH and the concentration of β-cyclodextrin for fenoprofen and ibupro-
fen. J Chromatogr A 1993;635:291–306.
1
1
1
1. 11.Hancu G, Kelemen H, Rusu A,Gyéresi A. Development of a capillary
electrophoresis method for the simultaneous determination of cephalo-
sporins. J Serb Chem Soc 2013;78(9):1413–1423.
2. Ali I, Kumerer K, Aboul-Enein HY. Mechanistic principles in chiral sepa-
rations using liquid chromatography and capillary electrophoresis.
Chromatographia 2006;63(7/8):295–307.
3. Eder AR, Chen JS, Arriaga EA. Separation of doxorubicin and
doxorubicinol by cyclodextrin-modified micellar electrokinetic capillary
chromatography. Electrophoresis 2006;27:3263–3270.
21. Danel C, Foulon C, Goossens JF, Bonte JP, Vaccher C. Validation of chiral
electrokinetic chromatography methods using highly sulfated cyclodex-
trins: determination of enantiomeric purity of aromatase inhibitors.
Chromatographia 2006;63(7-8):353–358.
22. Castro-Puyana M, Crego AL, Marina ML. Separation and quantitation
of the four stereoisomers of itraconazole in pharmaceutical formula-
tions by electrokinetic chromatography. Electrophoresis 2006;27(4):
887–895.
23. Wan Ibrahim WA, Hermawan D, Sanagi MM, Aboul-Enein HY. Cyclodex-
trin-modified MEKC for enantioseparation of hexaconazole, penconazole,
and myclobutanil. J Sep Sci 2009;32(3):466–471.
1
4. Nishi H, Terabe S. Micellar electrokinetic chromatography: perspectives
in drug analysis. J Chromatogr A 1996;735:3–27.
1
5. Zhu C, Lin X, Wei Y. Chiral separation of pemoline enantiomers by
24. Al Azzam KM, Saad B, Adnan R, Aboul-Enein HY. Enantioselective analy-
sis of ofloxacin and ornidazole in pharmaceutical formulations by capillary
electrophoresis using single chiral selector and computational calculation
of their inclusion complexes. Anal Chim Acta 2010;674(2):249–255.
25. Quirino JP, Dulay TM, Fu L, Mody TD, Zare RN. Capillary electrophore-
sis separation and native laser induced fluorescence detection of
metallotexaphyrins. J Sep Sci 2008;25:819–824.
26. Wan Ibrahim WA, Abd Wahib SM, Hermawan D, Sanagi MM. Separation
of selected imidazole enantiomers using dual cyclodextrin system in mi-
cellar electrokinetic chromatography. Chirality 2013;25:328–335.
cyclodextrin-modified micellar capillary chromatography.
Biomed Anal 2002;30(2):293–298.
J Pharm
1
1
1
1
2
6. Quaglia MG, Donati E, Desideri N, Fanali S, D’auria FD, Tecca M. Chiral
discrimination by HPLC and CE and antifungal activity of racemic
fenticonazole and its enantiomers. Chirality 2002;14(5):449–454.
7. Bendazzoli C, Mileo E, Lucarini M, Olmo S, Cavrini V, Gotti R. Capillary
electrophoretic study on the interaction between sodium dodecyl sulfate
and neutral cyclodextrins. Microchim Acta 2010;171(1-2):23–31.
8. Domínguez A, Fernández A, González N, Iglesias E, Montenegro L. De-
termination of critical micelle concentration of some surfactants by three
techniques. J Chem Educ 1997;74(10):1227–1231.
9. Liu Y, Fu X, Ma C, Zhong J, Liao Y, Liu H. Chiral separation of raltitrexed
by cyclodextrin-modified micellar electrokinetic chromatography. Anal
Bioanal Chem 2009;393:321–326.
27. Mayer BX. How to increase precision in capillary electrophoresis. J
Chromatogr A 2001;907(1-2):21–37.
28. Altria KD. Enhanced pharmaceutical analysis by CE using dynamic sur-
face coating system. J Pharmaceut Biomed 2003;31:447–453.
2
9. Javida FS, Shafaatia A, Zarghi A. Improvement of capillary electro-
phoretic enantioseparation of fluoxetine by a cationic additive. IJPR
2013;12:71–76.
0. Rawjee YY, Staerk DU, Vigh G. Capillary electrophoretic chiral separa-
tions with cyclodextrin additives: I. acids: Chiral selectivity as a function
Chirality DOI 10.1002/chir