Establishment and Evaluation of the Novel Tetramethylammonium-L-Hydroxyproline Chiral Ionic Liquid Synergistic System Based on Clindamycin Phosphate for Enantioseparation by Capillary Electrophoresis

Article abstract of DOI:10.1002/chir.22463

Much attention has been paid to chiral ionic liquids (ILs) in analytical chemistry, especially its application in capillary electrophoresis (CE) enantioseparation. However, the investigation of chiral ionic liquids synergistic systems based on antibiotic chiral selectors has been reported in only one article. In this work, a novel chiral ionic liquid, tetramethylammonium-L-hydroxyproline (TMA-L-Hyp), was applied for the first time in CE chiral separation to evaluate its potential synergistic effect with clindamycin phosphate (CP) as the chiral selector. As observed, significantly improved separation was obtained in this TMA-L-Hyp/CP synergistic system compared to TMA-L-Hyp or a CP single system. Several primary factors that might influence the separation were investigated, including CP concentration, TMA-L-Hyp concentration, buffer pH, types and concentrations of organic modifier, applied voltage, and capillary temperature. The best results were obtained with a 40 mM borax buffer (pH 7.6) containing 30 mM TMA-L-Hyp, 80 mM CP, and 20% (v/v) methanol, while the applied voltage and temperature were set at 20 kV and 20°C, respectively. Chirality 27:598-604, 2015.

Full text of DOI:10.1002/chir.22463

CHIRALITY 27:598–604 (2015)
Establishment and Evaluation of the Novel Tetramethylammonium-L-
Hydroxyproline Chiral Ionic Liquid Synergistic System Based on
Clindamycin Phosphate for Enantioseparation by Capillary
Electrophoresis
GUANGFU XU, YINGXIANG DU,1,2,3* FAN DU, JIAQUAN CHEN, TAO YU, QI ZHANG, JINJING ZHANG, SHUAIJING DU, AND
1
1
1
1
1
1
4
1
ZIJIE FENG
1
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, P.R. China
2
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical
University, Nanjing, P.R. China
3
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P.R. China
4
College of Chemistry and Molecular Engineering, Peking University, Beijing, P.R. China
ABSTRACT
Much attention has been paid to chiral ionic liquids (ILs) in analytical chemistry,
especially its application in capillary electrophoresis (CE) enantioseparation. However, the inves-
tigation of chiral ionic liquids synergistic systems based on antibiotic chiral selectors has been
reported in only one article. In this work, a novel chiral ionic liquid, tetramethylammonium-L-
hydroxyproline (TMA-L-Hyp), was applied for the first time in CE chiral separation to evaluate
its potential synergistic effect with clindamycin phosphate (CP) as the chiral selector. As ob-
served, significantly improved separation was obtained in this TMA-L-Hyp/CP synergistic sys-
tem compared to TMA-L-Hyp or a CP single system. Several primary factors that might
influence the separation were investigated, including CP concentration, TMA-L-Hyp concentra-
tion, buffer pH, types and concentrations of organic modifier, applied voltage, and capillary tem-
perature. The best results were obtained with a 40 mM borax buffer (pH 7.6) containing 30 mM
TMA-L-Hyp, 80 mM CP, and 20% (v/v) methanol, while the applied voltage and temperature
were set at 20 kV and 20°C, respectively. Chirality 27:598–604, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: enantioseparation; synergistic effect; ionic liquids; capillary electrophoresis;
antibiotics
Nowadays, enantiomeric separation has gained more and
more attention since different enantiomeric forms of chiral
compounds have different physiological, toxicological, or
considerable attention. Since ILs were first introduced by
Armstrong et al.12 in 1999, they have been used as a suitable
13
stationary phase in GC and additive/stationary phase in
1
14
pharmacodynamic properties. Recognizing the importance
HPLC . ILs can be used as background electrolyte (BGE)
1
5,16
of chiral effects, the US Food and Drug Administration and
the European Medicines Agency have issued guidelines re-
quiring information on the enantiomeric purity of the optically
or additives in capillary zone electrophoresis (CZE)
and
nonaqueous capillary electrophoresis (NACE),17 as well as
supported coatings of the capillary wall in capillary
electrochromatography (CEC). Recently, several studies
2
18
active compounds prior to their marketing. For this reason,
many effective chiral separation techniques have been ex-
plored such as high-performance liquid chromatography
have reported the application of chiral ILs (CILs) as chiral se-
lectors alone or as additives to cyclodextrins (CDs) and their
derivatives for CE enantioseparation,1
9–26
and researchers
(
HPLC), gas chromatography (GC), etc. Capillary electropho-
resis (CE) has been proven to be an excellent method in this
have noted that the combination of CDs and CILs exhibited
field for its high separation efficiency, fast separation, low
a significant synergistic effect during the enantioseparation
3
21–25
sample consumptions, and feasibility. In CE, most enantio-
process.
As far as we know, however, the investigation
meric separations are carried out by the use of a buffer solu-
tion containing chiral selectors, mainly cyclodextrins (CDs)
of chiral ionic liquid synergistic systems based on antibiotics
chiral selectors has been reported in only one article.
24
4
5
and their derivatives, polysaccharides, macrocyclic antibi-
Tetramethylammonium-L-hydroxyproline (TMA-L-Hyp) IL
(Fig. 1a), belonging to the short-chain tetraalkylammonium-
based Ils, has never been previously investigated. Compared
with long-chain ammonium surfactants, short-chain
tetraalkylammonium-based ILs are relatively more hydro-
philic and, thus, can be used in high concentrations for low
6
,7
otics, etc. Unfortunately, sometimes the aforementioned
chiral selectors used alone may not provide adequate selecti-
vity and resolution. Hence, more than one chiral selector is
required to improve the enantioseparation.
Ionic liquids (ILs) are defined as materials consisting of
8
only ionic components with low melting points. They have
a number of unique chemical and physical properties includ-
ing negligible vapor pressure, low melting point, high solubi-
*
Correspondence to: Yingxiang Du, Department of Analytical Chemistry,
China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu
10009, P.R. China. E-mail: du_yingxiang@126.com
9
,10
lity power, and being air- and moisture-stable.
These
2
properties enabled ILs to be used in many fields such as or-
Received for publication 21 July 2014; Accepted 10 April 2015
DOI: 10.1002/chir.22463
1
1
ganic synthesis, electrochemistry, and so on. To date, the
application of ILs in separation analysis has received
Published online 11 June 2015 in Wiley Online Library
(wileyonlinelibrary.com).
©
2015 Wiley Periodicals, Inc.

SYNERGISTIC SYSTEM FOR CE ENANTIOSEPARATION
599
Experimental Procedures
Buffer solution was a 40 mM borax buffer containing methanol
20% v/v), if not stated otherwise. The running buffer was freshly prepared
(
by dissolving CP and tetramethylammonium-L-hydroxyproline IL in the
buffer solution with a specified pH, and then pH was adjusted exactly to
a desired value by adding a small volume of 1 M hydrochloric acid or so-
dium hydroxide solution using a microsyringe. Each buffer solution was
filtered through a 0.45 μm pore membrane filter prior to use. The racemic
drugs were dissolved in water. Final sample solutions for injection were all
at a concentration of 0.4 mg/ml.
Fig. 1. Structures of the TMA-L-Hyp IL (a) and CP (b).
Calculations
Separation factor (α) and chiral resolution (Rs.) were calculated by the
equations and , respectively:
conductivity. Recently, we reported the use of a new anti-
biotic, clindamycin phosphate (CP), which belongs to the
lincosamides family as a novel chiral selector in CE. CP con-
1
2
7
a ¼ t2=t1
(1)
(2)
sists of an amide portion, an amino portion (tertiaryamine), a
phosphate ester portion, and an aglycon portion in each mole-
cule (Fig. 1b). It possesses not only high solubility but also
low viscosity in both water and alcohols. Furthermore, due
to the lack of aromatic rings in the structure, it exhibits very
weak UV absorption. In this work, a novel TMA-L-Hyp IL syn-
ergistic system based on CP was established for the first
time. Under the same condition, the enantioseparation capa-
bility of the single system (TMA-L-Hyp IL or CP) and the dual
system (TMA-L-Hyp IL and CP) were investigated and com-
pared to search for possible synergistic effects. Several sepa-
ration conditions that might influence this dual system were
examined and optimized, including CP concentration, TMA-
L-Hyp concentration, buffer pH, types and concentrations of
organic modifier, applied voltage, and capillary temperature.
Rs ¼ 2ðt2 ꢀ t1Þ=ðw1 þ w2Þ
Where t
tiomer, respectively, and w
base. The electroosmotic flow (EOF) was expressed by equation :
1
, t
2
are the migration time of the first and second eluted enan-
, w are the corresponding widths at the peak
1
2
3
μeof ¼ lL=t0V
(3)
Where l is the effective capillary length, L is the total capillary length, t
0
is the migration time of neutral maker (thiourea), and V is the applied
voltage.
RESULTS AND DISCUSSION
Figure 2 shows the comparative electropherograms of the
dual system and the single CP system. It can be observed that
the R of the tested drugs in the dual system had a significant
s.
increase compared with the single system: the R of the PRO,
s.
NEF, CHL, and CIT increased from 2.21, 3.01, 2.53, 0.95 to
3.63, 4.21, 3.55, 1.35, respectively. This indicates the exis-
tence of synergistic effect with the combination of TMA-L-
Hyp and CP in the separation system. It is worth noting that
all the studied racemic drugs could not be enantioseparated
when the TMA-L-Hyp IL was used alone.
In order to evaluate the stereoselectivity of the dual system
toward the tested drugs, the effects of a series of separation
conditions on enantioseparation were investigated.
EXPERIMENTAL
Chemicals and Reagents
CP (purity > 99%) was supplied by Jiangsu Institute for Food and Drug
Control (Nanjing, China). Tetramethylammonium-L-hydroxyproline ionic
liquid (purity >99%) was purchased from Shanghai Cheng Jie Chemical
(
Shanghai, China). Propranolol hydrochloride (PRO) was purchased
from Sigma (St. Louis, MO). Nefopam hydrochloride (NEF), citalopram
hydrobromide (CIT), and chlorphenamine maleate (CHL) were supplied
by Jiangsu Institute for Food and Drug Control. All these drug samples
were racemic mixtures.
Methanol, isopropanol, and acetonitrile, all of HPLC grade, were
purchased from Jiangsu Hanbon Sci. & Tech. (Nanjing, China). Sodium
hydroxide, hydrochloric acid, and sodium tetraborate decahydrate, all
of analytical grade, were purchased from Nanjing Chemical Reagent
Effect of CP and TMA-L-Hyp IL Concentrations on
Enantioseparation
The concentration of chiral selector is critical to the
enantioseparation for the migration time and resolutions of
enantiomers. To obtain the optimum concentrations of CP
and TMA-L-Hyp IL, their effects on the resolution of tested
drugs were investigated.
(
Nanjing, China). Double-distilled water was used throughout all the
experiments.
The effect of CP concentration was investigated through
fixing the concentration of TMA-L-Hyp IL at 30 mM while
changing the CP concentration (20, 40, 60, 80, 100 mM) with
Apparatus
Electrophoretic experiments were performed with an Agilent 3D capil-
lary electrophoresis system (Agilent Technologies, Waldbronn,
Germany), which consisted of a sampling device, a power supply, a
photodiode array UV detector (wavelength range from 190 nm to 600 nm),
and a data processor. The whole system was driven by Agilent ChemStation
software (Revision B.02.01) for system control, data collection, and analysis.
It was equipped with a 50 cm (41.5 cm effective length) × 50 μm i.d. uncoated
fused-silica capillary (Hebei Yongnian County Reafine Chromatography,
Hebei, China). A new capillary was flushed for 20 min with 1 M NaOH,
0 min with 0.1 M NaOH, and 30 min with water. At the end of each day, it
was flushed successively with 0.1 M NaOH (10 min) and water (10 min).
Between consecutive injections, the capillary was rinsed with 0.1 M NaOH
2 min), water (2 min), and running buffer (5 min). Sample injections were
4
0 mM borax buffer solutions (pH 7.6) containing 20% v/v
methanol. As can be observed in Table 1, it was evident that
the R of all the tested drugs increased generally as the CP
s.
concentration ranged from 20 to 80 mM, then decreased with
the CP concentration ascending from 80 to 100 mM. More-
over, Table 1 shows that the migration time of all the analytes
increased in parallel with the increasing CP concentration
due to the strengthened interaction between chiral selectors
and the analytes as well as the increased viscosity of the run-
ning buffer. Thus, 80 mM CP was chosen to separate the
tested drugs.
2
(
performed by pressure (50 mbar, 5 s). Enantioseparations were performed
at a constant applied voltage in a range of 12–28 kV, and the temperature of
capillary was controlled at 14–28°C using an air-cooling system.
Furthermore, the CP concentration was kept at 80 mM
while the TMA-L-Hyp IL concentration was changed over
Chirality DOI 10.1002/chir

6
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XU ET AL.
Fig. 2. Electropherograms of PRO,NEF, CHL,CIT in the circumstances (A) 80 mM CP (B) 80 mM CP and 30 mM TMA-L-Hyp IL. Other Conditions: 40 mM
borax buffer solution (pH 7.6)with methanol (20% v/v); applied voltage, 20 kV; capillary temperature, 20°C; fused-silica capillary, 50 cm (41.5 cm effective length)
50 m id; UV absorption at 289 nm (PRO), 230 nm (NEF), 265 nm (CHL) and 237 nm (CIT).
×
the range of 10 mM to 50 mM. Table 2 shows the effect of
TMA-L-Hyp IL concentration on Rs. and α of the tested
analytes. The migration time of all the analytes increased with
the ascending IL concentration. This effect might be partly at-
tributed to the increase of the buffer viscosity and the adsorp-
tion of IL cation on the capillary wall, which led to the
Chirality DOI 10.1002/chir

SYNERGISTIC SYSTEM FOR CE ENANTIOSEPARATION
601
Chirality DOI 10.1002/chir

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02
XU ET AL.
ꢀ5
ꢀ
4
2
ꢀ1 ꢀ1
decrease of EOF from 1.17 × 10 cm V
cm V
s
to 5.41 × 10
the TMA-L-Hyp-drugs-CP combination, it can be supposed
that two kinds of main bindings exist among the chiral
selectors, CIL, and analytes according to the structures of
CP, TMA-L-Hyp IL, and the analytes. One might be the
hydrogen-bonding among the amino, hydroxyl groups in the
drug, the amino, hydroxyl groups in CP, and the hydroxyl
group in TMA-L-Hyp IL; the other one might be the electro-
static interaction among the amino group in the drug, the
phosphate group in CP, and the carboxyl group in TMA-L-
Hyp IL. Thus, the optimum pH value was 7.6 for the analytes.
2
ꢀ1 ꢀ1
s . The R of the analytes increased as the concen-
s.
tration added up to 30 mM, and subsequently tended to drop
with the concentration ascending from 30 mM to 50 mM. The
decrease may be explained by more Joule heat caused by the
increased ionic strength of the running buffer, which led to a
broad peak. It is worth noting that sometimes the reduced
EOF would also, to some extent, influence the R of drug en-
s.
antiomers because the prolonged migration time would pro-
vide more chances for the enantiorecognition during the
separation process. Thus, we investigated the influence of
EOF on enantioseparation by decreasing the applied voltage
from 20 kV to 12 kV in the single CP system. As expected,
the migration times of the four tested analytes increased by
about 6 to 8 min. However, the decreased applied voltage
did not result in significantly increased Rs. and α for the
analytes compared to the ILs synergistic system (Supporting
Information, Fig. S1). This observation indicated, from an-
other aspect, that the improved enantioseparation in the
CP/chiral ILs system was mainly attributed to the synergistic
effect between chiral selector and the CILs.
Effect of the Types and Concentrations of Organic Modifier
on Enantioseparation
The use of an organic modifier has a great effect on migra-
tion time and R . It not only affects the effective charge of the
s
enantiomers and chiral selectors, the viscosity of the BGE,
and the EOF, but also the complexation interaction involved
in the enantioseparation mechanism when antibiotics are
27
used as the chiral selectors.
In this work, methanol, isopropanol, and acetonitrile were
added separately to the running buffer with a concentration
of 20% (v/v). From the results of the experiment, when the
isopropanol was used, the peaks of the analytes and the sol-
vent were found to be mutually interfered. It can be observed
from Supporting Information, Figure S2. that methanol had
better results, with satisfactory resolutions and proper migra-
tion time compared to acetonitrile. Thus, methanol is the best
choice for the tested enantiomers.
Furthermore, the effect of the concentration of methanol
(0%–30% v/v) on the separation was studied. According to
the results shown in Table 3, the migration time was
prolonged with the increase of the methanol concentration
because of the changing of the buffer viscosity, the EOF,
and the interactions among the complexing antibiotic,
Taking the Rs. and proper migration time into consider-
ation, 80 mM CP and 30 mM TMA-L-Hyp IL were chosen as
the optimum values for the four analytes.
Effect of pH on Enantioseparation
In the CE enantioseparation, buffer pH is one of the most
important parameters because it determines the EOF of the
capillary, the extent of ionization and mobility of analytes,
CILs, and the chiral selector. In addition, it influences the in-
teractions among chiral selector, chiral ILs, and analytes and
results in the changing of resolution. In this study, the effect
of pH for the tested drugs on enantioseparation was studied
by varying the pH (7.2–7.8) of the 40 mM borax buffer (con-
taining 20% v/v methanol) with 80 mM CP and 30 mM
TMA-L-Hyp IL. It can be observed from Figure 3 that as the
analytes, and the TMA-L-Hyp IL. Additionally, the R of the
s.
four compounds increased generally when the methanol
buffer pH ascended from 7.2–7.6, the R of all studied enantio-
concentration increased from 0% to 20%; however, the R de-
s.
s.
mers tended to increase; however, when the pH was over 7.6,
creased with further increasing of the methanol concentra-
tion. As a result, the optimum methanol concentration for
the studied analytes was 20% (v/v).
the R of analytes decreased. This trend gave important infor-
s.
mation on the interactions among the CP, TMA-L-Hyp IL, and
the analytes. Since the degrees of protonation in drugs, CP,
and TMA-L-Hyp IL were dependent on pH, the optimum pH
would point to the most compatible state of the amino func-
tion in the drugs for binding or reacting to the groups in
TMA-L-Hyp IL (hydroxyl or amino) and the groups in CP
Effect of Applied Voltage and Capillary Temperature on
Enantioseparation
The applied voltage affects the enantioseparation through
three main aspects: column efficiency, migration time, and
Rs. Generally, an optimum voltage value is obtained during
the separation. In this study, the influence of applied voltage
was investigated from 12 to 28 kV. As Supporting Information,
Figure S3a describes, an increase of applied voltage resulted
in a general decrease of the migration time of all studied
(
amino, hydroxyl, or phosphate). In the present system of
analytes. As to R , when the voltage value was lower than
s.
2
0 kV, the R of the analytes increased with the increase of
s.
the voltage value. It can be illustrated by the improving of
the column efficiency. However, with the voltage value
increasing continually, the Rs. of the tested substances
decreased due to the generation of Joule heating, which can
result in the broadening of the peaks. Thus, the applied volt-
age was set at 20 kV.
Buffer viscosity, pKa, and complexation interaction among
the chiral selector, CILs, and the analytes are affected by
the capillary temperature. It can be observed from Supporting
Information, Figure S3b that when the capillary temperature
changed from 14 to 28°C, the enantiomeric migration time
Fig. 3. Effect of pH on the Rs. of the studied drugs. Conditions: 40 mM
borax buffer containing 80 mM CP, 30 mM TMA-L-Hyp ionic liquid and 20%
(
v/v) methanol; pH, 7.2 to 7.8. All other conditions as in Figure 2.
Chirality DOI 10.1002/chir

SYNERGISTIC SYSTEM FOR CE ENANTIOSEPARATION
TABLE 3. Effect of the concentration of methanol on the Rs of the studied drugs
Methanol concentration (v/v)
603
0
%
10%
(min)
20%
(min)
30%
(min)
Chiral
Compound
t
1
/ t
2
(min)
R
s.
t
1
/ t
2
R
s.
t
1
/ t
2
R
s.
t
1
/ t
2
R
s.
PRO
NEF
CHL
CIT
6.568/6.690
5.799/5.901
6.561/6.683
6.445
0.63
1.23
1.24
—
9.205/9.464
7.850/8.076
9.120/9.354
8.904/9.034
1.93
2.04
2.07
0.58
12.130/12.550
10.371/10.777
12.446/12.825
11.933/12.118
3.63
4.21
3.55
1.35
16.256/17.074
13.384/14.176
16.782/17.344
15.244/15.579
2.71
3.51
1.51
0.99
Conditions: 40 mM borax buffer (pH 7.6) containing CP 80 mM and 30 mM TMA-L-Hyp ionic liquid; Methanol concentration, 0% to 30% (v/v); “—” : single peak. All
other conditions as in Figure 2.
2
. Rousseau A, Florence X, Pirotte B, Varenne A, Gareil P, Villemin D, Chiap
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s.
ture ranging from 14 to 20°C, then decreased when the capil-
lary temperature ascended from 20 to 28°C. Therefore, 20°C
was chosen for enantioseparation of the studied drugs.
The run-to-run reproducibility was evaluated by five repli-
cate testings. All separations were performed under the opti-
mum separation conditions as illustrated in Figure 2. The
variations of migration times (the RSD values of the migration
times) were less than 2.8%. It can therefore be concluded that
the TMA-L-Hyp synergistic system based on CP can provide
satisfactory reproducibility.
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The authors declare no conflict of interest.
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