Journal of Chromatographic Science 2014;52:919 925
doi:10.1093/chromsci/bmt107 Advance Access publication August 1, 2013
Separation and Determination of Quinolone Antibacterials by Capillary Electrophoresis
Aura Rusu1, Gabriel Hancu1*, Gergely Volgyi2, Gergo Toth2, Bela Noszal2 and Arpad Gyeresi1
1
Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy, 540139 Targu Mures, 38 Gh, Marinescu, Romania,
and 2Department of Pharmaceutical Chemistry, Semmelweis University, Research Group of Drugs of Abuse and Doping Agents,
Hungarian Academy of Sciences, Hogyes Endre u. 9, Budapest H-1092, Hungary
*Author to whom correspondence should be addressed. Email: g_hancu@yahoo.com
Received 16 October 2012; revised 6 April 2013
The migration behavior and separation of 13 quinolone antibacterials
were investigated by capillary electrophoresis (CE). In order to
predict the electrophoretic mobility, the protonation macroconstants
of all the compounds were determined by pH-potentiometric titra-
tions. We proved that the electrophoretic mobility of ionized quino-
lones (QNs) can be described with Offords equation, and the
migration order depends on their charge-to-mass ratios. A buffer of
25 mM sodium tetraborate adjusted to pH 9.3 was an efficient elec-
trophoresis system for the separation of 12 QNs by capillary zone
electrophoresis. This method can be considered a general method
to separate quinolone derivatives. Ciprofloxacin, norfloxacin and
ofloxacin, fluoroquinoles with very similar structural characteristics,
were separated by micellar electrokinetic chromatography. Validation
parameters, including linearity and detection and quantification limits,
were also determined. Our results prove the applicability of CE for the
simultaneous determination of QNs from complex mixtures. Our
methods are environment-friendly replacement and improvement of a
common high-performance liquid chromatography determination with
rapid analysis time without using any organic solvents.
Introduction
Quinolones (QNs) are a class of synthetic chemotherapeutic
antibiotics of great therapeutic value, eradicating bacteria by
interfering with DNA replication. QNs contain an 1-substituted-
1,4-dihydro-4-oxopyridine-3-carboxyilic moiety, whereas uoro-
quinolones (FQs) bear a uorine atom in the C-6 position and a
piperazinyl or other heterocycle with basic nitrogen in the C-7
position. These motifs provided a broad spectrum of activity
against Gram-negative and some Gram-positive bacteria and pro-
duced better pharmacokinetic prole (1).
Quinolone antibacterial therapy is valuable both in human and
in veterinary medicine. In the last decade, a real danger is the in-
duction of resistance phenomena that occur in food of animal
origin contaminated with these compounds, including degrad-
ation products that reach the environment (2). To minimize this
phenomenon, it is a global effort to develop rapid and sensitive
methods for the determination of these compounds in raw mate-
rials, pharmaceutical formulations and also in biological, environ-
mental and food samples (3 7).
We have recently shown that FQs are triprotic molecules and
quantitated their site-specic basicities (8). At physiological pH,
FQs have two protonating sites, namely the N-40 nitrogen atom
and the carboxylate group. The N-10 nitrogen atom protonates in
very acidic media only, and it has no inuence on the biological
or chromatographic behavior of these compounds.
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Article
The most frequently applied technique for the analysis of QNs
is high-performance liquid chromatography (HPLC) (9, 10). A
permanent difculty of the HPLC separation is that QNs bear a
charge at all pHs which necessitates ion-pair formation in the
process of their separation (10, 11). Unlike in HPLC, the ionized
state is an advantageous property in capillary zone electrophor-
esis (CZE) where separation is based on the differences between
the own electrophoretic mobilities of the analytes (12). In the
past few years, capillary electrophoresis (CE) has, therefore,
emerged to be an important tool in the analysis of QN deriva-
tives, due to its separation efciency, low amount of sample and
reagent consumption, speed of analysis and applications to a
wider selection of analytes.
The main advantage of CZE separation is that the electrophor-
etic mobility can be predicted. Nevertheless, few studies have
been reported on the prediction of electrophoretic behavior of
QNs to nd the optimal separation conditions for various QNs
taking into consideration parameters such as pH, pKa values and
the own electrophoretic mobilities of the analytes (13, 14).
Despite the great advantages of CZE, the majority of the litera-
ture methods published so far describe separations for a few FQs
from different generations, having different physico-chemical
characteristics. The main shortcoming of the reported electro-
phoretic separations is that once the number of QNs in the
sample exceeds 3 or 4, the resolution and selectivity become
poor (15 18).
Separation of the structurally very similar analytes such as nor-
oxacin (NOR) and ciprooxacin (CIP) is still very challenging
and cannot be solved by simple aqueous CZE, due to the highly
similar N1 substituents (14, 15, 19, 20).
Besides CZE separation (16, 17), micellar electrokinetic chro-
matography (MEKC) has also been introduced for the separation
of QNs (21). Upon addition of surfactant and the concomitant
formation of micelles, the analytes are separated by differential
partitioning between the micelles ( pseudo-stationary phase)
and the surrounding aqueous buffer solution (mobile phase).
The aim of the present study was to perform a systematic
study regarding the electrophoretic behavior of a large number
of QN derivatives in order to develop simple, rapid and efcient
CE methods for the simultaneous separation of QN derivatives
from complex mixtures.
In addition, we proposed a complementary MEKC separation
method for the separation of closely related QNs (e.g., CIP and
NOR) that cannot be separated by CZE.
For this purpose, 13 antibacterial QNs of extensive therapeut-
ic use were selected from various generations, including deriva-
tives with different structural characteristics and others with
Figure 1. Constitutional formulas, numbering and abbreviations of the investigated quinolones. See Materials and reagents section to decode abbreviations.
closely related structures. The constitutional formulas of the
quinolones studied are shown in Figure 1.
Experimental
Instrumentation and reagents
GLpKa instrument
The protonation macroconstants (logK values) were determined
by pH-potentiometry using a GLpKa instrument (Sirius Analytical
Instruments) tted with a combination AgAgCl pH electrode.
The electrode was calibrated with the four-parameterTM calibra-
tion procedure, based on alkalimetric titration of 0.5 M HCl.
CE system
All CE experiments were conducted in an Agilent 6100 CE
system equipped with a diode-array detector, while the results
were recorded and processed using the Chemstation 7.01 soft-
ware (Agilent). Separations were performed using uncoated
fused-silica capillaries of 48 70 cm  50 mm I.D (effective
length 40 62 cm) (Agilent). In all measurements, hydrodynamic
sample injection was used by injecting the sample at the anodic
end of the capillary, with the detector at the cathodic end.
Materials and reagents
The QNs were purchased from the following suppliers: nalidixic
acid (NAL) and sparoxacin (SPA) from Sigma-Aldrich; ciproox-
acin hydrochloride (CIP) and OFL from Ranbaxy Laboratories
Limited; enoxacin (ENO) from Fluka; moxioxacin hydrochlor-
ide (MOX) from Bayer Schering Pharma AG; NOR from Smruthi
Organics Limited; dioxacin (DIF) and 30 -methyl-dioxacin
(30 M-DIF) from Orichem International Ltd; lomeoxacin (LOM),
8-uor-noroxacin (8F-NOR) and 8-uor-peoxacin (8F-PEF)
920 Rusu et al.
from Chinoin Pharmaceutical & Chemical Works and peoxacin
mesilate (PEF) from Laropharm, Romania.
All reagents and solvents were of analytical grade and were
obtained from commercial suppliers and used without further
purication. The deionized water was prepared with a Milli-Q
Direct 8 Millipore system.
Methods
Potentiometric titration
In most experiments, 10 mL of an 1 mM aqueous solution of
sample was preacidied to pH 2 with 0.5 M HCl, and then
titrated with 0.5 M KOH to pH 12. Titrations of SPA were per-
formed in the opposite direction. For NAL, the experiments
were carried out in methanol/water mixtures and the aqueous
protonation macroconstants were obtained by Yasuda
Shedlovsky extrapolation (22). The titrations were carried out
under N2 atmosphere at constant (0.15 M KCl) ionic strength
and at standard temperature 25.0 + 0.58C. The RenementProTM
software was used to calculate the protonation macroconstants.
Capillary zone electrophoresis
A background electrolyte (BGE) 25 mM borax at a pH of 9.3 was
selected. The detection was carried out in ultra-violet (UV) at
214 and 280 nm, taking into consideration the UV absorption
maxima of the studied analytes. All experiments were carried
out at room temperature.
At the beginning of each day, the capillary was conditioned
with 0.1 M NaOH (30 min), deionized water (5 min) and BGE
(20 min). The capillary was preconditioned before every run
with water (1 min) and BGE (3 min).
pH was adjusted using a Terminal 740 (Inolab) pH meter.
Micellar electrokinetic chromatography
Sodium dodecyl sulfate (SDS) as a surfactant was added in a BGE
25 mM borax. The capillary was conditioned with deionized
water (5 min), 0.1 M NaOH (5 min) and BGE (5 min) and pre-
conditioned before every run with BGE (3 min). Detection was
performed at a wavelength of 280 nm. Other parameters were
similar to those in the CZE method.
Calculation of electrophoretic mobility
The electrophoretic mobilities were calculated from the
observed migration times with the following equation:
 
Ld Lt 1 1 CZE method validation
mep m  mEOF  1
V tm tEOF
where mep is the electrophoretic mobility of the analyte investi-
gated, m is the apparent mobility, mEOF is the electroosmotic mo-
bility, tm is the migration time of the quinolone in question, tEOF is
the migration time for an unchanged solute, methanol in our case,
Lt is the total length of the capillary, Ld is the length of capillary
between injection and detection and V is the applied voltage.
Preparation of stock and standard solutions
The QN stock solutions were prepared by dissolving the sub-
stance in methanol at 10 mg/mL concentration. They were
stored in the refrigerator at 48C, and later diluted to an appro-
priate concentration. The solutions were treated in an ultrasonic
bath for 10 min and ltered through a syringe lter of 0.45 mm
pore size.
Results
CZE method
Twelve QNs can be separated with the optimized parameters
within 10 min. The electropherogram of 12 QNs is shown in
Figure 2. To achieve the best separation with shortest analysis
time, 25 mM sodium tetraborate, 20 kV voltage and 258C were
chosen as optimum parameters.
Our method was validated and validation parameters were calcu-
lated. Table I presents migration time, relative standard deviation
(RSD), resolution, the results for linear regression equations, cor-
relation coefcient (r 2), precision, limit of detection (LOD), limit
of quantication (LOQ) and number of theoretical plates (N).
The linearity response for QNs was assessed in the range
5 200 mg/mL. The linear regression equations were calculated
using six concentration levels and three replicates per concen-
tration. All correlations were over 0.99, which demonstrates a
very good linearity of the method. The reproducibility of migra-
tion time and peak-area measurement was performed by six rep-
licate injections containing 67 mg/mL of each analyte. The RSD
values were all below 1%, indicating a good precision. LOD and
LOQ were calculated by using signal-to-noise ratios of 3:1 and
10:1, respectively.

Figure 2. Separation of the 12 studied ONs by CZE ( parameters: buffer 25 mM sodium tetraborate, pH 9.3, capillary: 70 cm (62 cm effective length)  50 mm, applied voltage:
20 kV, temperature: 258C, injection pressure: 30 mbar, 5 s, UV detection wavelength: 280 nm).

Table I.
The Migration and Validation Parameters of 12 QNs

Compound Migration time (RSD %) Resolution Regression equation r2 Precision (% area) LOD (mg/mL) LOQ (mg/mL) N
MOX 7.428 (0.001) y 0.2856x 2 0.2505 0.998 0.41 8.448 28.161 162,180
SPA 7.847 (0.002) 6.44 y 0.2932x 2.0064 0.995 0.28 16.183 53.943 349,291
8F-NOR 7.992 (0.003) 2.72 y 0.4605x 2 2.835 0.991 0.58 21.426 71.422 368,931
LOM 8.078 (0.007) 1.22 y 0.5397x 2 1.7982 0.992 0.64 21.089 70.298 140,507
30 M-DIF 8.417 (0.004) 5.02 y 0.3711x 2 0.6312 0.997 0.49 12.716 42.387 362,834
ENO 8.524 (0.003) 1.14 y 0.577x 2 3.2803 0.993 0.59 18.629 62.097 112,293
CIP 8.621 (0.003) 0.99 y 0.497x 2.2817 0.997 0.61 12.454 41.514 103,666
DIF 8.707 (0.005) 1.09 y 0.4167x 2 1.7377 0.997 0.40 12.699 42.332 227,726
OFL 8.787 (0.006) 1.46 y 0.2309x 4.6011 0.993 0.75 18.878 62.927 346,418
8F-PEF 8.859 (0.005) 1.09 y 0.359x 1.031 0.991 0.69 21.977 73.259 371,866
PEF 8.949 (0.006) 1.61 y 0.4701x 2 1.098 0.995 0.33 15.245 50.818 291,181
NAL 10.172 (0.012) 16.70 y 0.391x 2 1.9646 0.996 0.25 14.082 46.940 276,869

Separation and Determination of Quinolone Antibacterials 921

 These results verify that our CZE method can be used for sep-
aration, identication and quantitative determination for a large
number of QNs. Our validated method is, therefore, the most
general and reliable one so far, with the potential to separate
QNs in environmental and food samples. Also, this separation
can be achieved in purely aqueous BGE, which is also unique in
the literature.
MEKC method
Using this method, the three important QNs can be separated
within 9 min (Figure 3). The applied parameters were identical
to those in the CZE, and 100 mM SDS was added to the BGE. The
migration data with the validation parameters are summarized in
Table II.
MEKC method validation
Our separation method of the three quinolones was validated.
The linearity response for QNs was assessed in the range
10 100 mg/mL. The linear regression equations were calculated
using six concentration levels and three replicates per concen-
tration. All correlations were over 0.99, which demonstrates a
very good linearity of the method. The reproducibility of migra-
tion time and peak-area measurement was performed by six rep-
licate injections containing 100 mg/mL of each analyte. The RSD
values were all below 1%, indicating a good precision. We proved
that MEKC can be used for the separation of structure-related
derivatives. MEKC can be especially useful for the determination
of drugs in samples having high protein content (clinical sample,
body uids) by reducing the disadvantageous matrix effects
caused by organic materials.
Figure 3. Separation of OFL, NOR and CIP by MEKC ( parameters: buffer 25 mM sodium tetraborate 100 mM SDS, capillary: 48.5 cm (40 cm effective length)  50 mm, applied
voltage: 20 kV, temperature: 208C, injection pressure: 50 mbar, 5 s, UV detection wavelength: 280 nm).
Table II.
MEKC Separation of OFL, NOR and CIP Using 100 mM SDS
Compound Migration time (RSD %) Resolution Regression equation
OFL 7.32 (0.05) y 0.7734x 28.340
NOR 8.22 (0.05) 4.61 y 0.5416x 20.715
CIP 8.54 (0.05) 1.21 y 0.3862x 15.195
922 Rusu et al.
Discussion
Preliminary study
Besides CZE separation, our aim was also to predict the electro-
phoretic mobility of every compound. Determination of proton-
ation constants and then their use to predict the pH-dependent
electrophoretic mobilities of the analytes is a valuable systematic
approach in the development of a CZE separation. The electro-
phoretic mobility is related to structural parameters such as
charge and mass (mpH vs. q/Ma). This relationship can be
described by various semiempirical methods which differ in the
a value, which is 1/3 in Stokes law, 1/2 in the classical polymer
model and 2/3 in Offords approach (23).
For the exact calculation of the average charge, the protonation
macroconstants of QNs were determined by pH-potentiometric
titrations. The logK values along with the isoelectric points
(pHIEP) are listed in Table III.
Based on the prediction methods, the relative mobility (q/Ma)
of the compounds studied was calculated as a function of pH.
The curves predicted by Offords law are presented in
Figure 4. Using these curves, the optimal pH range of the separ-
ation can be selected, which is the most important parameter in
CZE separation.
The values of protonation macroconstants and the calculated
mobilities show that NAL, as a naphtiridine derivative with one
single protonation center, differs from all other compounds. The
logK2 values are very similar differing only by up to 0.24 logK
units from the average. The logK1 values are more different,
which can be harnessed in CZE separation. At pH .8, more ef-
cient separations can be achieved. As BGE, borate buffer was,
therefore, chosen and the experimental electrophoretic mobili-
ties were measured one by one for all compounds between pH 8
and 12.5. Using the experimental electrophoretic mobility
r2 Precision (% area) LOD (mg/mL) LOQ (mg/mL) N
0.996 0.045 13.188 43.962 32,979
0.996 0.863 14.186 47.287 19,036
0.993 0.820 20.759 69.198 17,867
Table III.
The logK Values and the Isoelectric Points (pHIEP) of the QNs Studied

Compound logK1 logK2 pHIEP

NAL 6.13 + 0.01
CIP 8.53 + 0.01 6.18 + 0.01 7.36
DIF 7.45 + 0.01 5.76 + 0.01 6.61
30 M-DIF 8.42 + 0.01 5.87 + 0.01 7.15
ENO 8.69 + 0.01 6.16 + 0.01 7.43
LOM 8.93 + 0.01 5.83 + 0.01 7.38
MOX 9.32 + 0.01 6.28 + 0.01 7.80
NOR 8.52 + 0.01 6.29 + 0.01 7.41
8F-NOR 9.00 + 0.01 5.82 + 0.01 7.41
OFL 8.14 + 0.01 6.03 + 0.01 7.09
PEF 7.51 + 0.01 6.26 + 0.01 6.89
8F-PEF 7.97 + 0.01 5.80 + 0.01 6.89
SPA 8.97 + 0.01 6.32 + 0.01 7.65

Figure 5. The calculated electrophoretic mobilities of QNs as a function of pH,

contrasted to a plot of the experimental mobilities in the 8 12.5 pH range.

be explained with the intermolecular association, was also

observed (8, 25). The possible reason of this pH-dependent
interaction is that near pH 8 the compounds occur partly in
zwitterionic form, enhancing the propensity of intermolecular
associations.
The largest number of compounds with shortest analysis time
can be separated at pH 9.3, which proved to be the optimum pH
for our subsequent investigations.

CZE method optimization

Figure 4. The curves predicted by Offords equation.
values, the applicability of the prediction method could also be
checked. The experimental and calculated mobilities were,
therefore, plotted between pH 8 and 12.5. The value of the cor-
relation coefcient (r 2) is 0.84, indicating that the migration
order depends on the charge-to-mass ratio. We found best cor-
relation with Offords law, which is in agreement with the
results of Benavente et al. (13). The regression equation is
shown in Figure 5. We also observed that the correlation
between pH 8 and pH 10.5 is much better than at the higher pH.
Above pH 11, unpredictable parameters play a signicant role in
the capillary electrophoretic separation that deteriorate the cor-
relation between calculated and measured mobilities and
worsen the reproducibility in the CZE system.
Based on the preliminary data, we presumed that optimal sep-
aration could be achieved near pH 8.
However, only a few compounds could be separated in a
complex mixture at pH 8. During the experiments, the peaks
were broad, resulting in an insufcient resolution. This phenom-
enon can be assumed to be a consequence of interaction
between the capillary wall and the analytes and/or among the
QNs. Michaleas and Antoniodu-Vyza (24) took advantage of this
phenomenon and developed a simple method for the quantita-
tive analysis of FQs based on their concentration/association-
dependent 1H nuclear magnetic resonance chemical shift in
D2O over a wide range of concentrations. In our previous works,
the concentration-dependent chemical shift of FQs, which can
Electrophoretic parameters (buffer concentration, buffer addi-
tives, applied voltage, temperature, injection time and pressure)
inuencing separation performance were studied and optimized.
The migration times of the analytes increased with the increase
in the BGE concentration, because of the decrease of electro-
osmotic ow with the increase in ionic strength. Regarding
voltage and temperature parameters, the migration times
decreased with the increase in those, the limiting factors here
being the Joule heating and viscosity of BGE directly related to
temperature.
Injection pressure and time inuence the migration time
slightly only, but they have an effect on the shape of the peaks.
Thus, a high injection pressure and a short injection time were
chosen to avoid peak broadening or splitting.
Various buffer additives (organic solvents and surfactants)
were used to change the selectivity of the separation, in order to
obtain a better resolution. Addition of organic solvents (metha-
nol and acetonitrile) could not improve the resolution, probably
because the studied QNs exhibit very close electrophoretic
mobilities that cannot be differentiated by adding a small
amount of additive.
Migration times are obvious consequences of some structural
and physico-chemical properties. NAL, a naphtyridine derivative
with the lowest logK1 value, has the longest migration time,
while MOX, with pyrrolidino-piperidine ring and the largest
logK1 value, has the shortest migration time. However, it is also
obvious that the average charge is not the only property that
inuences mobility. Although QN derivatives are compounds

Separation and Determination of Quinolone Antibacterials 923

of high structural similarity, differences also exist in their mo-
lecular shape, hydration and association capability with the BGE,
capillary wall or the other investigated analytes. These proper-
ties also inuence the electrophoretic mobility, but their para-
meters are mostly unknown and are not involved in Offords
equation, causing in some cases unexpected sequences even for
very similar molecules.
MEKC method optimization
Preliminarily, SDS as a surfactant was added to BGE. Separation of
several QNs from a complex mixture is beyond the capacity of
MEKC. Nevertheless, this method can be useful as an auxiliary
technique to separate those QNs that cannot be separated by
CZE ( e.g., CIP and NOR), from a mixture of a few components.
The addition of surfactants in a BGE over the critical micellar
concentration promotes spontaneous aggregation of surfactant
molecules forming micelles in the running buffer, causing
changes in the apparent mobility of the analytes due to hydro-
phobic interactions (26, 27). The advantage of this method is
that some compounds that could not be separated by CZE can
be separated by MEKC.
Our aim in this investigation was to develop a method for the
separation of CIP, NOR and OFL, the three most frequently used
therapeutic QNs (28, 29).
Table II shows that the MEKC migration order (OFL, NOR and
CIP) is different from CZE separation (CIP and OFL). The migra-
tion order in MEKC is inuenced not only by the average charge
but also by the hydrophobicity of the analytes. Compounds with
higher micelle afnity have slower migration, compared with
molecules that linger in the bulk phase. OFL migrates faster than
the other two components because its tricyclic skeleton can be
less accommodated in the micelles.
Conclusions
CZE was proved to be an appropriate method for the fast and
efcient separation of the same generation members of the
quinolone family of drugs that are highly similar in every
physico-chemical property, including electrophoretic mobility.
In addition, MEKC is veried to be a tool for the separation of
QNs with very similar structural characteristics.
For each method, the analytical parameters were optimized to
achieve the best separation. Validation parameters, including
precision, linearity, LOD and LOQ, for each compound were also
determined, providing the basis for further improvements in sep-
aration of even larger numbers of analytes and also for determin-
ation of trace contaminants in pharmacopoeial monographs.
Acknowledgments
The authors are grateful to Bayer Schering Pharma AG for provid-
ing the moxioxacin hydrochloride, and to Laropharm, Romania,
for providing peoxacin mesylate.
Funding
This work was supported by TAMOP 4.2.1.B-09/1/KMR and the
National Research Fund of Hungary (OTKA T 73804).
924 Rusu et al.
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