- Email: [email protected]
Chiral capillary electrophoresis
Journal Pre-proof Chiral Capillary Electrophoresis Samuel Bernardo-Bermejo, Elena Sánchez-López, María Castro-Puyana, María Luisa Marina PII:
S0165-9936(19)30518-7
DOI:
https://doi.org/10.1016/j.trac.2020.115807
Reference:
TRAC 115807
To appear in:
Trends in Analytical Chemistry
Received Date: 8 September 2019 Revised Date:
20 December 2019
Accepted Date: 5 January 2020
Please cite this article as: S. Bernardo-Bermejo, E. Sánchez-López, M. Castro-Puyana, M.L. Marina, Chiral Capillary Electrophoresis, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2020.115807. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
1
CHIRAL CAPILLARY ELECTROPHORESIS
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Samuel Bernardo-Bermejo1, Elena Sánchez-López1,2, María Castro-Puyana1,2, María
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Luisa Marina1,2*
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Ciencias, Universidad de Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de
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Henares (Madrid), Spain.
Departamento de Química Analítica, Química Física e Ingeniería Química, Facultad de
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Instituto de Investigación Química Andrés M. del Río (IQAR), Universidad de Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de Henares (Madrid), Spain.
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16
*
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Email: [email protected]
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Tel: (+34)-918854935
Corresponding author: Maria Luisa Marina
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1
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ABSTRACT
23
The implications of chirality in different environments are already well known and reported
24
extensively in the literature. Capillary Electrophoresis, a separation technique that only
25
requires few nanoliters of sample, has demonstrated its potential for chiral analysis in the
26
past years. The aim of this article is to provide an overview on the fundamentals and
27
characteristics of Chiral Capillary Electrophoresis as well as the main advances and trends
28
in this topic. Special attention is paid to the most recent technological and methodological
29
developments achieved mainly in the most employed separation mode (Electrokinetic
30
Cromatography). The most noteworthy and recent applications reported on the
31
enantiomeric separation and determination of compounds in pharmaceutical, food,
32
biomedical, environmental or forensic samples will also be critically overviewed. The
33
characteristics of the developed methodologies will be detailed in Tables and future trends
34
will also be discussed.
35
36
Keywords:
Chiral
Capillary
Electrophoresis;
Enantiomers;
37
Chromatography; pharmaceutical formulations; food; biological samples.
Electrokinetic
38
39
Abbreviations: A-β-CD, acetyl-β-CD; APFO, ammonium perfluorooctanoate; CFSE, 5-
40
carboxyfluorescein succinimidyl ester; CILs, chiral ionic liquids; CNTs, carbon nanotubes;
41
CM-β-CD, carboxymethyl-β-CD; CSF, cerebrospinal fluid; DM-β-CD, 2,6-dimethyl-β-CD;
42
EBC, exhaled breath condensate; FASI, field-amplified sample injection; FASS, field-
43
amplified sample stacking; FMOC, 9-fluorenylmethoxycarbonyl chloride; FLEC, (+)-1-(92
44
fluorenyl)
ethyl
chloroformate;
FITC,
fluorescein
45
hydroxypropyl-γ-CD; LEKC, liposome EKC; M-β-CD, methyl-β-CD; NPs, nanoparticles;
46
OS-γ-CD, octa (6-O-sulfo)-γ-CD; LE-CEC, open tubular ligand exchange CEC; succ-γ-CD,
47
succinyl-γ-CD; S-α-CD, sulfated α-CD; S-β-CD, sulfated β-CD; S-γ-CD, sulfated γ-CD;
48
TM-β-CD, (2,3,6-tri-O-methyl)-β-CD; SBE-β-CD, sulfobutyl ether-β-CD.
49
50
51
52
53
54
55
56
57
58
59
60
61
3
isothiocyanate;
HP-γ-CD,
2-
62
1. Introduction
63
Chiral analysis is nowadays one of the most interesting areas within Analytical
64
Chemistry. This interest arises from the different properties that the enantiomers of a chiral
65
compound may have which originates the need to achieve their individual determination
66
[1]. As enantiomers of chiral drugs, food components or additives, agrochemicals or
67
pollutants can exhibit different biological activity, toxicity, degradation rates, persistence,
68
and other properties, the relevance of chiral analysis includes very different fields such as
69
the pharmaceutical, biomedical, food or environmental, among others [2-4].
70
Separation techniques are very powerful tools to achieve chiral analyses. Among
71
them, liquid chromatography (LC), gas chromatography (GC), supercritical fluid
72
chromatography (SFC) and capillary electrophoresis (CE) have been the most employed
73
[5]. Although LC has been the most frequently used, CE has shown to have very interesting
74
characteristics to carry out enantiomeric separations [1] and it has had an important impact
75
in the last years as it can be observed in Figure 1 which shows the number of publications
76
dealing with chiral analysis using different analytical techniques from 2015.
77
On the one hand, chiral analysis benefits from some inherent characteristics of CE
78
such as its high efficiency and resolution, low consumption of reagents and samples or
79
short
80
electrophoretic principles can be applied to increase the sensitivity of the developed
81
methodologies which can also be enhanced using detection systems such as fluorescence or
82
mass spectrometry (MS), among others. On the other hand, the possibility of adding the
83
chiral selector directly to the separation medium in the so-called Electrokinetic
84
Chromatography (EKC) mode confers a high flexibility to chiral CE since the nature of the
analysis
times.
Also,
in-capillary
4
preconcentration
techniques
based
on
85
selector (or mixture of chiral selectors) and also its concentration can easily be changed
86
favoring the separation of enantiomers and reducing the economic costs derived from the
87
use of chiral chromatographic columns. The big variety of chiral selectors that can be
88
employed in EKC also contributes to increase the flexibility of CE to achieve an
89
enantiomeric separation [6]. In addition, the low amounts of reagents, solvents and samples
90
needed in CE fits well in the principles of green chemistry and reduces the environmental
91
impact of the developed methodologies. Although a chiral stationary phase can also be
92
employed in Capillary Electrochromatography (CEC) [7], EKC is by far the most employed
93
mode to perform enantiomeric separations in the format of CE as it will be shown in next
94
sections.
95
Numerous reviews devoted to chiral analysis by CE have been published in the last
96
years showing the high interest of this subject in different research fields. Table 1 groups
97
some of the most representative and recent review articles focused on general aspects of
98
enantiomeric separations by CE such as the current trends and future directions [8], the
99
history and contemporary theory of this technique as well as the study of the mechanisms
100
of non-covalent (enantioselective) interactions in different disciplines [9] or the potential of
101
different chiral selectors such as sugar-, amino acid-, nucleic acid-based polymers and ionic
102
liquids, among others [6, 10-12]. Although not included in this Table, other articles devoted
103
to review more specific and applied aspects of chiral CE have also been recently published
104
such as its application in food, environmental and pharmaceutical analysis [2-4, 13].
105
The aim of this article is to provide an overview of the most relevant contributions
106
of CE to chiral separations paying special attention to the most innovative and recent
107
developments and improvements achieved. The fundamentals and characteristics of chiral 5
108
CE are also included and the most noteworthy applications to the analysis of real samples
109
in different fields reported in the last years will also be critically discussed.
110
2. Fundamentals and characteristics of chiral CE
111
CE separation principle relies on differences in the effective mobilities of the given
112
species in a narrow silica capillary (e.g. 50 µm internal diameter) under an electric field.
113
This effective mobility is defined as the sum vector of the electrophoretic mobility and the
114
mobility of the so-called electroosmotic flow (EOF). Silanol groups from the inner surface
115
of a fused-silica capillary are ionized above pH 3. At this or higher pH, positive ions from
116
the electrolyte solution, more widely known as background electrolyte (BGE), will then be
117
attracted by the negative wall generated by the silanol groups, forming a double layer.
118
Applying an electric voltage to the capillary ends will result in a flow of cations towards the
119
cathode. This flow, i.e. the EOF, will drag the bulk solution on this direction. Thus,
120
considering a cathodic detection, positively charged molecules will reach the detector first,
121
followed by neutral species (null electrophoretic mobility) and negatively charged
122
molecules will then follow. Note that both the size and charge of the molecule will also
123
play a role in the effective mobility since the electrophoretic mobility is proportional to the
124
effective charge and indirectly proportional to the radius. Detection of negatively charged
125
species will only be achieved if the EOF is able to counteract their electrophoretic mobility
126
towards the anode. It is clear that this is the most simplistic way, but the enormous
127
flexibility of CE opens a wide array of possibilities. This simple scenario can be further
128
modified, if, for instance, coated capillaries are used. There is a high number of works
129
reported in literature showing the different capillary coatings [14]. This coating can be
6
130
temporary, meaning that it will have to be renewed in every analytical run, or can be
131
permanent, when the coat is covalently attached to the capillary inner surface.
132
CE offers higher efficiency obtaining narrower peaks typically with higher
133
resolution because of the flat flow occurring in the capillary opposed to the parabolic flow
134
found in LC. This also results in a larger peak capacity in CE, having a million theoretical
135
plates. Among the other different advantages of CE over LC, it is of relevance to highlight
136
the minimal requirements for sample and solvent consumption. Typically, sample injection
137
in CE is in the nanoliter range compared to the microliter range for LC. The inherent
138
dimensions of CE make it a miniaturized technique with minimal waste and therefore,
139
reduced environmental impact.
140
However, when one wants to separate enantiomers a more difficult mechanism
141
needs to be applied. Enantiomers have identical physicochemical properties, hence, their
142
electrophoretic mobility will be exactly the same, and enantioseparation will only be
143
possible under a chiral environment, e.g. interaction with a chiral selector. Typically, this
144
interaction is non-covalent (usually, van der Waals forces or hydrogen bonding are
145
involved), which origins the formation of temporary diastereoisomers complexes which are
146
then separated based on their different mobilities in the so-called direct mode. On the other
147
hand, and used to a much lower extent is the indirect mode in which each enantiomer is
148
covalently linked to an enantiopure chiral derivatization reagent. These two new molecules
149
are now diastereoisomers, thus, the differences in their physicochemical properties will
150
enable their separation under achiral conditions. The less popularity of this later approach is
151
due to the fact that the availability of these chiral derivatization reagents is limited and they
152
must be of high enantiomeric purity. Their high cost and also the time-consuming step 7
153
consequence of the derivatization makes it a not desired approach. Considering that the
154
direct mode is the approach most used in CE, we will further discuss its features.
155
Overall, chiral separations in CE are based on two mechanisms, one
156
chromatographic and the other electrophoretic. The chromatographic mechanism arises
157
from the differences in the complexation constants between the enantiomers and the chiral
158
selector. The electrophoretic mechanism is based on the different electrophoretic mobilities
159
that the two enantiomer-chiral selector complexes might have due to subtle differences in
160
size of these complexes, if an analyte is more or less “embedded” in the chiral selector. For
161
further details on mechanisms on enantioseparations in CE, readers are redirected to
162
previous contributions [15].
163
Now, it is important to describe the two approaches used to carry out chiral
164
separations in CE: EKC and CEC. In EKC, which is by far the most used mode, the chiral
165
selector is solved in the BGE forming a “pseudo-stationary phase” making the interaction
166
with the analyte happen in the liquid state and, if it shows enantiorecognition, it will enable
167
the enantioseparation. Several chiral selectors are currently available in CE but nowadays
168
there is an ongoing search to find more CE suitable chiral selectors with exceptional
169
properties. Among the available chiral selectors, cyclodextrins (CDs) deserve a special
170
mention. CDs are cyclic oligosaccharides composed of mainly 6 (α-CD), 7 (β-CD) or 8 (γ-
171
CD) α-D-glucopyranoside units linked via 1-4 bonds and are produced from starch via
172
enzymatic treatment. These “donut”-shaped molecules have secondary and primary
173
hydroxyl groups exposed on the outside, making the outside hydrophilic and the inside
174
hydrophobic. Depending on the interaction, enantiorecognition can be performed both in
175
the inside and/or in the outside. Several research groups have aimed their efforts on 8
176
obtaining a deeper understanding on the mechanisms of this interaction. Nuclear magnetic
177
resonance (NMR), often in combination with molecular modelling, is typically used to
178
study the nature of such interaction [16]. Native CDs can be chemically modified via
179
hydroxyl-derivatization to change their enantioselectivity, enhancing the applicability of
180
these molecules as chiral selectors. Note that the use of single-isomer CD derivatives is
181
advantageous compared to the ones with random substitution for reproducibility reasons.
182
However, single-isomer CDs prices are still somehow prohibited.
183
Other chiral selectors used in EKC are chiral surfactants. When working above the
184
so-called critical micelle concentration (CMC) they will be in the micelle state, i.e.
185
spherical-like aggregates of surfactant molecules in which the hydrophilic heads are in
186
direct contact with the solvent whereas the hydrophobic tails are embedded in the middle of
187
the micelle aggregate. Other chiral selectors falling under EKC mode are ligand-exchange
188
compounds, proteins, bile salts, polysaccharides, ionic liquids, chiral crown ethers,
189
nucleotides, and antibiotics, among others. Readers are encouraged to go to different
190
reviews for detailed description on this topic [10, 17-20].
191
Unlike in EKC, in CEC, chiral stationary phases are employed. CEC is considered
192
to be in between electrophoretic and chromatographic techniques since it combines
193
characteristics of both. The mechanisms of separation of enantiomers in CEC are due to the
194
electrophoretic mobility of the charged analytes and their partition between the chiral
195
stationary phase and their free state in the mobile phase. There are three types of columns
196
in CEC: packed, open tubular, and monolithic columns, depending on how the chiral
197
stationary phase is placed in the capillary. Since this CE mode has not been widely reported
198
in last years, readers are redirected to previously published contributions on the matter [21]. 9
199
Even though the most used media in chiral separations are based on aqueous
200
solvents, CE also enables the possibility to work under non-aqueous conditions in the so
201
called non-aqueous capillary electrophoresis (NACE). This mode is indicated when the
202
analytes of interest or the chiral selectors are not soluble in water. The most common chiral
203
selectors employed in NACE are neutral and charged cyclodextrins as well as ion-pair
204
selectors and antibiotics [15]. Some advantages of NACE include shorter analysis times
205
with higher reproducibility thanks to the less Joule heat produced even at high voltages, as
206
well as more suitable coupling to MS detection [22]. Although there is a wide array of non-
207
aqueous solvents used, methanol is the most popular one. To favor charge of the analytes, it
208
is also needed to add an electrolyte to the non-aqueous solvent, such as formic or acetic
209
acid with or without the corresponding salt (ammonium formate or acetate). Although
210
NACE enables to achieve chiral separations, as we will see in coming sections, this CE
211
mode has scarcely been employed compared with EKC.
212
3. Advances in chiral CE
213
From the middle of 1980s when the first applications of CE in the field of chiral
214
separations were published, this technique has shown its attractive capabilities to achieve
215
highly efficient separations of enantiomers. Although the use of CE is not an easy task (it
216
effectively requires trained and skilled researchers and, above all, a lot of patience and
217
dedication as Fanali and Chankvetadze have recently pointed out [8]), from 1990 to 2010s,
218
there was a high interest in the scientific community to explore and exploit its inherent
219
advantages so that, great efforts were focused on the development of technological and
220
methodological improvements. Since then, other areas have attracted the researchers’s
221
attention affecting the development and applications of chiral CE. Nevertheless, even 10
222
though nowadays CE is a well-established separation technique (mainly in the EKC mode),
223
its full potential has not yet been reached and there are still significant challenges to face.
224
Currently, one of the most emerging research areas in chiral CE is the search of
225
novel chiral selectors. In fact, a considerable number of reviews have examined this topic
226
(some representative and recent examples are included in Table 1). Although, as it will be
227
illustrated in section 4, the use of single CDs or combinations of CDs remains the most
228
popular separation system in chiral CE, numerous researchers have focused their efforts on
229
the evaluation of other alternatives. Sonnendecker et al. demonstrated in an interesting
230
work, the discrimination power of large-ring CDs (single CDs composed of 10 to 12
231
glucose units) in the CE separation of chiral drugs [23]. On the other hand, one of the most
232
popular area in the search of novel chiral selectors is the use of chiral ionic liquids (CILs)
233
which have either a chiral cation, a chiral anion, or both in their structure. From the first
234
enantioseparations obtained using CILs as chiral selectors [24, 25], the number of
235
applications in this field has kept growing. Although some works have shown the
236
possibility of using CILs as single chiral selectors [26], they are mainly used in
237
combination with other chiral selectors (mostly CDs or macrocyclic antibiotics). In this
238
way, they can bring extra enantiorecognition ability while retaining the system modification
239
capability of achiral ionic liquids, so that the enantioseparations can be significantly
240
improved due to the cooperation between both selectors through a synergistic effect. In this
241
point, it should be mentioned that CILs have generated a considerable controversy among
242
the scientists since some of them claim that the addition of the synthesized CIL to the
243
separation buffer implies that it is no longer a CIL but a mixture of independent cations and
244
anions. Therefore, the addition of these anions or cations should work in the same manner
11
245
as the synthesized CIL [11]. However, some works have reported that the addition of CILs
246
gave rise to better results than the addition of their separated ions [27]. More research work
247
is necessary to provide a higher understanding on this subject. Nevertheless, regardless of
248
the mechanisms involved, the fact is that an important number of recent works reported the
249
synthesis of new CILs and their evaluation as chiral selectors in CE [28-30]. Readers
250
interested on gaining deeper insight on the use of CILs in chiral CE are referred to some
251
excellent recent review papers [10, 11, 31]. In addition to CILs, other compounds have also
252
been evaluated as chiral selectors over the last years. For instance, some articles have
253
shown for the first time the enantioseparation abilities of rifampicin [32], doxycycline [33],
254
clarithromycin lactobionate in combination with neutral CD derivatives [34], erythromycin
255
through the use of a carbamoylated erythromycin-zirconia hybrid monolithic column in
256
CEC [35], amino triazolium-modified lactobionic acid [36], or chondroitin sulfate D in
257
combination with a β-CD derivative [37].
258
It is worthy to mention that the use of nanoparticles (NPs) (quantum dots, gold NPs,
259
silica NPs, or carbon nanotubes (CNTs), among others) to improve chiral CE separations in
260
terms of efficiency and resolution, is receiving a significant attention [38]. Chiral selector
261
modified NPs can be bound to a capillary column in CE [39] or added to the buffer solution
262
[40] which confers a high flexibility to the choice of the chiral selector. Between these two
263
approaches, the second one may be, a priori, of greater interest due to some advantages
264
such as easier performance, no need to prepare a column, and no residual effect of
265
stationary phase. In any case, this research area is still far from reaching its full potential so
266
that it is foreseeable that new methodologies based on the use of NPs will be developed in
267
the next years.
12
268
Other important aspect to be considered is the limited concentration sensitivity that
269
can be obtained when using UV detection that is the most employed detection system in
270
chiral CE. The main strategies used to overcome this limitation are focused on the use of
271
preconcentration strategies based on electrophoretic principles or alternative detection
272
systems. Of course, chiral CE can also benefit from the advances and new developments
273
achieved in the field of off-line sample preparation techniques but these techniques will not
274
be commented here. The preferred preconcentration techniques based on electrophoretic
275
principles to improve the detection sensitivity are in-line (carried out within the capillary)
276
or on-line (in a completely integrated and automated manner) sample treatment. Among
277
them, field-amplified sample stacking (FASS), field-amplified sample injection (FASI),
278
and sweeping are usually the most employed in chiral CE analysis to improve the
279
sensitivity not only when UV detection is employed but also with other detectors such as
280
MS. The fundamentals of these preconcentration strategies are already well-known so the
281
developments in this area are being related mainly with the finding of the appropriate
282
combination of some of them in order to achieve the highest sensitivity as illustrated in
283
section 4. Regarding the use of alternative detection systems, laser induced fluorescence
284
(LIF) or MS detectors continue being the preferred ones although LIF detection implies the
285
need of molecule labeling as an essential step prior to CE analysis. By adding a
286
derivatization step, sample treatment becomes more tedious, leading in most cases to
287
derivatization problems when complex matrices are analyzed. Even so, different works
288
published in the last years demonstrated the potential of LIF as detection mode to carry out
289
the chiral analysis of different compounds in real samples enabling to achieve LODs in the
290
nM and sub-pM range [41, 42]. With respect to the CE-MS coupling, two interesting
291
review articles have been published in the last years. On the one hand, Jiang et al. described 13
292
the recent advances of CE-MS instrumentation and methodology in a general way [43]
293
whereas Liu and Shamsi´s review was focused directly on chiral CE-MS [44]. The main
294
limitation of MS detection in chiral CE is due to the incompatibility of nonvolatile chiral
295
selectors frequently employed that may cause ion suppression and contamination of the
296
ionization source leading to a sensitivity decrease. In some cases, it is possible to use a low
297
concentration of chiral selector to achieve a complete enantioseparation without a loss in
298
MS sensitivity [45, 46]. However, in many cases, the presence of nonvolatile chiral
299
selectors in the MS chamber should be avoided. In this line, different authors proposed
300
MEKC-MS methodologies based on the use of (+)-1-(9-fluorenyl) ethyl chloroformate
301
(FLEC) as chiral derivatization reagent to form stable diastereomers with the enantiomers
302
of
303
perfluorooctanoate (APFO) as a volatile pseudostationary phase [47, 48]. Other possibility
304
when the presence of a chiral selector is imperative is the use of chiral stationary phases
305
(CEC mode) or volatile chiral selectors such as polymeric micelles [49]. Nevertheless, the
306
most used strategies to improve the applicability of CE-MS to chiral analysis are those
307
based on the use of the counter migration or partial filling techniques [50]. The
308
combination of a preconcentration strategy with LIF or MS detection appears as the most
309
powerful way to reach a high sensitivity in chiral analysis by CE. This fact has been
310
illustrated in different works such as those reported by Patel et al. and Piestansky et al. who
311
developed chiral methodologies for the ultra-trace chiral determination of two protein
312
amino acids or pheniramine and its metabolite in single neurons or human urine,
313
respectively [41, 51].
the
chiral
compounds
and
their
subsequent
14
separation
using
ammonium
314
The combination of chiral CE with NMR spectroscopy, MS and computation
315
methodologies (molecular modeling and molecular mechanics calculations) is another
316
interesting and relevant area through which it is possible to obtain data related to the
317
mechanistic aspects of selector-selectand interactions [8, 9]. The high peak efficiency that
318
can be achieved in CE makes this separation technique a powerful tool for studying non-
319
covalent intermolecular interactions. In this way, a higher understanding of the enantiomer
320
separation mechanisms on the molecular level is provided. Even though this research is
321
constantly evolving and a high number of works have been published in the last five years
322
to determine the intermolecular interactions between different chiral compounds and chiral
323
selectors [36, 46, 52, 53], we are still far from understanding the nature of the forces
324
involved in the chiral recognition, especially when dual chiral systems are employed.
325
326
4. Applications of CE to chiral analysis
327
Due to its great versatility, CE enables the analysis of numerous chiral compounds
328
in a wide variety of samples in different fields such as the pharmaceutical, biomedical, or
329
food analysis, among others. Here, the most relevant and recent applications of CE to the
330
analysis of chiral compounds in real samples are included.
331
332
4.1. Pharmaceutical analysis
333
In the last years, new CE methods enabling the enantiomeric determination of drugs
334
in pharmaceutical formulations were developed and validated according to the International
335
Conference on Harmonisation guidelines (ICH guidelines). Some of the most relevant and 15
336
recent works published are grouped in Table 2. EKC using CDs as chiral selectors is the
337
most frequent approach, and the most frequent detection mode was UV, used in all cases
338
except in one in which LIF detection was employed.
339
The enantiomeric analysis of drugs marketed as pure enantiomers continues being a
340
challenge because a low amount of the enantiomeric impurity has to be detected in the
341
presence of the majority enantiomer. In fact, ICH guidelines establish that the presence of
342
the impurity enantiomer cannot be higher than 0.1% of the majority enantiomer [76]. This
343
requires a considerable enantiomeric resolution to avoid the overlapping of the majority
344
peak and the enantiomeric impurity peak, and a high sensitivity enabling the detection of
345
the enantiomeric impurity at such low concentrations. In this context, there is another
346
interesting factor affecting the determination of the enantiomeric impurity, which is the
347
enantiomer migration order. In fact, unless high enantiomeric resolutions are possible, it is
348
desirable that the enantiomeric impurity is the first-migrating enantiomer, this avoiding the
349
overlapping of the majority peak with that corresponding to the impurity. Taking into
350
account the importance of the enantiomeric resolution obtained and that the “one factor at
351
time” method to optimize the experimental conditions that influence the chiral separation is
352
time-consuming, the use of experimental designs is recommended. Some methodologies
353
combined both strategies, i.e. the “one factor at time” approach was employed to fix some
354
variables influencing the enantiomeric resolution and subsequently a multivariate
355
optimization was used to select others. For example, the development of a stereoselective
356
MEKC method for the simultaneous determination of montelukast enantiomers and
357
diastereomers and its degradation products was based on the evaluation of the influence of
358
some parameters by the trial-and-error approach and the use of a full factorial design to
16
359
investigate the effect of buffer pH and voltage on the resolution between montelukast
360
enantiomers [54]. Under the best separation conditions (see Table 2), and after evaluating
361
the analytical characteristics and the robustness of the method, the developed methodology
362
was applied to the determination of the above-mentioned isomers in bulk drugs, pouches
363
and tablets. The combination of the “one factor at time” approach and a central composite
364
face centered design was also applied to the optimization of the enantiomeric separation of
365
tramadol, a drug marketed as racemate [55].
366
A relevant point in the pharmaceutical industry for the development of CE
367
methodologies suitable to evaluate the enantiomeric purity of drugs marketed as pure
368
enantiomers is the application of Quality by Design (QbD) approaches. In fact, better
369
analytical methods can be developed applying the QbD principles. Briefly, QbD involves
370
the definition of the analytical target profile (for example, the determination of the main
371
compound and its chiral impurity at the 0.1% level with a resolution value > 2.0 in an
372
analysis time < 10 min), the identification of the critical process variables (buffer
373
concentration and pH, chiral selector concentration, voltage, temperature, etc) affecting the
374
critical quality attributes (resolution and analysis time), and the establishment of the design
375
space (that reflects the experimental conditions under which the analytical target is
376
reached). Fractional factorial resolution designs are usually employed for the identification
377
of the critical parameters. The most relevant are subsequently optimized using central
378
composite face centered designs and Monte Carlo simulations to define the design space.
379
Different works published in the last years reported the application of QbD approaches to
380
the development of CE methodologies for the chiral purity determination of drugs such as
381
ambrisentan [56], levomepromazine [57], dextromethorphan [58], dapoxetine [59], and
17
382
levosulpiride [60]. In some of these works, the analytical target profile comprises not only
383
the determination of the enantiomeric impurity but also its simultaneous analysis with other
384
impurities [57, 59]. The experimental conditions selected (using the above-mentioned
385
experimental designs) in all these CE methodologies are detailed in Table 2. Placket-
386
Burman designs were performed to assess the robustness, and all the developed
387
methodologies were validated according to ICH guidelines. In all cases, the application of
388
these methodologies to pharmaceutical formulations allowed the determination of
389
enantiomeric impurities at levels of 0.1% [56, 57, 58], 0.27% [60] or in the 0.05-1.0%
390
range [59].
391
Despite the application of experimental designs is advisable, different works
392
reported systematic screenings using the principle of “one factor at time” to select the most
393
adequate experimental conditions for the determination of the enantiomeric purity of drugs
394
marketed as pure enantiomers. For instance, using acetyl-β-CD (A-β-CD) as chiral selector
395
in a basic buffer, it was possible to determine the chiral purity of valsartan in tablets at the
396
0.01% level without interferences from the excipients [61]. In spite of the migration order
397
was not the most adequate (the minor component migrated after the major one), this is the
398
contribution of chiral CE to pharmaceutical analysis reporting the lowest relative LOD in
399
the last five years. In addition, the application of this methodology to two commercial
400
brands of tablets enabled to determine the enantiomeric impurity at percentages of 0.12%
401
and 0.22% revealing that ICH guidelines were not accomplished [61]. In the case of the
402
enantiomeric purity control of R-cinacalcet in tablets, the use of an EKC methodology
403
based on the use of 2-hydroxypropyl-γ-CD (HP-γ-CD) as chiral selector allowed for the
404
first time the separation of these enantiomers allowing a favorable migration order, i.e. the
18
405
S-enantiomer was the first-migrating enantiomer [62]. All the tablets analyzed by this
406
method accomplished the ICH regulations since the enantiomeric impurity was in all cases
407
below a 0.1%.
408
As it has been mentioned in section 3, the combination of chiral CE with NMR and
409
molecular modeling studies makes possible to obtain data related to the mechanistic aspects
410
of selector-selectand interactions. Two works published in the last five years reported the
411
development, optimization and validation of EKC-UV methodologies for the enantiomeric
412
quality control of enantiomerically pure drugs and applied NMR and molecular modeling
413
for the characterization of intermolecular interactions between drugs and CDs [63, 64]. On
414
the one hand, Menéndez-López et al. developed an EKC method using two different CDs,
415
succinyl-γ-CD (succ-γ-CD) and S-γ-CD, as chiral selectors for the first CE separation of
416
colchicine enantiomers [63]. Although the enantiomeric impurity migrated in the first place
417
using S-γ-CD, just the use of succ-γ-CD allowed to detect this impurity at a level of 0.1%.
418
Apparent and averaged equilibrium constants for the enantiomer-Succ-γ-CD complexes
419
were calculated by NMR, suggesting that the electrophoretic mobility of these complexes
420
was the predominant factor in the enantiomer migration order. On the other hand, Szabó et
421
al. developed a pressure-assisted EKC-UV with sulfobutyl ether-β-CD (SBE-β-CD) as
422
chiral selector to carry out for the first time the determination of the enantiomeric purity of
423
rasagiline [64]. Binding affinities of the individual enantiomers towards the CD were
424
investigated using both NMR and molecular modeling demonstrating the formation of a
425
more stable inclusion complex between the CD and the enantiomeric impurity which was in
426
accordance with the enantiomer migration order obtained by CE analysis.
19
427
Although EKC with UV detection is the preferred system to perform the evaluation
428
of enantiomeric purity in pharmaceutical formulations (see Table 2), LIF has also been
429
used for the purity control of magnesium L-aspartate dihydrate in two batch samples and
430
three drugs products [65]. Data obtained by the developed EKC-LIF methodology, based
431
on the use of a basic buffer containing HP-β-CD, were compared to those obtained by using
432
a LC-fluorescence method with chiral derivatization. Although the LC method had a higher
433
sensitivity (LOQ of D-aspartate: 0.006% vs 0.03%), both methodologies were suitable for
434
the control of the enantiomeric purity.
435
It is worthy to note that even if some drugs are nowadays marketed as racemates,
436
the studies carried out to achieve their enantiomeric separation when the biological activity
437
of both enantiomers differs, present a high interest taking into account that they could be
438
marketed as pure enantiomers in a near future. Drug racemates enantiomerically separated
439
in the last years include the antihypertensive amlodipine [66], carvedilol [67] and
440
lercanidipine [68] using neutral CDs as chiral selectors such as methyl-β-CD (M-β-CD), β-
441
CD, (2,3,6-tri-O-methyl)-β-CD (TM-β-CD), respectively. The enantiomeric separation of
442
the β-blocker pindolol was also achieved by EKC using an octa(6-O-sulfo)-γ-CD (OS-γ-
443
CD) [69]. Analyte-chiral selector complexation constants were determined, and a software
444
tool was proposed to predict the separation time and other variables from the complexation
445
constants and mobilities of complexes of both enantiomers. Although these four drugs are
446
marketed as racemates, a higher pharmaceutical activity was reported for their (S)-
447
enantiomers. The antiparasitic praziquantel is also commercialized as racemate even though
448
the (R)-enantiomer is the only responsible for its activity. This drug is employed in the
449
prevention and treatment of the tropical disease schistosomiasis (bilharziasis). 20
The
450
quantitation of (R)-praziquantel in tablets was achieved by EKC using S-β-CD as chiral
451
selector [70]. Other antiparasitic compounds present in the samples did not cause
452
interferences.
453
CDs were not the only chiral selectors employed for the enantiomeric determination
454
of drug racemates. In fact, the use of a maltodextrin as chiral selector enabled the
455
simultaneous stereoselective separation of tramadol and methadone not only in tablets but
456
also in biological fluids such as urine and plasma [71]. This was the first time that this
457
chiral selector was used to separate methadone. Researchers claim that the use of
458
maltodextrins shows some advantages over other chiral selectors because they are cost-
459
effective and allow efficient chiral separations.
460
An interesting approach to determine the enantiomeric excess of chiral drugs is the
461
use of the velocity gap mode of CE (VGCE) [72]. This strategy enables to carry out the
462
enantiomeric excess measurement even when the chiral selector does not provide enough
463
resolving power. This is of high relevance in those cases where the content of one
464
enantiomer is significantly higher than the other one. VGCE is based on the fractionation of
465
a small part of the mixture that contains both enantiomers from the main component (which
466
is already enantiopure). Then, the enantioseparation of the small fraction can be achieved
467
due to less longitudinal dispersion. The suitability of this methodology was demonstrated
468
by analyzing levamlodipine besytate tablet as it is illustrated in Figure 2 [72].
469
The potential of a liposome electrokinetic capillary chromatography (LEKC) was
470
evaluated for the enantioseparation of different model drugs and applied to test the chiral
471
impurity of naproxen samples [73]. The chiral separation was carried out employing
472
liposomes comprised of phosphatidylcholine and cholesterol as pseudo-stationary phase 21
473
and SBE-β-CD as chiral selector. The results obtained demonstrated that the
474
enantioseparation increased by using this separation system in comparison with the use of
475
single SBE-β-CD system and SBE-SDS-MEKC system.
476
Even though the most popular approach in chiral separations by CE is based on
477
aqueous solvents, NACE has also been used in drug analysis. Different β-blockers and β -
478
agonists were enantioseparated using a NACE methodology based on the use of boric acid
479
derivatives as new chiral selectors. Lactobionic acid-boric 9 acid complex and D-(+)-
480
xylose-boric acid complex were applied to the chiral separation of propranolol [74] whereas
481
diacetone-D-mannitol-boric acid complex was employed to carry out the enantioseparation
482
of seven β-agonists [75]. In both cases, the chiral selectors were in situ synthesized. The
483
developed NACE methods were successfully applied to the chiral separation of propranolol
484
in tablets [74] and the determination of clenbuterol in an oral solution [75].
485
486
4.2. Bioanalysis
487
CE has also proven its powerfulness in the chiral separation of analytes present in
488
biological samples such as plasma, urine, or even single neurons. These remarkable
489
research works are included in Table 3 and will be further detailed paying attention to
490
different aspects as the chiral selector used or the sensitivity reached. UV, MS and LIF
491
have been the detector systems used in the analysis of biological samples. It is relevant to
492
highlight that in most cases at least a preconcentration strategy was employed.
493
EKC-UV is typically used in combination with preconcentration strategies in the
494
bioanalysis field. This mode has been used to analyze mainly urine and plasma but analysis 22
495
of exhaled breath condensate (EBC) has also been reported [77]. There, authors separated
496
methadone enantiomers using carboxymethyl-β-CD (CM-β-CD) in an acidic medium,
497
included a FASS procedure, reaching LOQs in the µg/mL range. Authors applied the
498
method to analyze EBC collected from patients following methadone maintenance therapy.
499
Concentrations of both enantiomers of methadone in EBC poorly correlated to the ones
500
obtained in urine and serum [77].
501
An API derived from pyroglutamic acid, containing two chiral centers, was also
502
enantioseparated by EKC-UV [78]. Researchers employed S-β-CD as chiral selector and
503
validated the method according to the ICH guidelines. Application was conducted to
504
quality control in bulk material and in the investigation of in vivo inversion in rat plasma, to
505
evaluate the degradation of this drug occurring upon metabolization. LODs as low as ppb
506
were obtained thanks to the utilization of cation-selective exhaustive injection (CSEI)-
507
sweeping. Results showed that there were no in vivo racemization upon the intake of this
508
drug, which confirmed the viability of the commercialization of this product as a single
509
enantiomer. Another metabolization study, this time of ketamine and its metabolites was
510
also conducted by EKC-UV using S-γ-CD [79]. Dogs treated with sevoflurane or
511
medetomidine co-administered with ketamine (racemic or (S)-ketamine). Results showed
512
stereoselective metabolism occurring for metabolites 6-hydroxynorketamine and 5,6-
513
dehydronorketamine in plasma but not for ketamine and norketamine, its main metabolite.
514
Using another sulfated CD, this time sulfated-β-CD (S-β-CD), researchers separated
515
pheniramine enantiomers in rat plasma employing an EKC-UV setup [80]. Large volume
516
sample stacking (LVSS) and CSEI-stacking gave rise to a considerable improvement in the
23
517
sensitivity of 600 and 4000 times, respectively, when compared to not using
518
preconcentration approach.
519
Bioanalysis is the application field in which EKC-MS has been used to a larger
520
extent. This is because higher sensitivity is needed in the analysis of biological samples.
521
Sensitivity is often times not met by EKC-UV even if preconcentration strategies are used.
522
This is the case of a work reporting the development of a method based on ITP and MS
523
detection. The LODs in the pg/mL level were enough to enable its application to study the
524
enantioselective metabolism of pheniramine and its metabolite, desmethyl pheniramine, in
525
human urine samples obtained at different times after administration of racemic
526
pheniramine [51]. This method achieved about 125 times better sensitivity than the above-
527
mentioned method for pheniramines, based on EKC-UV [80]. Note that together with Patel
528
et al. contribution [41], this is the lowest LODs reported for a bioanalysis application and
529
all applications included in the present article altogether. In a recent and very
530
comprehensive publication [46], Liu et al. used EKC-MS to enantioseparate the four
531
stereoisomers
532
hydroxyaspartate. Low concentration of chiral selector (β-CD) was used to minimize MS-
533
source contamination. LVSS with polarity switching enabled a 10-fold increase in
534
sensitivity, reaching LODs as low as 87 nM in rat cerebrospinal fluid samples (CSF).
of
9-fluorenylmethoxycarbonyl
chloride
(FMOC)
labeled
3-
535
Protein amino acids are paramount metabolites with high importance in living
536
organisms. A MEKC-MS approach using derivatization with FLEC for amino acid analysis
537
was developed by Prior et al. to analyze human CSF where levels of endogenous L-amino
538
acids were quantified [48]. Although low, levels of D-serine and D-glutamine were also
539
found in these samples. Another work by Prior et al. also reported the enantioseparation of 24
540
amino acids in CSF, this time, using FMOC as labelling agent and since it is not a chiral
541
labelling compound, a chiral selector, β-CD, was used [81]. Working with a concentration
542
of 10 mM β-CD did not cause much sensitivity decrement and was used filling up
543
completely the separation capillary. Another contribution focusing on amino acids and
544
related compounds was also reported [82]. There authors enantioseparated the constituents
545
of the phenylalanine-tyrosine metabolic pathway using an EKC-MS/MS platform combined
546
with LVSS. LODs of 40-150 nM were found. The method was validated in rat plasma in
547
which endogenous levels of L-phenylalanine and L-tyrosine could be measured. This
548
method is the one based on the use of largest amount of CD (180 mM M-β-CD + 40 mM
549
HP-β-CD). This might be considered as a disadvantage but thanks to the versatility and low
550
consumption of reagents in CE, especially when working in the partial filling technique
551
capillary, this drawback is reduced. The fact that molecules from the same metabolic
552
pathway are analyzed, i.e. closely structural-like compounds could have had an effect on
553
needing such large quantities of chiral selector and favor the enantiorecognition [82].
554
As anticipated earlier in this review article, CDs are by far the most used chiral
555
selectors, but some researchers also used alternative chiral selectors. For example,
556
Svidrnoch et al. who used vancomycin, a macrocyclic antibiotic having 18 chiral centers,
557
for the chiral separation of 2-hydroxyglutaric acid in an EKC-MS configuration [83].
558
Atypical levels of D-2-hydroxyglutaric acid were found in urine from child diagnosed with
559
hydroxyglutaric aciduria. This is a rapid and sensitive method to distinguish between D-
560
and L-2-hydroxyglutaraciduria. Another example is the work by Liu et al. developed a
561
MEKC-MS/MS method using poly-L,L-SULA as chiral selector, to study the
562
pharmacokinetics and pharmacodynamics of the enantiomers of both antidepressant drugs
25
563
venlafaxine and O-desmethylvenlafaxine in human plasma [49]. The results suggested a
564
potential drug to drug interaction between indinavir (an inhibitor drug enabling to prevent
565
the breakage of polyproteins in patients with HIV) and these two antidepressants. This is
566
the only work reporting the use of chiral surfactants in the last years. These polymeric
567
surfactants are promising chiral selectors that are MS compatible but due to their limited
568
commercialization their application might be somehow compromised.
569
LIF detection offers high sensitivity and selectivity but demands sample
570
derivatization with a fluorescent tag. An EKC-LIF method by Patel et al. combined with
571
online preconcentration by LVSS improved the sensitivity 480 times also for the amino
572
acids aspartate and glutamate, reaching LODs in the sub-pM range [41] (Figure 3). This is
573
indeed the lowest LOD reported for chiral separations from last five years, together with
574
Pietansky et al [51]. Thanks to the high sensitivity of this approach it has been possible to
575
determine levels of D-aspartic and D-glutamic acids in single neurons isolated from the
576
neuronal model Aplysia californica. Differences in concentrations of these excitatory amino
577
acids were found depending on the clusters where the neurons were isolated from.
578
4.3. Food analysis
579
Chirality of a great variety of food components makes that their enantioselective
580
analysis has a significant role in food science and technology since it enables to obtain
581
information related to food quality, food processing, storage, or adulterations, among
582
others. Table 4 summarizes the characteristics of the most relevant and recent chiral CE
583
methodologies developed for food analysis in the last years. As this table shows, most of
584
these works are based on the use of CDs as chiral selectors and sometimes SDS is also
585
present in the BGE. As in pharmaceutical analysis, UV is the preferred detection system. 26
586
Different EKC methodologies have demonstrated their potential for the quality
587
control of food supplements [84, 85]. For instance, the enantioselective determination of
588
non-protein amino acids can provide information related to adulterations (the use of D-
589
enantiomers in the elaboration of dietary supplements is not allowed by legal regulations)
590
or food processing (fermentation, storage, etc). In this sense, different EKC methods based
591
on the use of anionic CDs (sulfated-α-CD (S-α-CD) or sulfated-γ-CD (S-γ-CD) depending
592
on the amino acid) were developed to carry out the enantiomeric separation of eight non-
593
protein amino acids previously labeled with FMOC [84]. After studying the effect of
594
different parameters, an optimized methodology using S-γ-CD as chiral selector was
595
applied to the enantiomeric analysis of citrulline in six food supplements. This method
596
enabled to reach LODs in the 10-7 M range. Data obtained demonstrated that the storage
597
time gave rise to a decrease in the amount of the L-enantiomer with respect to the labeled
598
content, but this effect could not be attributed to a racemization process since the D-
599
enantiomer was not detected in any of the samples analyzed [84]. Food supplements quality
600
can also be determined analyzing the origin of their constituents. A clear example of this
601
perspective is the enantioselective separation of 1,3-dimethylamylamine (DMAA) [85].
602
Using the combination of S-α-CD and S-β-CD in a chiral dual system and
603
benzyltriethylammonium chloride as chromophoric additive (to carry out an indirect UV
604
detection) it was possible to achieve the separation of the four DMMA diastereoisomers.
605
The application of this chiral method to the analysis of DMMA in dietary supplements
606
enabled to assume that DMMA probably was not of natural origin because its
607
diastereoisomeric ratios were identical to synthetic DMMA.
27
608
Geographical origin is also an important factor to determine the quality of
609
commercial tea products. One way to do that is using the content of cathechins and
610
methylxanthines as indicators. To do that, a MEKC method with HP-β-CD combined with
611
chemometric analysis was developed [86]. The presence of HP-β-CD in the BGE allowed
612
the enantioselective separation of some of the catechins ((+)-catechin, (‒)-catechin and (‒)-
613
epicatechin). Data obtained were evaluated using principal component and hierarchical
614
cluster analyses as exploratory techniques and by using discriminant models built using
615
linear and quadratic discriminant analyses. The results obtained demonstrated that the
616
developed methodology has a high potential to discriminate green tea samples according to
617
their geographical origin using catechines and methylxanthines as phytomarkers [86]. On
618
the other hand, the presence of (‒)-catechin and D-theanine (both considered as non-native
619
enantiomers) provides information of tea leaves treatment (fermentation, thermal treatment,
620
etc) which also enables to establish a tea classification. In this research line, Fiori et al.
621
developed a MEKC-UV methodology based on the use of SDS and 2,6-di-O-methyl-β-CD
622
(DM-β-CD) for the simultaneous enantioseparation of six major catechins and D,L-
623
theanine (previously derivatized with o-phtaldialdehyde in the presence of N-acetyl-L-
624
cystein) in green tea samples [87]. Once a set of different tea samples were analyzed, it was
625
possible to assign the presence of (‒)-catechin as indicator of a thermal degradation, and D-
626
theanine as marker of microbial or enzymatic processes.
627
The enantiomeric determination of protein amino acid in rice wine is an interesting
628
topic to obtain information about the wine age. For this reason, Miao et al. developed a
629
MEKC method for the determination of D-glutamic acid and D-aspartic acid (previously
630
derivatized with FMOC) in rice wine [88]. The method was based on the use of a dual
28
631
chiral system composed of β-CD and HP-β-CD, SDS, D-fructose (as additive) and
632
isopropanol (as organic modifier). Certain amounts of both D-amino acids were found in
633
the analyzed samples, but their percentage did not show a significant correlation with wine
634
age.
635
The potential of CE for the chiral analysis of lipids has also been demonstrated.
636
Analysis of hydroxyeicosatetraenoic acids requires separating both regioisomers and
637
enantiomers which was achieved in 35 min using a BGE containing HP-γ-CD as chiral
638
selector and SDS [89]. To demonstrate the viability of this MEKC methodology, the chiral
639
analysis of 8-, and 12-hydroxyeicosatetraenoic acids in two species of red algae was carried
640
out.
641
It is interesting to highlight that even though CDs are the most employed chiral
642
selectors and UV is the preferred detection system, an EKC methodology based on the use
643
of a (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) as selector and MS as
644
detection system has also been reported for the enantiomeric determination of underivatized
645
D,L-amino acids in three varieties of vinegar samples [50]. To avoid the contamination of
646
the ionization source due to the presence of the crown ether and make possible EKC-MS
647
hyphenation, the partial filling technique was employed. This provided LODs in the µg/mL
648
level and enabled the analysis of L and D enantiomers which gave information on the
649
fermentation degrees during the manufacturing process. The applicability of this
650
methodology could be extended to the sensitive determination of D,L-amino acids in other
651
samples.
652
The possibilities of ligand exchange, both in CE and in CEC (LE-CE and LE-CEC),
653
for the chiral analysis of short chain organic acids have also been demonstrated in the last 29
654
years. Aydogan et al., applied the ligand exchange principle in an open tubular CEC
655
column using copper (II) as central ion and L-histidine as chiral ligand. Using this
656
approach, it was possible to carry out the determination of D- and L-malic acid enantiomers
657
in apple juice with different dilutions, as Figure 4 shows [90]. On the other hand,
658
Kamencev et al. developed an alternative LE-CE method based on the combination of
659
copper (II)/aluminum (III)/D-quinic acid system and hexadecyltrimethylammonium
660
hydroxide (CTA-OH) to revert the EOF for the simultaneous enantiomeric separation of
661
DL-tartaric and DL-malic acids in wines [91]. While D-malic acid was determined in a
662
broad range of concentrations in different wines, D-tartaric acid was detected just in two of
663
the samples analyzed (at concentrations of 90 and 200 mg/L). It is worth noting that
664
whereas the use of DL-malic acid is allowed for wine acidification, the use of DL-tartaric
665
acid is not allowed. For this reason, the developed methodology acquires a high relevance,
666
especially from the winemaking point of view, since it enables the wine quality control
667
through the enantioselective determination of these short chain organic acids.
668
Until now, all the mentioned methodologies are based on direct approaches, which
669
as highlighted in the section 2, are the most commonly used in chiral separations. However,
670
two indirect strategies have also been described in the last five years. In one of them, D,L-
671
aldoses
672
dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole, were separated
673
in phenylboronate buffer containing SDS [92]. Under these conditions, and using LIF as
674
detection system, the enantiomeric separation of D,L-galactose in the hydrolysate of
675
commercial red seaweed samples was performed. The other work was focused on the
676
derivatization of selenomethionine with FLEC and their enantioselective separation
diastereomers,
formed
by
30
derivatization
with
(S)-(+)-4-(N,N-
677
employing APFO as pseudostationary phase [93]. The developed MEKC-UV methodology
678
was subsequently applied to the determination of L-selenomethionine in food supplements.
679
These two works demonstrate the usefulness of MEKC indirect approaches to the quality
680
control of edible marina algae and food supplements.
681
4.4. Environmental and forensic analysis
682
Chiral CE has also demonstrated its potential in the development of analytical
683
methodologies to be applied in other fields such as environmental and forensic analysis.
684
Table 5 groups some of the most relevant works published in the last years in these fields.
685
In the environmental field, different types of environmental waters (lake, river, ground,
686
wastewater) were the samples most frequently analyzed. Amino acids, drugs and herbicides
687
were enantiomerically determined. Native and derivatized CDs were the preferred chiral
688
selectors in these works to be used as the sole selectors or in dual systems based on
689
mixtures with other CDs or bile salts. In general, UV detection was employed although the
690
use of LIF detection was also reported. Due to the limited concentration sensitivity of UV
691
detection in CE and the low levels of these compounds in the analyzed samples,
692
preconcentration methods were employed to achieve the required sensitivity. With this aim,
693
new periodic mesoporous organosilica materials were synthesized and evaluated as
694
sorbents to carry out the solid-phase extraction of drugs and herbicides from water samples.
695
These novel mesoporous materials were synthesized with neutral ligands to be applied to
696
the preconcentration of a mixture of seven drugs of different characteristics [94] considered
697
as emergent pollutants and with cationic ligands to achieve the preconcentration of a group
698
of six herbicides [95]. The simultaneous separation of the fourteen enantiomers of these
699
drugs and the twelve enantiomers of the herbicides by CE enabled the application of the 31
700
developed methodologies to the determination of these compounds in different water
701
samples at µg/L levels. S-β-CD was employed as chiral selector for the simultaneous
702
enantiomeric determination by EKC of the seven studied drugs in 16 min and this method
703
was further optimized and applied to the evaluation of the enantiomer stability and the
704
toxicity of duloxetine and econazole on Daphnia magna [96]. The stability of these drugs
705
was studied under abiotic and biotic conditions. Figure 5 represents the analysis of
706
mixtures of both drugs. Figure 5A corresponds to an aqueous standard solution and Figure
707
5B to culture medium at zero time. In Figure 5C it can be observed that econazole
708
concentration was unstable and disappeared at 72 h of culture media incubation, the same
709
as in presence of Dapnhnia magna as shown in Figure 5D. Duloxetine concentration
710
decreased in presence of Dapnhnia magna (Figure 5D) [96]. On the other hand, the
711
simultaneous enantiomeric separation by EKC of the six herbicides studied in 11 min
712
required the use of a dual chiral system based on a mixture of TM-β-CD and HP-β-CD.
713
Other mesoporous silica materials were also developed to make possible the chiral analysis
714
of β-blockers by CE in water samples after their preconcentration by solid-phase extraction
715
[97, 98]. M-β-CD was employed as chiral selector enabling the simultaneous determination
716
of propranolol, atenolol, metoprolol and pindolol enantiomers in spiked water samples at
717
µg/l levels.
718
LIF detection was employed in the analysis of different amino acids in water
719
samples from the Mono Lake in USA that can be considered as an analogue for
720
astrobiologically revelant targets [42]. Two different methods were developed. One of them
721
was a MEKC method based on the use of a mixture of γ-CD and sodium taurocholate as
722
chiral system to study neutral amino acids and the other one was an EKC methodology
32
723
consisting of the use of γ-CD to analyze acidic amino acids. Sample preparation was easy
724
since no desalting or preconcentration procedures were necessary due to a derivatization
725
step with 5-carboxyfluorescein succinimidyl ester (CFSE). The use of CFSE increased the
726
sensitivity 2 orders of magnitude compared to fluorescein isothiocyanate (FITC)
727
derivatization.
728
Other interesting application of chiral CE is forensic analysis. CE can provide
729
relevant information related to the consumption of legal or illicit drugs and their
730
toxicological effects or about their origin and synthesis, among others [13]. Table 5 groups
731
detailed information of some of the most recent works published in relation with this type
732
of analysis. Seized samples of stimulant drugs, human hair and human blood are the
733
samples attracting most interest. CDs were the preferred chiral selectors and UV and MS
734
detection systems were employed. An EKC-MS method using S-γ-CD as chiral selector
735
was developed to carry out the analysis of eight amphetamine-like stimulants [99]. A
736
chemically modified capillary containing sulfonated groups was employed to improve the
737
migration times repeatability. This method was applied to two types of seized samples of
738
methamphetamine in order to detect impurities. (-)-ephedrine and (+)-ephedrine were
739
detected as impurities in both samples. The development of other EKC-MS method also
740
based on the use of S-γ-CD as chiral selector was reported and applied to the analysis of
741
twenty seized methamphetamine samples [45]. Results enabled to conclude that three of the
742
twenty samples were a mixture of R-methamphetamine and S-methamphetamine while the
743
rest of the samples contained only the S-enantiomer. This method could be used in the
744
analysis
745
methylenedioxyamphetamine, among others, thanks to the use of TOF-MS which provides
of
other
chiral
abuse
drugs
33
such
as
amphetamine
and
3,4-
746
unequivocal analyte identification. Seized samples were also analyzed by EKC-UV using
747
HP-β-CD as chiral selector. The regioisomeric and enantiomeric analysis of 24 design
748
cathinones and phenethylamines in these samples was reported [100]. The method
749
developed in this work was compared to a LC methodology with UV detection, using a
750
BEH Phenyl column as chiral stationary phase. The CE method was more advantageous, as
751
it resolved all 24 regioisomers while the LC method resolved 18 out of 24.
752
Regarding human hair and blood samples, an EKC-UV method using S-γ-CD as
753
chiral selector was proposed for the chiral separation of ketamine and norketamine which is
754
its principal metabolite, in hair samples from twelve ketamine abusers [101]. Ketamine is a
755
dissociative drug with analgesic, anesthetic and sedative properties. In order to carry out the
756
analysis of these compounds, LLE was used prior to CE. This work provides a fast method
757
for the study of the enantioselective metabolism of ketamine (see Figure 6). Moreover, an
758
EKC-UV method was developed for the determination of methorphan and its most
759
important metabolites in post-mortem blood samples from ten subjects who died due to
760
heroin overdose [102]. Dextromethorphan which is the D-form, can be used as anti-cough
761
medications, the L-enantiomer, levomethorphan, is an opiate agonist and unlike the D-
762
enantiomer, it has narcotic activity. Firstly, a LLE was necessary to extract the compounds
763
for the analysis. HP-β-CD was used as chiral selector which allowed baseline
764
enantioseparation of methorphan enantiomers in 20 min.
765
5. Conclusions and future trends
766
The attractive features of CE make this analytical technique one of the best options
767
to carry out a chiral separation. The research works published demonstrate the relevant role
768
of chiral CE in different fields such as the pharmaceutical, food analysis, biomedical, 34
769
environmental or forensic. This fact is possible thanks to the use of different CE modes
770
such as EKC, MEKC or NACE, and a wide variety of commercially available chiral
771
selectors. In the last years, EKC using a single CD or a combination of CDs has been the
772
most popular choice to develop chiral methodologies although other chiral selectors such as
773
maltodextrins, crown ethers or CILs have also been used to a lesser extent. Even though the
774
search of novel chiral selectors has received a significant attention in the last years and a
775
high number of research articles have been published in this area, until now the applications
776
of novel chiral selectors in the enantiomeric analysis of real samples are scarce. New
777
developments mainly related to the use of novel CILs and NPs as chiral selectors will be
778
expected in the near future.
779
UV detection being the most common system in chiral CE is followed by the
780
coupling with other detectors such as LIF or MS. The combination of these two detection
781
systems with preconcentration strategies has enabled the development of the most sensitive
782
chiral methodologies by CE with LODs in the pg/mL and sub-nM level. It is not surprising
783
that the lowest LODs were reached in the bioanalysis field as it is in this case where the
784
highest sensitivity is usually required. Even though CE-MS is a very powerful tool, its
785
application in chiral separations is limited by the incompatibility of nonvolatile chiral
786
selectors with the MS source. The use of the partial filling and countercurrent techniques as
787
well as of volatile selectors enables to increase the applicability of CE-MS to perform a
788
chiral analysis. Future trends should be redirected to the development of MS compatible
789
chiral selectors. In this way and using more versatile and efficient preconcentration
790
strategies, it would be possible to determine a higher number of chiral compounds at trace
791
levels in a big variety of matrices.
35
792
One of the main applications in pharmaceutical analysis has been the determination
793
of the enantiomeric purity of different drugs in pharmaceutical formulations with LODs at
794
the µg/mL level or relative LODs as low as 0.01%. To obtain low relative LODs in
795
pharmaceutical analysis is essential to ensure proper quality control of the enantiomeric
796
impurity to minimize detrimental effects of non-active substances. Bioanalysis is the field
797
in which more researchers have developed CE methods using MS detection. Amino acids
798
and drugs, among other compounds, are the most analyzed ones, being plasma and urine
799
from human or animal models the most analyzed samples. The low LODs achieved by
800
EKC-MS to study the enantioselective metabolism of pheniramine in urine (LOD of 80
801
pg/mL) or by EKC-LIF to determine D-glutamate and D-aspartate in single neurons (LODs
802
in the sub-pM range) can be highlighted. Although biofluids, EBC samples, and even single
803
neurons were analyzed in the last years, the analysis of tissues has not been reported. This
804
could be due to a more difficult sample preparation in the metabolite extraction from
805
tissues. This is something to keep in mind and hopefully future trends will also focus on
806
tissue analysis. This will definitely give insight on the chiral composition of the different
807
organs. Amino and organic acids were the compounds most frequently determined in food
808
samples. The lowest LODs achieved in this case were at the µg/mL and µM level. Several
809
types of water were analyzed in the environmental field to study the chiral separation of
810
different drugs and amino acids. LODs in the nM order were reached using MEKC or EKC
811
with LIF detection. Researchers working in the forensic field mainly focused their work on
812
the study of different drugs in seized samples as well as in human biological samples, being
813
ng/mL the lowest LODs achieved.
36
814
A notable number of research works published in the last years were focused on the
815
combined use of CE with NMR and molecular modeling studies in order to provide a
816
deeper knowledge on the enantioselective noncovalent intermolecular interactions taking
817
place in the chiral recognition mechanism. This is a very interesting area in which the full
818
potential of CE has not been demonstrated yet so new developments are expected in next
819
years.
820
821
Acknowledgements
822
Authors thank financial support from the Spanish Ministry of Economy and
823
Competitiveness (project CTQ2016-76368-P) and the Comunidad of Madrid and European
824
funding from FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL-II-
825
CM). S.B.B and M.C.P. also thank the Spanish Ministry of Economy and Competitiveness
826
for their predoctoral (BES-2017-082458) and “Ramón y Cajal” (RYC-2013-12688)
827
research contracts, respectively. E.S.L. thanks the University of Alcalá for her postdoctoral
828
contract.
829 830
37
831
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987
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990
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991
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996
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999
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1009
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1025
electrokinetic chromatography. Method development and quantitative analysis, J. Pharm.
1026
Biomed. Anal. 138 (2017) 189-196.
1027
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1028
rasagiline using sulfobutylether-β-cyclodextrin: capillary electrophoresis, NMR and
1029
molecular modeling study, Electrophoresis 40 (2019) 1897-1903.
1030
[65] O. Wahl, U. Holzgrabe, Evaluation of enantiomeric purity of magnesium-L-aspartate
1031
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1032
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1033
separation of amlodipine enantiomers by capillary electrophoresis, Adv. Pharm. Bull. 5
1034
(2015) 35-40.
1035
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1036
chiral separation of carvedilol by capillary electrophoresis, Iran J. Pharm. Res. 14 (2015)
1037
425-433.
47
1038
[68] L.P. Lourenco, F.A. Aguiar, A.R.M. de Oliveira, C.M. Gaitani, Quantitative
1039
determination of lercanidipine enantiomers in commercial formulations by capillary
1040
electrophoresis, J. Anal. Methods Chem. (2015) 294270.
1041
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1042
sulfo)-β-cyclodextrin for fast capillary zone electrophoretic enantioseparation of pindolol:
1043
Determination of complexation constants, software-assisted optimization, and method
1044
validation, J. Chromatogr. A 21 (2018) 214-221.
1045
[70] Z.I. Szabó, R. Gál, L. Szőcs, R. Ludmerczki, D.L. Muntean, B. Noszál, G. Tóth,
1046
Validated capillary electrophoretic method for the enantiomeric quality control of R-
1047
praziquantel, Electrophoresis 38 (2017) 1886-1894.
1048
[71] E. Nagdhdi, A.R. Fakhari, Simultaneous chiral separation of tramadol and methadone
1049
in tablets, human urine, and plasma by capillary electrophoresis using maltodextrin as the
1050
chiral selector, Chirality 30 (2018) 1161-1168.
1051
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1052
of the enantiomeric excess at the extreme case by capillary electrophoresis, J. Chromatogr.
1053
A 1408 (2015) 205-254.
1054
[73] X. Li, Y. Du, Z. Feng, X. Sun, Z. Huang, A novel enantioseparation approach based
1055
on liposome electrokinetic capillary chromatography, J. Pharm. Biomed. Anal. 145 (2017)
1056
186-194.
1057
[74] N. An, L. Wang, J. Zhao, L. Lv, N. Wanga, H. Guo, Enantioseparation of fourteen
1058
amino alcohols by nonaqueous capillary electrophoresis using lactobionic acid/D-(+)-xylos
1059
e–boric acid complexes as chiral selectors, Anal. Methods 8 (2016) 1127-1134. 48
1060
[75] L. Lv, L. Wang, J. Li, Y. Jiao, S. Gao, J. Wang, H. Yan, Enantiomeric separation of
1061
seven β-agonists by NACE-Study of chiral selectivity with diacetone-d-mannitol-boric acid
1062
complex, J. Pharm. Biomed. Anal. 145 (2017) 399-405.
1063
[76] ICH Harmonised Tripartite Guideline. Impurities in New Drug Products Q3B(R2)
1064
(2006) International Conference on Harmonisation of technical requirements for
1065
registration
1066
https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3B_R
1067
2/Step4/Q3B_R2__Guideline.pdf Accessed Aug 2019.
1068
[77] S. Hamidi, M. Khoubnasabjafari, K. Ansarin, V. Jouyban-Gharamaleki, A. Jouyban,
1069
Chiral separation of methadone in exhaled breath condensate using capillary
1070
electrophoresis, Anal. Methods 9 (2017) 2342-2350.
1071
[78] A.B. Martínez-Girón, M.L. Marina, A.L. Crego, Chiral separation of a basic drug with
1072
two chiral centers by electrokinetic chromatography for its pharmaceutical development, J.
1073
Chromatogr. A 1467 (2016) 427-435.
1074
[79] F.A. Sandbaumhüter, R. Theurillat, R.N. Bektas, A.P.N. Kutter, R. Bettschart-
1075
Wolfensberger, W. Thormann, Pharmacokinetics of ketamine and three metabolites in
1076
Beagle
1077
enantioselective capillary electrophoresis, J. Chromatogr. A 1467 (2016) 436-444.
1078
[80] Y. Yao, B. Zhang, S. Li, J. Yu, X. Guo, Enantioselective analysis of pheniramine in rat
1079
using large volume sample stacking or cation-selective exhaustive injection and sweeping
1080
coupled with cyclodextrin modified electrokinetic chromatography, Talanta 192 (2019)
1081
226-232.
dogs
of
under
pharmaceuticals
sevoflurane
vs.
for
medetomidine
49
human
comedication
use.
assessed
by
1082
[81] A. Prior, L. Sánchez-Hernández, J. Sastre-Toraño, M.L. Marina, G.J. de Jong, G.W.
1083
Somsen, Enantioselective analysis of proteinogenic amino acids in cerebrospinal fluid by
1084
capillary electrophoresis–mass spectrometry, Electrophoresis 37 (2016) 2410-2419.
1085
[82] E. Sánchez-López, A. Marcos, E. Ambrosio. M.L. Marina, A.L. Crego,
1086
Enantioseparation of the constituents involved in the phenylalanine-tyrosine metabolic
1087
pathway by capillary electrophoresis tandem mass spectrometry, J. Chromatogr. A 1467
1088
(2016) 372-382.
1089
[83] M. Svidrnoch, A. Pribylka, V. Bekarek, J. Sevcik, V. Smolka, V. Maier,
1090
Enantioseparation of D,L-2-hydroxyglutaric acid by capillary electrophoresis with tandem
1091
mass spectrometry. Fast and efficient tool for D- and L-2-hydroxyglutaracidurias diagnosis,
1092
J. Chromatogr. A 1467 (2016) 383-390.
1093
[84] R. Pérez-Míguez, M.L. Marina, M. Castro-Puyana, Enantiomeric separation of non-
1094
protein amino acids by electrokinetic chromatography. J. Chromatogr. A 1467 (2016) 409-
1095
416.
1096
[85] A. Pribylka, M. Svidrnoch, J. Sevcık, V. Maier, Enantiomeric separation of 1,3-
1097
dimethylamylamine by capillary electrophoresis with indirect UV detection using a dual-
1098
selector system. Electrophoresis 36 (2015) 2866-2873.
1099
[86] B. Pasquini, S. Orlandini, M. Goodarzi, C. Caprini, R. Gotti, S. Furlanetto, Chiral
1100
cyclodextrin-modified micellar electrokinetic chromatography and chemometric techniques
1101
for green tea samples origin discrimination, Talanta 150 (2016) 7-13.
1102
[87] J. Fiori, B. Pasquini, C. Caprini, S. Orlandini, S. Furlanetto, R. Gotti, Chiral analysis
1103
of theanine and catechin in characterization of greentea by cyclodextrin-modified micellar 50
1104
electrokinetic
chromatographyand
high
performance
1105
Chromatogr. A 1562 (2018) 115-122.
1106
[88] Y.N. Miao, Q. Liu, W. Wang, L. Liu, L. Wang, Enantioseparation of amino acids by
1107
micellar capillary electrophoresis using binary chiral selectors and determination of D-
1108
glutamic acid and D-aspartic acid in rice wine, J. Liq. Chromatogr. Relat. Technol. 40
1109
(2017) 783-789.
1110
[89] S. Kodama, S. Nakajima, H. Ozaki, R. Takemoto, Y. Itabashi, A. Kuksis,
1111
Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl-γ-cyclodextrin
1112
modified micellar electrokinetic chromatography. Electrophoresis 37 (2016) 23-24.
1113
[90] C. Aydogan, V. Karakoc, A. Denizli, Chiral ligand-exchange separation and
1114
determination of malic acid enantiomers in apple juice by open-tubular capillary
1115
electrochromatography. Food Chem. 187 (2015) 130-134
1116
[91] M. Kamencev, N. Komarova, O. Morozova, Enantioseparation of tartaric and malic
1117
acids in wines by Ligand Exchange Capillary Electrophoresis using uncoated fused silica
1118
capillary. Chromatographia 79 (2016) 927-93.
1119
[92] S. Yamamoto, Y. Tamata, K. Sejima, M. Kinoshita, S. Suzuki, Chiral separation of
1120
D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent
1121
and a phenylboronic acid complex. Anal. Bioanal. Chem. 407 (2015) 6201-6206.
1122
[93] R. Pérez-Míguez, M.L. Marina, M Castro-Puyana, A micellar electrokinetic
1123
chromatography approach using diastereomeric derivatization and a volatile surfactant for
1124
the enantioselective separation of selenomethionine, Electrophoresis 40 (2019) 1951-1958.
51
liquid
chromatography,
J.
1125
[94] J. Valimaña-Traverso, S. Morante-Zarcero, D. Pérez-Quintanilla, M.A. García, I.
1126
Sierra, M.L. Marina, Periodic mesoporous organosilica materials as sorbents for solid-
1127
phase extraction of drugs prior to simultaneous enantiomericseparation by capillary
1128
electrophoresis, J. Chromatogr. A 1566 (2018) 135-145.
1129
[95] J. Valimaña-Traverso, S. Morante-Zarcero, D. Pérez-Quintanilla, M.A. García, I.
1130
Sierra, M.L. Marina, Cationic amine-bridged periodic mesoporous organosilica materialsfor
1131
off-line solid-phase extraction of phenoxy acid herbicides from water samples prior to their
1132
simultaneous enantiomericdetermination by capillary electrophoresis, J. Chromatogr. A
1133
1566 (2018) 146-157.
1134
[96] J. Valimaña-Traverso, G. Amarieri, K. Boltes, M.A. García, I. Sierra, M.L. Marina,
1135
Enantiomer stability and combined toxicity of duloxetine and econazole on Daphnia magna
1136
using real concentrations determined by capillary electrophoresis, Sci. Total Environ. 670
1137
(2019) 770-778.
1138
[97] M. Silva, S. Morante-Zarcero, D. Pérez-Quintanilla, M.L. Marina, I. Sierra,
1139
Preconcentration of β -blockers using functionalized ordered mesoporous silica as sorbent
1140
for SPE and their determination in waters by chiral CE, Electrophoresis 38 (2017) 1905-
1141
1912.
1142
[98] M. Silva, S. Morante-Zarcero, D. Pérez-Quintanilla, M. L. Marina, I. Sierra,
1143
Environmental chiral analysis of β-blockers: evaluation of different n-alkyl-modified SBA-
1144
15 mesoporous silicas as sorbents in solid-phase extraction, Environ. Chem. 15 (2018) 362-
1145
371.
52
1146
[99] T. Mikuma, Y.T. Iwata, H. Miyaguchi, K. Kuwayama, K. Tsujikawa, T. Kanamori, H.
1147
Inouse, The use of a sulfonated capillary on chiral capillary electrophoresis/mass
1148
spectrometry of amphetamine-type stimulants for methamphetamine impurity profiling,
1149
Forensic Sci. Int. 249 (2015) 59-65.
1150
[100] L. Li, I.S. Lurie, Regioisomeric and enantiomeric analyses of 24 designer cathinones
1151
and phenethylamines using ultra high performance liquid chromatography and capillary
1152
electrophoresis with added cyclodextrins, Forensic Sci. Int. 254 (2015) 148-157.
1153
[101] N. Porpiglia, G. Musile, F. Bortolotti, E.F. De Palo, F. Tagliaro, Chiral separation
1154
and determination of ketamine and norketamine in hair by capillary electrophoresis,
1155
Forensic Sci. Int. 266 (2016) 304-310.
1156
[102] A. Bertaso, G. Musile, R. Gottardo, C. Seri, F. Tagliaro, Chiral analysis of
1157
methorphan in opiate-overdose related deaths by using capillary electrophoresis, J
1158
Chromatogr. B 1000 (2015) 130-135.
1159
1160
1161
53
1162
Figure Captions
1163 1164
Figure 1. Percentage of the number of research papers published from 2015 to 2019
1165
dealing with chiral analysis using different separation techniques obtained from Web of
1166
Science Thomson Reuters database (Accessed August 2019). The keywords employed were
1167
“chiral analysis” and “liquid chromatography”, “capillary electrophoresis”, “gas
1168
chromatography”, or “supercritical fluid chromatography”.
1169
Figure 2. Electropherogram corresponding to enantiomeric excess measurement of
1170
levamlodipine besylate tablets (4.312 mg/mL) obtained using 3.0 % (w/v) α-CD as chiral
1171
selector in velocity gap mode of CE. Reproduced with permission from Ref. [72].
1172
Figure 3. Comparison of EKC-LIF and LVSS-EKC-LIF in the analysis of D,L-aspartate
1173
(Asp) and D,L-glutamate (Glu). CE conditions: 110 mg/mL QA-β-CD in 60 mM 2-(N-
1174
morpholino)ethanesulfonic acid (pH 6.0) plus 10 mM KBr; applied voltage, -26 kV;
1175
temperature, 18°C; LVSS injection, 20 psi × 1 min; detection, emission wavelength at 440
1176
± 8 nm. Peaks in the electropherograms: (1) L-Asp, (2) D-Asp, (3) D-Glu, and (4) L-Glu.
1177
Adapted with permission from Ref. [41].
1178
Figure 4. Electropherograms corresponding to the chiral analysis of malic acid by OT-
1179
CEC. (A) Enantiomeric mixture of malic acid, (B, C, and D) the standard solution from
1180
apple juice diluted 10, 20 and 40-fold, respectively. CEC conditions: ACN/5.0 mM CuSO4,
1181
20.0 mM (NH4)2SO4 (60/40%); applied voltage, 20 kV; injection: -12 kV during 0.05 min;
1182
detection, 214 nm. Reproduced with permission from Ref. [90]
1183
Figure 5. Analysis of duloxetine and econazole racemates (both at a racemic concentration
1184
of 20 mg/L) in (A) an aqueous standard solution; (B) culture medium at zero time; (C) 54
1185
culture medium at 72 h incubation (abiotic conditions); (D) culture medium in presence of
1186
Daphnia magna at 72 h incubation. EKC conditions: 1.5% S-β-CD in 25 mM phosphate
1187
buffer (pH 3.0); applied voltage, -20 kV; temperature, 30°C; injection, 50 mbar × 10 s,
1188
detection, 210 nm. Reproduced with permission from Ref. [96].
1189
Figure 6. Analysis of ketamine and norketamine in three different hair sample from
1190
ketamine abusers. EKC conditions: 0.1% S-γ-CD in 15 mM Tris phosphate (pH 2.5);
1191
applied voltage, 20 kV; temperature, 20°C; injection, 7 kV × 20 s, detection, 200 nm.
1192
Reproduced with permission from Ref. [101].
55
Table 1. Some of the most representative and recent review articles on general aspects of enantiomeric separations by CE. Year
Title
Ref.
2019
Some thoughts about enantioseparations in capillary electrophoresis
[8]
2019
Chiral selectors in capillary electrophoresis: trends during 2017–2018
[6]
2018
Contemporary theory of enantioseparations in capillary electrophoresis
[9]
2018 2018 2017
Enantioseparation by capillary electrophoresis using ionic liquids as chiral selectors Ionic liquids in capillary electrophoresis for enantioseparation Chiral selectors in CE: recent development and applications (mid-2014 to mid2016)
[10] [11] [12]
]Table 2. Characteristics of the most representative CE methodologies developed for pharmaceutical analysis. Analyte
Sample
Montelukast enantiomeric and diastereoisomeric forms
Bulk product, chewable tablets and oral granules (pouches)
Tramadol
Pharmaceutical formulations (capsules and tablets)
CE modedetection
MEKC-UV
EKC-UV
BGE
LOD
Application
Ref.
CS: 10 mM SBE-β-CD +10 mM TM-β-CD B: 10 mM SDS + 20 mM borate (pH 9.0)
0.30 µg/mL (RLOD 0.02%)
Determination of montelukast enantiomeric and diastereoisomeric forms and its main degradation product in bulk product, chewable tablets and oral granules
[54]
CS: 5 mM CM-β-CD B:25 mM Borate pH (11.0)
0.02 mg/mL ((S,S)tramado) 0.024 mg/mL ((R,R)tramadol)
Enantioseparation of two tramadol enantiomers
[55]
Ambrisentan
A chemical supplier sample
EKC-UV
CS: 30 mM γ-CD B: 50 mM acetate (pH 4.0)
0.2 µg/mL
Levomepromazine
Injection solution and a reference substance of the European Pharmacopoeia
EKC-UV
CS: 3.6 mg/mL HP-γ-CD B: 100 mM citric acid (pH 2.85)
0.03% (dextromepromazine)
Dextromethorphan
Capsules
EKC-UV
Dapoxetine
Tablets
EKC-UV
CS: 20 mg/mL S-β-CD + 10 mg/mL M-α-CD B: 50 mM phosphate (pH 7.0)
0.3 µg/mL (0.02%) (levomethorphan)
CS: 45 mg/mL S-γ-CD and
0.66 µg/mL ((R)-
Evaluation of the enantiomeric purity of (S)-ambrisentan in a chemical supplier sample Determination of dextromepromazine and the oxidation product levomepromazine sulfoxide in levomepromazine Determination of the enantiomeric purity of dextromethorphan in capsules Determination of the
[56]
[57]
[58] [59]
40.2 mg/mL DM-β-CD (50%) B: 50 mM phosphate (pH 6.3) CS: 10 mM S-β-CD + 34 mM M-β-CD B: 5 mM Britton Robinson (pH 3.45)
dapoxetine) 1.2 µg/mL for enantiomeric impurity (R-levosulpiride)
Levosulpiride
Pharmaceutical formulations
EKC-UV
Valsartan
Tablets
EKC-UV
CS: 10 mM A-β-CD B: 25 mM phosphate (pH 8)
0.01% (R-valsartan)
Cinacalcet
Tablets
EKC-UV
CS: 0.5% (w/v) HP-γ-CD B: 150 mM phosphate (pH 2.5) + 20% (v/v) of methanol
0.1% ((S)-cinacalcet)
Colchicine
Pharmaceutical formulations
EKC-UV
CS: 7 mM Succ-γ-CD B: 50 mM borate (pH 9.0)
0.3 mg/L ((R)colchicine)
Rasagiline
Tablets
EKC-UV
2 µg/mL ((S)rasagiline)
Mn-L-Asp
Batch samples and drug products
EKC-LIF
CS: 30 mM SBE-β-CD B: 50 mM glycine-HCl (pH 2) CS: 18 mM HP-β-CD B: 50 mM phosphate (pH 7.0) + 18% (v/v) DMSO
Amlodipine
Tablets
EKC-UV
CS: 20 mM M-β-CD B: 50 mM phosphate (pH 3.0)
Carvedilol
Tablets
EKC-UV
CS: 10 mM β-CD B: 25 mM phosphate (pH 2.5)
Lercanidipine
Tablets
EKC-UV
CS: 10 mM of TM-β-CD B: 200 mM acetate (pH 4.0)
LOQ: 0.03 % (Mn-DAsp) 2.31 µg/mL ((R)amlodipine) 2.43 µg/mL ((S)amlodipine) 1.13 µg/mL ((R)carvedilol) 1.18 µg/mL ((S)carvedilol) 0.8 µg/mL ((S)lercanidipine)
purity of dapoxetine in tablets Quantification of levosulpiride in pharmaceutical formulations Determination of the (R)-enantiomer of valsartan in tablets Quantification of cinacalcet in tablets Quantification of colchicine in pharmaceutical formulations Determination of (S)rasagiline in tablets Quantification of Mn-LAsp in batch samples and drug products
[60]
[61]
[62]
[63]
[64] [65]
Determination of amlodipine enantiomers in tablets
[66]
Quantification of carvedilol in tablets
[67]
Quantification of lercanidipine in tablets
[68]
Pindolol
Pills
EKC-UV
CS: 6 mM OS-γ-CD B: 40/80 mM Sodium/MOPS pH (7.12)
Praziquantel
Tablets
EKC-UV
CS: 15 mM S-β-CD B: 50 mM phosphate (pH 2.0)
Tramadol and methadone
Tablets
EKC-UV
Levamlodipine besylate
Tablets
EKC-UV
Naproxene
Bulk sample
LEKC-UV
Propranolol
Tablets
NACE-UV
Clenbuterol
Oral solution
NACE-UV
0.6 µg/mL ((R)lercanidipine) 0.6 µg/mL ((R)pindolol) 0.6 µg/mL ((S)pindolol)
Enantioseparation of pindolol enantiomers in pills
[69]
0.75 µg/mL
Separation of the Praziquantel enantiomers in tablets
[70]
CS: 20% (w/v) Maltodextrin (DE=4-7) B: 100mM phosphate (pH 8.0)
2 µg/mL (tramadol) 1.5 µg/mL (methadone)
Determination of tramadol and methadone in different samples
[71]
CS: 3.0% (w/v) α-CD B: not stated
1 % ((R)levamlodipine)
Quantification of levamlodipine besylate in tablets
[72]
4.5 µg/mL
Enantioseparation of Naproxene in bulk sample
[73]
0.25 µg/mL (((R)propranolol) 0.50 µg/mL ((S)propranolol)
Quantification of propranolol in tablets
[74]
0.25 µg/mL
Separation of clenbuterol agonists in oral solution
[75]
CS: 2% SBE-β-CD (w/v) + 1.2% (w/v) nanoliposome B: 20 mM phosphate buffer (pH 5.2) CS: 8 mM lactobionic acid, 100 mM boric acid B: 14.4 mM triethylamine in methanol CS:80 mM diacetone-Dmannitol B: 100 mM boric acid + 50.4 mM triethylamine in methanol
Abbreviations: A-β-CD, acetyl-β-CD; B, buffer; CM-β-CD, carboxymethyl-β-CD; CS, chiral selector; DE, dextrose equivalent; DM-β-CD, 2,6-dimethyl-βCD; HP-γ-CD, 2-hydroxypropyl-γ- CD; γ-CD,γ -CD; M-α-CD, methyl-α-CD; M-β-CD, methyl-β- CD; MOPS, 3-(N-morpholino) propanesulphonic acid;
OS-γ-CD, octa (6-O-sulfo) γ- CD; Succ-γ-CD, succinyl-γ- CD; S-β-CD: sulfated β- CD; S-γ-CD, sulfated γ- CD; SBE-β-CD, sulfobutyl ether-β-CD; TM-βCD, 2,3,6-tri-O-methyl-β-CD; SDS, sodium dodecyl sulphate.
Table 3. Characteristics of the most representative methodologies developed by CE for bioanalysis. Analyte
Sample
CE modedetection
BGE
LOD
Methadone
EBC
EKC-UV
CS: (0.8%) CM-β-CD B: 100 mM phosphoric acidTEA (pH 2.5)
LLOQ: 0.15 µg/mL
API derived from pyroglutamic acid
Rat plasma
EKC-UV
CS: 2.5% (w/v) S-β-CD B: 50 mM phosphate (pH 2.5)
0.15-0.17 µg/mL
CS: 0.66 % (w/v) S-γ-CD B: disodium hydrogenphosphate buffer (pH 3.0)
3 ng/mL
Ketamine and metabolites
Dog plasma
EKC/UV
Pheniramine
Rat plasma
EKC/UV
Pheniramine and metabolites
Human urine
EKC/MS
3-hydroxyaspartate
Rat CSF
EKC/MS
CS: 6 mM β-CD B: 49 mM NH4Ac + 15% (v/v) isopropanol
87 nM
Proteinogenic amino acids
Human CSF
EKC/MS
CS: 10 mM β-CD B: 50 mM ammonium bicarbonate (pH 8) + 15%
0.5-84.3 µM
CS: 30 mg/mL S-β-CD B: 30 mM phosphate buffer (pH 3.0) CS: 5 mg/mL CM-β-CD B: 25 mM ε-aminocaproic acid + 25 mM acetic acid (pH 4.5) + 0.05 % (w/v) methylhydroxyethylcellulose
LLOQ: 10 ng/mL
80 pg/mL
Application Quantification of methadone enantiomers in EBC Study on drug enantiomeric inversion in vivo Determination of ketamine and metabolites in dog plasma under sevoflurane or medetomidine and ketamine intake Pharmacokinetic study on rats treated with racemic pheniramine Determination of pheniramine and its metabolites in urine of healthy patients after pheniramine intake Simultaneous determination of four isomers of 3hydroxyaspartate in rat CSF Enantioselective analysis of proteinogenic amino
Ref. [77]
[78]
[79]
[80]
[51]
[46]
[81]
Phenylalanine, tyrosine, DOPA, dopamine, norepinephrine, epinephrine
2-hydroxyglutaric acid
Rat plasma
Human urine
EKC-MS/MS
(v/v) isopropanol
acids in CSF
CS: 180 mM M-β-CD + 40 mM HP-β-CD B: 2 M formic acid (pH 1.2)
Chiral separation of the constituents of the PheTyr metabolic pathway in rat plasma
40-150 nM
EKC/MS
CS: 25 mM vancomycin B: 50 mM ammonium acetate (pH 4.5)
31-38 nM
10.5-15 ng/mL 15 ng/mL
Sub-pM
Venlafaxine and metabolites
Human plasma
MEKC/MS
CS: 15 mM poly-L,L-SULA B: 20 mM ammonium acetate + 25 mM TEA (pH 8.5)
Aspartate and glutamate
Single neurons from Aplysia californica
EKC/LIF
CS: 110 mg/mL QA-β-CD B:60 mM MES (pH 6.0) + 10 mM KBr
Determination of 2hydroxyglutaric acid in urine of healthy patients and children with abnormal excretion of 2hydroxyglutaric acid Determination of venlafaxine and metabolites in plasma of healthy patients after venlafaxine intake with or without indinavir Study on excitatory amino acids in neurons isolated from different neurological clusters of sea slug
[82]
[83]
[49]
[41]
Abbreviations: API, active pharmaceutical ingredient; B, buffer; CM-β-CD, carboxymethyl-β-CD; CS: chiral selector; CSF, cerebrospinal fluid; EBC, exhaled breath condensate; HP-β-CD, hydroxypropyl-β-CD; S-β-CD: sulfated-β-CD; S-γ-CD, highly sulfated-γ-CD; LLOQ, lower limit of quantification; MES, 2-(N-morpholino)ethanesulfonic acid; M-β-CD, methyl-β-CD, poly-L,L-SULA, poly-sodium N-undecenoyl-l,l-leucylalaninate; QA-β-CD, quaternary ammonium β-cyclodextrin; S-β-CD, sulfated-β-CD; TEA, triethylamine.
Table 4. Characteristics of the most representative methodologies developed by CE for food analysis. Analyte
Sample
CE modedetection
BGE
LOD
D,L-Citrulline
Food supplements
EKC-UV
CS: 10 mM S-γ-CD B: 100 mM formate (pH 3.0)
0.21 µM (D-Cit) 0.18 µM (L-Cit)
7.82-9.24 µg/mL
Application Analysis of D- and Lcitrulline in food supplements Determination of the stereoisomeric composition of DMAA in food supplements to verify their potential natural origin Analysis of the principal catechins and methylxanthines in green tea samples Chiral analysis of theanine and catechin in the characterization of green tea samples
Ref. [84]
1,3Dimethylamylamine
Food supplements
EKC-UV
CS: 1.1% (w/v) S-α-CD + 0.2 % (w/v) S-β-CD B: 5 mM phosphate/ Tris (pH 3.0) and 10 mM BTEAC
Catechins
Green tea
MEKC-UV
CS: 25 mM HP-β-CD B: 90 mM SDS + 25 mM borate-phosphate (pH 2.5)
-
Theanine and catechins
Green tea
MEKC-UV
CS: 28 mM DM-β-CD B: 65 mM SDS + 25 mM borate-phosphate (pH 2.5)
0.1-02 µg/mL
MEKC-UV
CS: 35 mM β-CD + 6 mM HPβ-CD + 25 mM D-fructose B: 30 mM SDS + 100 mM borate (pH 9.5) + 15% IPA (v/v)
2.5 µM
Determination of D-Glu and D-Asp in rice wine
[88]
MEKC-UV
CS: 30 mM HP-γ-CD B: 75 mM SDS + 30 mM phosphate-15 mM borate (pH 9.0)
0.95-0.99 µg/mL
Determination of the enantiomeric composition of the hydroxyeicosatetraenoic acids found in red algae
[89]
EKC-MS
CS: 30 mM (18C6H4)
0.07-1.03 µg/mL
Determination of D- and
[50]
Glutamic acid and aspartic acid
8- and 12Hydroxyeicosatetrae noic acids Proteinogenic amino
Rice wine
Marine Red Algae (Gracilaria vermiculophylla and Gracilaria arcuata) Vinegars
[85]
[86]
[87]
acids
B: 1M formate
D- and L-Malic acid
Apple juice
LE-CEC-UV
D- and L-Tartaric acid and D- and Lmalic acid
Wine
LECE-UV
CS: 5 mM Cu (II) sulfate B: 20 mM ammonium sulfate (60:40 v/v) (pH 3.0) CS: 100 mM D-quinic acid, 10 mM Cu (II), 0.5 mM Al (III) and 0.5 mM CTA-OH B: 20 mM acetate
1.5 mg/L (D-tartaric acid) 3 mg/L (D-malic acid)
L-amino acids in vinegars Determination of malic acid enantiomers in apple juice Determination of Denantiomers of both acids in wines.
[90]
[91]
Abbreviations: B, buffer, BTEAC: benzyltriethylammonium chloride, CS: chiral selector, Cit: citrulline, CTA-OH, hexadecyltrimethylammonium hydroxide; 18C6H4, (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid; DM-β-CD, 2,6-di-O-methyl-β-cyclodextrin; HP-β-CD, 2-hydroxypropyl-βcyclodextrin; IPA, isopropanol; LECE, ligand exchange capillary electrophoresis; LE-CEC, ligand exchange capillary electrochromatography;, SDS, sodium dodecyl sulfate; S-α-CD, sulfated-α- CD; S-β-CD, sulfated-β-CD.
Table 5. Characteristics of the most representative methodologies by CE in other fields. Analyte Duloxetine, terbutaline, econazole, propranolol, verapamil, metoprolol and betaxolol Fenoprop, mecoprop, dichloroprop, 2-(4chlorophenoxy) propionic acid, 2-(3chlorophenoxy)propi onic acid and 2phenoxypropionic acid Duloxetine and econazole Pindolol, atenolol, propranolol and metoprolol Pindolol, atenolol, propranolol and metoprolol
Sample
Wastewater of different treatment plants
River water and wastewater of effluent treatment plants from different Spanish regions
CE modedetection
BGE
LOD
Application
Ref.
EKC-UV
CS: 2% (w/v) S-β-CD B: 25 mM phosphate (pH 3.0)
0.4-1.5 mg/L
Enantiomeric determination of drugs in waste water
[94]
EKC-UV
CS: 20 mM of TM-β-CD + 7 mM of HP-β-CD B: 50 mM phosphate (pH 7.0)
0.1-4.3 µg/L
Enantiomeric determination of phenoxy acid herbicides in water samples
[95]
Daphnia magna
EKC-UV
CS: 15% (w/v) S-β-CD B: 25 mM phosphate (pH 3.0)
0.3-1,1 mg/L
River and ground water
EKC-UV
CS: 1.25% (w/v) M-β-CD B: 50 mM phosphate (pH 2.5)
1-1.6 µg/L
EKC-UV
CS: 1.25% (w/v) M-β-CD B: 50 mM phosphate (pH 2.5)
0.4- 0.6 µg/L
River water and sewage water of the waste water treatment plant (effluent water)
Enantiomer stability and combined toxicity of duloxetine and econazole on Daphnia magna Determination of βblockers in environmental waters Chiral analysis of β blockers in river and sewage water
[96]
[97]
[98]
Neutral amino acids CS: 30 mM γ-CD and 30 mM sodium taurocholate B: 80 mM tetraborate (pH 9.2) and 5% (v/v) ACN Acidic amino acids CS: 30 mM γ-CD B: 80 mM tetraborate (pH 9.2) CS: 20 mM S-γ-CD B: 10 mM formic acid (pH 2.5)
Amino acids
Lake water
Neutral amino acids: MEKCLIF Acidic amino acids: EKC-LIF
8 Amphetaminerelated stimulants
Seized samples
EKC-MS
Amphetamine‐type stimulants and ephedrine
Seized samples
EKC-MS
CS: 0.26% (w/v) S-γ-CD B: 50 mM ammonium formate (pH 2.2)
3.24-8.57 µg/mL
EKC-UV
CS: 80 mM HP-β-CD B: Celixir initiator solution (pH 2.5)
5 µg/mL
24 Design cathinones and phenethylamines
Ketamine and norketamine
Methorphan
Seized samples
Human hair
Human blood
EKC-UV
EKC-UV
CS: 0.1% (w/v) S-γ-CD B: 15 mM Tris-phosphate (pH 2.5) CS: 5 mM HP-β-CD B: 150 mM phosphate (pH 4.4) containing 20% (v/v) of MeOH
Neutral amino acids: 5-100 nM Acidic amino acids: 500-750 nM
2 µg/mL (for (-)ephedrine and (+)pseudoephedrine)
Chiral separation of DLamino acids in lake water
Analysis of seized samples of methamphetamine Analysis of seized samples of amphetamine‐type stimulants and ephedrine Regioisomeric and enantiomeric analysys of cathinones and phenethylamines in seized samples
[42]
[99]
[45]
[100]
0.08 ng/mL
Determination of ketamine in hair from ketamine abusers
[101]
8 ng/mL
Analysis of post-mortem blood samples of corpses overdosed by heroin
[102]
Abbreviations: ACN, acetonitrile; B, buffer; CS, chiral selector; S-γ-CD, sulfated-γ- CD; HP-β-CD, (2-hydroxypropyl)-β-cyclodextrin; M-β-CD, methylated-β- CD; LIF, laser-induced fluorescence; S-β-CD: sulfated-β-CD; TM-β-CD, (2,3,6-tri-O-methyl)-β-CD.
Figure 1.
73
Figure 2.
74
Figure 3.
75
Figure 4.
76
Figure 5.
77
Figure 6.
78
Highlights • Most relevant and recent contributions of Chiral CE were reviewed • Fundamentals and characteristics of Chiral CE were described • Most recent technological and methodological developments were presented • Applications in the pharmaceutical, food, biomedical or other fields were included
Conflicts of Interest: The authors declare no conflict of interest.
S0165-9936(19)30518-7
DOI:
https://doi.org/10.1016/j.trac.2020.115807
Reference:
TRAC 115807
To appear in:
Trends in Analytical Chemistry
Received Date: 8 September 2019 Revised Date:
20 December 2019
Accepted Date: 5 January 2020
Please cite this article as: S. Bernardo-Bermejo, E. Sánchez-López, M. Castro-Puyana, M.L. Marina, Chiral Capillary Electrophoresis, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2020.115807. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
1
CHIRAL CAPILLARY ELECTROPHORESIS
2
Samuel Bernardo-Bermejo1, Elena Sánchez-López1,2, María Castro-Puyana1,2, María
3
Luisa Marina1,2*
4
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Ciencias, Universidad de Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de
6
Henares (Madrid), Spain.
Departamento de Química Analítica, Química Física e Ingeniería Química, Facultad de
2
7
Instituto de Investigación Química Andrés M. del Río (IQAR), Universidad de Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de Henares (Madrid), Spain.
8 9
10
11
12
13
14
15
16
*
17
Email: [email protected]
18
Tel: (+34)-918854935
Corresponding author: Maria Luisa Marina
19
20
21
1
22
ABSTRACT
23
The implications of chirality in different environments are already well known and reported
24
extensively in the literature. Capillary Electrophoresis, a separation technique that only
25
requires few nanoliters of sample, has demonstrated its potential for chiral analysis in the
26
past years. The aim of this article is to provide an overview on the fundamentals and
27
characteristics of Chiral Capillary Electrophoresis as well as the main advances and trends
28
in this topic. Special attention is paid to the most recent technological and methodological
29
developments achieved mainly in the most employed separation mode (Electrokinetic
30
Cromatography). The most noteworthy and recent applications reported on the
31
enantiomeric separation and determination of compounds in pharmaceutical, food,
32
biomedical, environmental or forensic samples will also be critically overviewed. The
33
characteristics of the developed methodologies will be detailed in Tables and future trends
34
will also be discussed.
35
36
Keywords:
Chiral
Capillary
Electrophoresis;
Enantiomers;
37
Chromatography; pharmaceutical formulations; food; biological samples.
Electrokinetic
38
39
Abbreviations: A-β-CD, acetyl-β-CD; APFO, ammonium perfluorooctanoate; CFSE, 5-
40
carboxyfluorescein succinimidyl ester; CILs, chiral ionic liquids; CNTs, carbon nanotubes;
41
CM-β-CD, carboxymethyl-β-CD; CSF, cerebrospinal fluid; DM-β-CD, 2,6-dimethyl-β-CD;
42
EBC, exhaled breath condensate; FASI, field-amplified sample injection; FASS, field-
43
amplified sample stacking; FMOC, 9-fluorenylmethoxycarbonyl chloride; FLEC, (+)-1-(92
44
fluorenyl)
ethyl
chloroformate;
FITC,
fluorescein
45
hydroxypropyl-γ-CD; LEKC, liposome EKC; M-β-CD, methyl-β-CD; NPs, nanoparticles;
46
OS-γ-CD, octa (6-O-sulfo)-γ-CD; LE-CEC, open tubular ligand exchange CEC; succ-γ-CD,
47
succinyl-γ-CD; S-α-CD, sulfated α-CD; S-β-CD, sulfated β-CD; S-γ-CD, sulfated γ-CD;
48
TM-β-CD, (2,3,6-tri-O-methyl)-β-CD; SBE-β-CD, sulfobutyl ether-β-CD.
49
50
51
52
53
54
55
56
57
58
59
60
61
3
isothiocyanate;
HP-γ-CD,
2-
62
1. Introduction
63
Chiral analysis is nowadays one of the most interesting areas within Analytical
64
Chemistry. This interest arises from the different properties that the enantiomers of a chiral
65
compound may have which originates the need to achieve their individual determination
66
[1]. As enantiomers of chiral drugs, food components or additives, agrochemicals or
67
pollutants can exhibit different biological activity, toxicity, degradation rates, persistence,
68
and other properties, the relevance of chiral analysis includes very different fields such as
69
the pharmaceutical, biomedical, food or environmental, among others [2-4].
70
Separation techniques are very powerful tools to achieve chiral analyses. Among
71
them, liquid chromatography (LC), gas chromatography (GC), supercritical fluid
72
chromatography (SFC) and capillary electrophoresis (CE) have been the most employed
73
[5]. Although LC has been the most frequently used, CE has shown to have very interesting
74
characteristics to carry out enantiomeric separations [1] and it has had an important impact
75
in the last years as it can be observed in Figure 1 which shows the number of publications
76
dealing with chiral analysis using different analytical techniques from 2015.
77
On the one hand, chiral analysis benefits from some inherent characteristics of CE
78
such as its high efficiency and resolution, low consumption of reagents and samples or
79
short
80
electrophoretic principles can be applied to increase the sensitivity of the developed
81
methodologies which can also be enhanced using detection systems such as fluorescence or
82
mass spectrometry (MS), among others. On the other hand, the possibility of adding the
83
chiral selector directly to the separation medium in the so-called Electrokinetic
84
Chromatography (EKC) mode confers a high flexibility to chiral CE since the nature of the
analysis
times.
Also,
in-capillary
4
preconcentration
techniques
based
on
85
selector (or mixture of chiral selectors) and also its concentration can easily be changed
86
favoring the separation of enantiomers and reducing the economic costs derived from the
87
use of chiral chromatographic columns. The big variety of chiral selectors that can be
88
employed in EKC also contributes to increase the flexibility of CE to achieve an
89
enantiomeric separation [6]. In addition, the low amounts of reagents, solvents and samples
90
needed in CE fits well in the principles of green chemistry and reduces the environmental
91
impact of the developed methodologies. Although a chiral stationary phase can also be
92
employed in Capillary Electrochromatography (CEC) [7], EKC is by far the most employed
93
mode to perform enantiomeric separations in the format of CE as it will be shown in next
94
sections.
95
Numerous reviews devoted to chiral analysis by CE have been published in the last
96
years showing the high interest of this subject in different research fields. Table 1 groups
97
some of the most representative and recent review articles focused on general aspects of
98
enantiomeric separations by CE such as the current trends and future directions [8], the
99
history and contemporary theory of this technique as well as the study of the mechanisms
100
of non-covalent (enantioselective) interactions in different disciplines [9] or the potential of
101
different chiral selectors such as sugar-, amino acid-, nucleic acid-based polymers and ionic
102
liquids, among others [6, 10-12]. Although not included in this Table, other articles devoted
103
to review more specific and applied aspects of chiral CE have also been recently published
104
such as its application in food, environmental and pharmaceutical analysis [2-4, 13].
105
The aim of this article is to provide an overview of the most relevant contributions
106
of CE to chiral separations paying special attention to the most innovative and recent
107
developments and improvements achieved. The fundamentals and characteristics of chiral 5
108
CE are also included and the most noteworthy applications to the analysis of real samples
109
in different fields reported in the last years will also be critically discussed.
110
2. Fundamentals and characteristics of chiral CE
111
CE separation principle relies on differences in the effective mobilities of the given
112
species in a narrow silica capillary (e.g. 50 µm internal diameter) under an electric field.
113
This effective mobility is defined as the sum vector of the electrophoretic mobility and the
114
mobility of the so-called electroosmotic flow (EOF). Silanol groups from the inner surface
115
of a fused-silica capillary are ionized above pH 3. At this or higher pH, positive ions from
116
the electrolyte solution, more widely known as background electrolyte (BGE), will then be
117
attracted by the negative wall generated by the silanol groups, forming a double layer.
118
Applying an electric voltage to the capillary ends will result in a flow of cations towards the
119
cathode. This flow, i.e. the EOF, will drag the bulk solution on this direction. Thus,
120
considering a cathodic detection, positively charged molecules will reach the detector first,
121
followed by neutral species (null electrophoretic mobility) and negatively charged
122
molecules will then follow. Note that both the size and charge of the molecule will also
123
play a role in the effective mobility since the electrophoretic mobility is proportional to the
124
effective charge and indirectly proportional to the radius. Detection of negatively charged
125
species will only be achieved if the EOF is able to counteract their electrophoretic mobility
126
towards the anode. It is clear that this is the most simplistic way, but the enormous
127
flexibility of CE opens a wide array of possibilities. This simple scenario can be further
128
modified, if, for instance, coated capillaries are used. There is a high number of works
129
reported in literature showing the different capillary coatings [14]. This coating can be
6
130
temporary, meaning that it will have to be renewed in every analytical run, or can be
131
permanent, when the coat is covalently attached to the capillary inner surface.
132
CE offers higher efficiency obtaining narrower peaks typically with higher
133
resolution because of the flat flow occurring in the capillary opposed to the parabolic flow
134
found in LC. This also results in a larger peak capacity in CE, having a million theoretical
135
plates. Among the other different advantages of CE over LC, it is of relevance to highlight
136
the minimal requirements for sample and solvent consumption. Typically, sample injection
137
in CE is in the nanoliter range compared to the microliter range for LC. The inherent
138
dimensions of CE make it a miniaturized technique with minimal waste and therefore,
139
reduced environmental impact.
140
However, when one wants to separate enantiomers a more difficult mechanism
141
needs to be applied. Enantiomers have identical physicochemical properties, hence, their
142
electrophoretic mobility will be exactly the same, and enantioseparation will only be
143
possible under a chiral environment, e.g. interaction with a chiral selector. Typically, this
144
interaction is non-covalent (usually, van der Waals forces or hydrogen bonding are
145
involved), which origins the formation of temporary diastereoisomers complexes which are
146
then separated based on their different mobilities in the so-called direct mode. On the other
147
hand, and used to a much lower extent is the indirect mode in which each enantiomer is
148
covalently linked to an enantiopure chiral derivatization reagent. These two new molecules
149
are now diastereoisomers, thus, the differences in their physicochemical properties will
150
enable their separation under achiral conditions. The less popularity of this later approach is
151
due to the fact that the availability of these chiral derivatization reagents is limited and they
152
must be of high enantiomeric purity. Their high cost and also the time-consuming step 7
153
consequence of the derivatization makes it a not desired approach. Considering that the
154
direct mode is the approach most used in CE, we will further discuss its features.
155
Overall, chiral separations in CE are based on two mechanisms, one
156
chromatographic and the other electrophoretic. The chromatographic mechanism arises
157
from the differences in the complexation constants between the enantiomers and the chiral
158
selector. The electrophoretic mechanism is based on the different electrophoretic mobilities
159
that the two enantiomer-chiral selector complexes might have due to subtle differences in
160
size of these complexes, if an analyte is more or less “embedded” in the chiral selector. For
161
further details on mechanisms on enantioseparations in CE, readers are redirected to
162
previous contributions [15].
163
Now, it is important to describe the two approaches used to carry out chiral
164
separations in CE: EKC and CEC. In EKC, which is by far the most used mode, the chiral
165
selector is solved in the BGE forming a “pseudo-stationary phase” making the interaction
166
with the analyte happen in the liquid state and, if it shows enantiorecognition, it will enable
167
the enantioseparation. Several chiral selectors are currently available in CE but nowadays
168
there is an ongoing search to find more CE suitable chiral selectors with exceptional
169
properties. Among the available chiral selectors, cyclodextrins (CDs) deserve a special
170
mention. CDs are cyclic oligosaccharides composed of mainly 6 (α-CD), 7 (β-CD) or 8 (γ-
171
CD) α-D-glucopyranoside units linked via 1-4 bonds and are produced from starch via
172
enzymatic treatment. These “donut”-shaped molecules have secondary and primary
173
hydroxyl groups exposed on the outside, making the outside hydrophilic and the inside
174
hydrophobic. Depending on the interaction, enantiorecognition can be performed both in
175
the inside and/or in the outside. Several research groups have aimed their efforts on 8
176
obtaining a deeper understanding on the mechanisms of this interaction. Nuclear magnetic
177
resonance (NMR), often in combination with molecular modelling, is typically used to
178
study the nature of such interaction [16]. Native CDs can be chemically modified via
179
hydroxyl-derivatization to change their enantioselectivity, enhancing the applicability of
180
these molecules as chiral selectors. Note that the use of single-isomer CD derivatives is
181
advantageous compared to the ones with random substitution for reproducibility reasons.
182
However, single-isomer CDs prices are still somehow prohibited.
183
Other chiral selectors used in EKC are chiral surfactants. When working above the
184
so-called critical micelle concentration (CMC) they will be in the micelle state, i.e.
185
spherical-like aggregates of surfactant molecules in which the hydrophilic heads are in
186
direct contact with the solvent whereas the hydrophobic tails are embedded in the middle of
187
the micelle aggregate. Other chiral selectors falling under EKC mode are ligand-exchange
188
compounds, proteins, bile salts, polysaccharides, ionic liquids, chiral crown ethers,
189
nucleotides, and antibiotics, among others. Readers are encouraged to go to different
190
reviews for detailed description on this topic [10, 17-20].
191
Unlike in EKC, in CEC, chiral stationary phases are employed. CEC is considered
192
to be in between electrophoretic and chromatographic techniques since it combines
193
characteristics of both. The mechanisms of separation of enantiomers in CEC are due to the
194
electrophoretic mobility of the charged analytes and their partition between the chiral
195
stationary phase and their free state in the mobile phase. There are three types of columns
196
in CEC: packed, open tubular, and monolithic columns, depending on how the chiral
197
stationary phase is placed in the capillary. Since this CE mode has not been widely reported
198
in last years, readers are redirected to previously published contributions on the matter [21]. 9
199
Even though the most used media in chiral separations are based on aqueous
200
solvents, CE also enables the possibility to work under non-aqueous conditions in the so
201
called non-aqueous capillary electrophoresis (NACE). This mode is indicated when the
202
analytes of interest or the chiral selectors are not soluble in water. The most common chiral
203
selectors employed in NACE are neutral and charged cyclodextrins as well as ion-pair
204
selectors and antibiotics [15]. Some advantages of NACE include shorter analysis times
205
with higher reproducibility thanks to the less Joule heat produced even at high voltages, as
206
well as more suitable coupling to MS detection [22]. Although there is a wide array of non-
207
aqueous solvents used, methanol is the most popular one. To favor charge of the analytes, it
208
is also needed to add an electrolyte to the non-aqueous solvent, such as formic or acetic
209
acid with or without the corresponding salt (ammonium formate or acetate). Although
210
NACE enables to achieve chiral separations, as we will see in coming sections, this CE
211
mode has scarcely been employed compared with EKC.
212
3. Advances in chiral CE
213
From the middle of 1980s when the first applications of CE in the field of chiral
214
separations were published, this technique has shown its attractive capabilities to achieve
215
highly efficient separations of enantiomers. Although the use of CE is not an easy task (it
216
effectively requires trained and skilled researchers and, above all, a lot of patience and
217
dedication as Fanali and Chankvetadze have recently pointed out [8]), from 1990 to 2010s,
218
there was a high interest in the scientific community to explore and exploit its inherent
219
advantages so that, great efforts were focused on the development of technological and
220
methodological improvements. Since then, other areas have attracted the researchers’s
221
attention affecting the development and applications of chiral CE. Nevertheless, even 10
222
though nowadays CE is a well-established separation technique (mainly in the EKC mode),
223
its full potential has not yet been reached and there are still significant challenges to face.
224
Currently, one of the most emerging research areas in chiral CE is the search of
225
novel chiral selectors. In fact, a considerable number of reviews have examined this topic
226
(some representative and recent examples are included in Table 1). Although, as it will be
227
illustrated in section 4, the use of single CDs or combinations of CDs remains the most
228
popular separation system in chiral CE, numerous researchers have focused their efforts on
229
the evaluation of other alternatives. Sonnendecker et al. demonstrated in an interesting
230
work, the discrimination power of large-ring CDs (single CDs composed of 10 to 12
231
glucose units) in the CE separation of chiral drugs [23]. On the other hand, one of the most
232
popular area in the search of novel chiral selectors is the use of chiral ionic liquids (CILs)
233
which have either a chiral cation, a chiral anion, or both in their structure. From the first
234
enantioseparations obtained using CILs as chiral selectors [24, 25], the number of
235
applications in this field has kept growing. Although some works have shown the
236
possibility of using CILs as single chiral selectors [26], they are mainly used in
237
combination with other chiral selectors (mostly CDs or macrocyclic antibiotics). In this
238
way, they can bring extra enantiorecognition ability while retaining the system modification
239
capability of achiral ionic liquids, so that the enantioseparations can be significantly
240
improved due to the cooperation between both selectors through a synergistic effect. In this
241
point, it should be mentioned that CILs have generated a considerable controversy among
242
the scientists since some of them claim that the addition of the synthesized CIL to the
243
separation buffer implies that it is no longer a CIL but a mixture of independent cations and
244
anions. Therefore, the addition of these anions or cations should work in the same manner
11
245
as the synthesized CIL [11]. However, some works have reported that the addition of CILs
246
gave rise to better results than the addition of their separated ions [27]. More research work
247
is necessary to provide a higher understanding on this subject. Nevertheless, regardless of
248
the mechanisms involved, the fact is that an important number of recent works reported the
249
synthesis of new CILs and their evaluation as chiral selectors in CE [28-30]. Readers
250
interested on gaining deeper insight on the use of CILs in chiral CE are referred to some
251
excellent recent review papers [10, 11, 31]. In addition to CILs, other compounds have also
252
been evaluated as chiral selectors over the last years. For instance, some articles have
253
shown for the first time the enantioseparation abilities of rifampicin [32], doxycycline [33],
254
clarithromycin lactobionate in combination with neutral CD derivatives [34], erythromycin
255
through the use of a carbamoylated erythromycin-zirconia hybrid monolithic column in
256
CEC [35], amino triazolium-modified lactobionic acid [36], or chondroitin sulfate D in
257
combination with a β-CD derivative [37].
258
It is worthy to mention that the use of nanoparticles (NPs) (quantum dots, gold NPs,
259
silica NPs, or carbon nanotubes (CNTs), among others) to improve chiral CE separations in
260
terms of efficiency and resolution, is receiving a significant attention [38]. Chiral selector
261
modified NPs can be bound to a capillary column in CE [39] or added to the buffer solution
262
[40] which confers a high flexibility to the choice of the chiral selector. Between these two
263
approaches, the second one may be, a priori, of greater interest due to some advantages
264
such as easier performance, no need to prepare a column, and no residual effect of
265
stationary phase. In any case, this research area is still far from reaching its full potential so
266
that it is foreseeable that new methodologies based on the use of NPs will be developed in
267
the next years.
12
268
Other important aspect to be considered is the limited concentration sensitivity that
269
can be obtained when using UV detection that is the most employed detection system in
270
chiral CE. The main strategies used to overcome this limitation are focused on the use of
271
preconcentration strategies based on electrophoretic principles or alternative detection
272
systems. Of course, chiral CE can also benefit from the advances and new developments
273
achieved in the field of off-line sample preparation techniques but these techniques will not
274
be commented here. The preferred preconcentration techniques based on electrophoretic
275
principles to improve the detection sensitivity are in-line (carried out within the capillary)
276
or on-line (in a completely integrated and automated manner) sample treatment. Among
277
them, field-amplified sample stacking (FASS), field-amplified sample injection (FASI),
278
and sweeping are usually the most employed in chiral CE analysis to improve the
279
sensitivity not only when UV detection is employed but also with other detectors such as
280
MS. The fundamentals of these preconcentration strategies are already well-known so the
281
developments in this area are being related mainly with the finding of the appropriate
282
combination of some of them in order to achieve the highest sensitivity as illustrated in
283
section 4. Regarding the use of alternative detection systems, laser induced fluorescence
284
(LIF) or MS detectors continue being the preferred ones although LIF detection implies the
285
need of molecule labeling as an essential step prior to CE analysis. By adding a
286
derivatization step, sample treatment becomes more tedious, leading in most cases to
287
derivatization problems when complex matrices are analyzed. Even so, different works
288
published in the last years demonstrated the potential of LIF as detection mode to carry out
289
the chiral analysis of different compounds in real samples enabling to achieve LODs in the
290
nM and sub-pM range [41, 42]. With respect to the CE-MS coupling, two interesting
291
review articles have been published in the last years. On the one hand, Jiang et al. described 13
292
the recent advances of CE-MS instrumentation and methodology in a general way [43]
293
whereas Liu and Shamsi´s review was focused directly on chiral CE-MS [44]. The main
294
limitation of MS detection in chiral CE is due to the incompatibility of nonvolatile chiral
295
selectors frequently employed that may cause ion suppression and contamination of the
296
ionization source leading to a sensitivity decrease. In some cases, it is possible to use a low
297
concentration of chiral selector to achieve a complete enantioseparation without a loss in
298
MS sensitivity [45, 46]. However, in many cases, the presence of nonvolatile chiral
299
selectors in the MS chamber should be avoided. In this line, different authors proposed
300
MEKC-MS methodologies based on the use of (+)-1-(9-fluorenyl) ethyl chloroformate
301
(FLEC) as chiral derivatization reagent to form stable diastereomers with the enantiomers
302
of
303
perfluorooctanoate (APFO) as a volatile pseudostationary phase [47, 48]. Other possibility
304
when the presence of a chiral selector is imperative is the use of chiral stationary phases
305
(CEC mode) or volatile chiral selectors such as polymeric micelles [49]. Nevertheless, the
306
most used strategies to improve the applicability of CE-MS to chiral analysis are those
307
based on the use of the counter migration or partial filling techniques [50]. The
308
combination of a preconcentration strategy with LIF or MS detection appears as the most
309
powerful way to reach a high sensitivity in chiral analysis by CE. This fact has been
310
illustrated in different works such as those reported by Patel et al. and Piestansky et al. who
311
developed chiral methodologies for the ultra-trace chiral determination of two protein
312
amino acids or pheniramine and its metabolite in single neurons or human urine,
313
respectively [41, 51].
the
chiral
compounds
and
their
subsequent
14
separation
using
ammonium
314
The combination of chiral CE with NMR spectroscopy, MS and computation
315
methodologies (molecular modeling and molecular mechanics calculations) is another
316
interesting and relevant area through which it is possible to obtain data related to the
317
mechanistic aspects of selector-selectand interactions [8, 9]. The high peak efficiency that
318
can be achieved in CE makes this separation technique a powerful tool for studying non-
319
covalent intermolecular interactions. In this way, a higher understanding of the enantiomer
320
separation mechanisms on the molecular level is provided. Even though this research is
321
constantly evolving and a high number of works have been published in the last five years
322
to determine the intermolecular interactions between different chiral compounds and chiral
323
selectors [36, 46, 52, 53], we are still far from understanding the nature of the forces
324
involved in the chiral recognition, especially when dual chiral systems are employed.
325
326
4. Applications of CE to chiral analysis
327
Due to its great versatility, CE enables the analysis of numerous chiral compounds
328
in a wide variety of samples in different fields such as the pharmaceutical, biomedical, or
329
food analysis, among others. Here, the most relevant and recent applications of CE to the
330
analysis of chiral compounds in real samples are included.
331
332
4.1. Pharmaceutical analysis
333
In the last years, new CE methods enabling the enantiomeric determination of drugs
334
in pharmaceutical formulations were developed and validated according to the International
335
Conference on Harmonisation guidelines (ICH guidelines). Some of the most relevant and 15
336
recent works published are grouped in Table 2. EKC using CDs as chiral selectors is the
337
most frequent approach, and the most frequent detection mode was UV, used in all cases
338
except in one in which LIF detection was employed.
339
The enantiomeric analysis of drugs marketed as pure enantiomers continues being a
340
challenge because a low amount of the enantiomeric impurity has to be detected in the
341
presence of the majority enantiomer. In fact, ICH guidelines establish that the presence of
342
the impurity enantiomer cannot be higher than 0.1% of the majority enantiomer [76]. This
343
requires a considerable enantiomeric resolution to avoid the overlapping of the majority
344
peak and the enantiomeric impurity peak, and a high sensitivity enabling the detection of
345
the enantiomeric impurity at such low concentrations. In this context, there is another
346
interesting factor affecting the determination of the enantiomeric impurity, which is the
347
enantiomer migration order. In fact, unless high enantiomeric resolutions are possible, it is
348
desirable that the enantiomeric impurity is the first-migrating enantiomer, this avoiding the
349
overlapping of the majority peak with that corresponding to the impurity. Taking into
350
account the importance of the enantiomeric resolution obtained and that the “one factor at
351
time” method to optimize the experimental conditions that influence the chiral separation is
352
time-consuming, the use of experimental designs is recommended. Some methodologies
353
combined both strategies, i.e. the “one factor at time” approach was employed to fix some
354
variables influencing the enantiomeric resolution and subsequently a multivariate
355
optimization was used to select others. For example, the development of a stereoselective
356
MEKC method for the simultaneous determination of montelukast enantiomers and
357
diastereomers and its degradation products was based on the evaluation of the influence of
358
some parameters by the trial-and-error approach and the use of a full factorial design to
16
359
investigate the effect of buffer pH and voltage on the resolution between montelukast
360
enantiomers [54]. Under the best separation conditions (see Table 2), and after evaluating
361
the analytical characteristics and the robustness of the method, the developed methodology
362
was applied to the determination of the above-mentioned isomers in bulk drugs, pouches
363
and tablets. The combination of the “one factor at time” approach and a central composite
364
face centered design was also applied to the optimization of the enantiomeric separation of
365
tramadol, a drug marketed as racemate [55].
366
A relevant point in the pharmaceutical industry for the development of CE
367
methodologies suitable to evaluate the enantiomeric purity of drugs marketed as pure
368
enantiomers is the application of Quality by Design (QbD) approaches. In fact, better
369
analytical methods can be developed applying the QbD principles. Briefly, QbD involves
370
the definition of the analytical target profile (for example, the determination of the main
371
compound and its chiral impurity at the 0.1% level with a resolution value > 2.0 in an
372
analysis time < 10 min), the identification of the critical process variables (buffer
373
concentration and pH, chiral selector concentration, voltage, temperature, etc) affecting the
374
critical quality attributes (resolution and analysis time), and the establishment of the design
375
space (that reflects the experimental conditions under which the analytical target is
376
reached). Fractional factorial resolution designs are usually employed for the identification
377
of the critical parameters. The most relevant are subsequently optimized using central
378
composite face centered designs and Monte Carlo simulations to define the design space.
379
Different works published in the last years reported the application of QbD approaches to
380
the development of CE methodologies for the chiral purity determination of drugs such as
381
ambrisentan [56], levomepromazine [57], dextromethorphan [58], dapoxetine [59], and
17
382
levosulpiride [60]. In some of these works, the analytical target profile comprises not only
383
the determination of the enantiomeric impurity but also its simultaneous analysis with other
384
impurities [57, 59]. The experimental conditions selected (using the above-mentioned
385
experimental designs) in all these CE methodologies are detailed in Table 2. Placket-
386
Burman designs were performed to assess the robustness, and all the developed
387
methodologies were validated according to ICH guidelines. In all cases, the application of
388
these methodologies to pharmaceutical formulations allowed the determination of
389
enantiomeric impurities at levels of 0.1% [56, 57, 58], 0.27% [60] or in the 0.05-1.0%
390
range [59].
391
Despite the application of experimental designs is advisable, different works
392
reported systematic screenings using the principle of “one factor at time” to select the most
393
adequate experimental conditions for the determination of the enantiomeric purity of drugs
394
marketed as pure enantiomers. For instance, using acetyl-β-CD (A-β-CD) as chiral selector
395
in a basic buffer, it was possible to determine the chiral purity of valsartan in tablets at the
396
0.01% level without interferences from the excipients [61]. In spite of the migration order
397
was not the most adequate (the minor component migrated after the major one), this is the
398
contribution of chiral CE to pharmaceutical analysis reporting the lowest relative LOD in
399
the last five years. In addition, the application of this methodology to two commercial
400
brands of tablets enabled to determine the enantiomeric impurity at percentages of 0.12%
401
and 0.22% revealing that ICH guidelines were not accomplished [61]. In the case of the
402
enantiomeric purity control of R-cinacalcet in tablets, the use of an EKC methodology
403
based on the use of 2-hydroxypropyl-γ-CD (HP-γ-CD) as chiral selector allowed for the
404
first time the separation of these enantiomers allowing a favorable migration order, i.e. the
18
405
S-enantiomer was the first-migrating enantiomer [62]. All the tablets analyzed by this
406
method accomplished the ICH regulations since the enantiomeric impurity was in all cases
407
below a 0.1%.
408
As it has been mentioned in section 3, the combination of chiral CE with NMR and
409
molecular modeling studies makes possible to obtain data related to the mechanistic aspects
410
of selector-selectand interactions. Two works published in the last five years reported the
411
development, optimization and validation of EKC-UV methodologies for the enantiomeric
412
quality control of enantiomerically pure drugs and applied NMR and molecular modeling
413
for the characterization of intermolecular interactions between drugs and CDs [63, 64]. On
414
the one hand, Menéndez-López et al. developed an EKC method using two different CDs,
415
succinyl-γ-CD (succ-γ-CD) and S-γ-CD, as chiral selectors for the first CE separation of
416
colchicine enantiomers [63]. Although the enantiomeric impurity migrated in the first place
417
using S-γ-CD, just the use of succ-γ-CD allowed to detect this impurity at a level of 0.1%.
418
Apparent and averaged equilibrium constants for the enantiomer-Succ-γ-CD complexes
419
were calculated by NMR, suggesting that the electrophoretic mobility of these complexes
420
was the predominant factor in the enantiomer migration order. On the other hand, Szabó et
421
al. developed a pressure-assisted EKC-UV with sulfobutyl ether-β-CD (SBE-β-CD) as
422
chiral selector to carry out for the first time the determination of the enantiomeric purity of
423
rasagiline [64]. Binding affinities of the individual enantiomers towards the CD were
424
investigated using both NMR and molecular modeling demonstrating the formation of a
425
more stable inclusion complex between the CD and the enantiomeric impurity which was in
426
accordance with the enantiomer migration order obtained by CE analysis.
19
427
Although EKC with UV detection is the preferred system to perform the evaluation
428
of enantiomeric purity in pharmaceutical formulations (see Table 2), LIF has also been
429
used for the purity control of magnesium L-aspartate dihydrate in two batch samples and
430
three drugs products [65]. Data obtained by the developed EKC-LIF methodology, based
431
on the use of a basic buffer containing HP-β-CD, were compared to those obtained by using
432
a LC-fluorescence method with chiral derivatization. Although the LC method had a higher
433
sensitivity (LOQ of D-aspartate: 0.006% vs 0.03%), both methodologies were suitable for
434
the control of the enantiomeric purity.
435
It is worthy to note that even if some drugs are nowadays marketed as racemates,
436
the studies carried out to achieve their enantiomeric separation when the biological activity
437
of both enantiomers differs, present a high interest taking into account that they could be
438
marketed as pure enantiomers in a near future. Drug racemates enantiomerically separated
439
in the last years include the antihypertensive amlodipine [66], carvedilol [67] and
440
lercanidipine [68] using neutral CDs as chiral selectors such as methyl-β-CD (M-β-CD), β-
441
CD, (2,3,6-tri-O-methyl)-β-CD (TM-β-CD), respectively. The enantiomeric separation of
442
the β-blocker pindolol was also achieved by EKC using an octa(6-O-sulfo)-γ-CD (OS-γ-
443
CD) [69]. Analyte-chiral selector complexation constants were determined, and a software
444
tool was proposed to predict the separation time and other variables from the complexation
445
constants and mobilities of complexes of both enantiomers. Although these four drugs are
446
marketed as racemates, a higher pharmaceutical activity was reported for their (S)-
447
enantiomers. The antiparasitic praziquantel is also commercialized as racemate even though
448
the (R)-enantiomer is the only responsible for its activity. This drug is employed in the
449
prevention and treatment of the tropical disease schistosomiasis (bilharziasis). 20
The
450
quantitation of (R)-praziquantel in tablets was achieved by EKC using S-β-CD as chiral
451
selector [70]. Other antiparasitic compounds present in the samples did not cause
452
interferences.
453
CDs were not the only chiral selectors employed for the enantiomeric determination
454
of drug racemates. In fact, the use of a maltodextrin as chiral selector enabled the
455
simultaneous stereoselective separation of tramadol and methadone not only in tablets but
456
also in biological fluids such as urine and plasma [71]. This was the first time that this
457
chiral selector was used to separate methadone. Researchers claim that the use of
458
maltodextrins shows some advantages over other chiral selectors because they are cost-
459
effective and allow efficient chiral separations.
460
An interesting approach to determine the enantiomeric excess of chiral drugs is the
461
use of the velocity gap mode of CE (VGCE) [72]. This strategy enables to carry out the
462
enantiomeric excess measurement even when the chiral selector does not provide enough
463
resolving power. This is of high relevance in those cases where the content of one
464
enantiomer is significantly higher than the other one. VGCE is based on the fractionation of
465
a small part of the mixture that contains both enantiomers from the main component (which
466
is already enantiopure). Then, the enantioseparation of the small fraction can be achieved
467
due to less longitudinal dispersion. The suitability of this methodology was demonstrated
468
by analyzing levamlodipine besytate tablet as it is illustrated in Figure 2 [72].
469
The potential of a liposome electrokinetic capillary chromatography (LEKC) was
470
evaluated for the enantioseparation of different model drugs and applied to test the chiral
471
impurity of naproxen samples [73]. The chiral separation was carried out employing
472
liposomes comprised of phosphatidylcholine and cholesterol as pseudo-stationary phase 21
473
and SBE-β-CD as chiral selector. The results obtained demonstrated that the
474
enantioseparation increased by using this separation system in comparison with the use of
475
single SBE-β-CD system and SBE-SDS-MEKC system.
476
Even though the most popular approach in chiral separations by CE is based on
477
aqueous solvents, NACE has also been used in drug analysis. Different β-blockers and β -
478
agonists were enantioseparated using a NACE methodology based on the use of boric acid
479
derivatives as new chiral selectors. Lactobionic acid-boric 9 acid complex and D-(+)-
480
xylose-boric acid complex were applied to the chiral separation of propranolol [74] whereas
481
diacetone-D-mannitol-boric acid complex was employed to carry out the enantioseparation
482
of seven β-agonists [75]. In both cases, the chiral selectors were in situ synthesized. The
483
developed NACE methods were successfully applied to the chiral separation of propranolol
484
in tablets [74] and the determination of clenbuterol in an oral solution [75].
485
486
4.2. Bioanalysis
487
CE has also proven its powerfulness in the chiral separation of analytes present in
488
biological samples such as plasma, urine, or even single neurons. These remarkable
489
research works are included in Table 3 and will be further detailed paying attention to
490
different aspects as the chiral selector used or the sensitivity reached. UV, MS and LIF
491
have been the detector systems used in the analysis of biological samples. It is relevant to
492
highlight that in most cases at least a preconcentration strategy was employed.
493
EKC-UV is typically used in combination with preconcentration strategies in the
494
bioanalysis field. This mode has been used to analyze mainly urine and plasma but analysis 22
495
of exhaled breath condensate (EBC) has also been reported [77]. There, authors separated
496
methadone enantiomers using carboxymethyl-β-CD (CM-β-CD) in an acidic medium,
497
included a FASS procedure, reaching LOQs in the µg/mL range. Authors applied the
498
method to analyze EBC collected from patients following methadone maintenance therapy.
499
Concentrations of both enantiomers of methadone in EBC poorly correlated to the ones
500
obtained in urine and serum [77].
501
An API derived from pyroglutamic acid, containing two chiral centers, was also
502
enantioseparated by EKC-UV [78]. Researchers employed S-β-CD as chiral selector and
503
validated the method according to the ICH guidelines. Application was conducted to
504
quality control in bulk material and in the investigation of in vivo inversion in rat plasma, to
505
evaluate the degradation of this drug occurring upon metabolization. LODs as low as ppb
506
were obtained thanks to the utilization of cation-selective exhaustive injection (CSEI)-
507
sweeping. Results showed that there were no in vivo racemization upon the intake of this
508
drug, which confirmed the viability of the commercialization of this product as a single
509
enantiomer. Another metabolization study, this time of ketamine and its metabolites was
510
also conducted by EKC-UV using S-γ-CD [79]. Dogs treated with sevoflurane or
511
medetomidine co-administered with ketamine (racemic or (S)-ketamine). Results showed
512
stereoselective metabolism occurring for metabolites 6-hydroxynorketamine and 5,6-
513
dehydronorketamine in plasma but not for ketamine and norketamine, its main metabolite.
514
Using another sulfated CD, this time sulfated-β-CD (S-β-CD), researchers separated
515
pheniramine enantiomers in rat plasma employing an EKC-UV setup [80]. Large volume
516
sample stacking (LVSS) and CSEI-stacking gave rise to a considerable improvement in the
23
517
sensitivity of 600 and 4000 times, respectively, when compared to not using
518
preconcentration approach.
519
Bioanalysis is the application field in which EKC-MS has been used to a larger
520
extent. This is because higher sensitivity is needed in the analysis of biological samples.
521
Sensitivity is often times not met by EKC-UV even if preconcentration strategies are used.
522
This is the case of a work reporting the development of a method based on ITP and MS
523
detection. The LODs in the pg/mL level were enough to enable its application to study the
524
enantioselective metabolism of pheniramine and its metabolite, desmethyl pheniramine, in
525
human urine samples obtained at different times after administration of racemic
526
pheniramine [51]. This method achieved about 125 times better sensitivity than the above-
527
mentioned method for pheniramines, based on EKC-UV [80]. Note that together with Patel
528
et al. contribution [41], this is the lowest LODs reported for a bioanalysis application and
529
all applications included in the present article altogether. In a recent and very
530
comprehensive publication [46], Liu et al. used EKC-MS to enantioseparate the four
531
stereoisomers
532
hydroxyaspartate. Low concentration of chiral selector (β-CD) was used to minimize MS-
533
source contamination. LVSS with polarity switching enabled a 10-fold increase in
534
sensitivity, reaching LODs as low as 87 nM in rat cerebrospinal fluid samples (CSF).
of
9-fluorenylmethoxycarbonyl
chloride
(FMOC)
labeled
3-
535
Protein amino acids are paramount metabolites with high importance in living
536
organisms. A MEKC-MS approach using derivatization with FLEC for amino acid analysis
537
was developed by Prior et al. to analyze human CSF where levels of endogenous L-amino
538
acids were quantified [48]. Although low, levels of D-serine and D-glutamine were also
539
found in these samples. Another work by Prior et al. also reported the enantioseparation of 24
540
amino acids in CSF, this time, using FMOC as labelling agent and since it is not a chiral
541
labelling compound, a chiral selector, β-CD, was used [81]. Working with a concentration
542
of 10 mM β-CD did not cause much sensitivity decrement and was used filling up
543
completely the separation capillary. Another contribution focusing on amino acids and
544
related compounds was also reported [82]. There authors enantioseparated the constituents
545
of the phenylalanine-tyrosine metabolic pathway using an EKC-MS/MS platform combined
546
with LVSS. LODs of 40-150 nM were found. The method was validated in rat plasma in
547
which endogenous levels of L-phenylalanine and L-tyrosine could be measured. This
548
method is the one based on the use of largest amount of CD (180 mM M-β-CD + 40 mM
549
HP-β-CD). This might be considered as a disadvantage but thanks to the versatility and low
550
consumption of reagents in CE, especially when working in the partial filling technique
551
capillary, this drawback is reduced. The fact that molecules from the same metabolic
552
pathway are analyzed, i.e. closely structural-like compounds could have had an effect on
553
needing such large quantities of chiral selector and favor the enantiorecognition [82].
554
As anticipated earlier in this review article, CDs are by far the most used chiral
555
selectors, but some researchers also used alternative chiral selectors. For example,
556
Svidrnoch et al. who used vancomycin, a macrocyclic antibiotic having 18 chiral centers,
557
for the chiral separation of 2-hydroxyglutaric acid in an EKC-MS configuration [83].
558
Atypical levels of D-2-hydroxyglutaric acid were found in urine from child diagnosed with
559
hydroxyglutaric aciduria. This is a rapid and sensitive method to distinguish between D-
560
and L-2-hydroxyglutaraciduria. Another example is the work by Liu et al. developed a
561
MEKC-MS/MS method using poly-L,L-SULA as chiral selector, to study the
562
pharmacokinetics and pharmacodynamics of the enantiomers of both antidepressant drugs
25
563
venlafaxine and O-desmethylvenlafaxine in human plasma [49]. The results suggested a
564
potential drug to drug interaction between indinavir (an inhibitor drug enabling to prevent
565
the breakage of polyproteins in patients with HIV) and these two antidepressants. This is
566
the only work reporting the use of chiral surfactants in the last years. These polymeric
567
surfactants are promising chiral selectors that are MS compatible but due to their limited
568
commercialization their application might be somehow compromised.
569
LIF detection offers high sensitivity and selectivity but demands sample
570
derivatization with a fluorescent tag. An EKC-LIF method by Patel et al. combined with
571
online preconcentration by LVSS improved the sensitivity 480 times also for the amino
572
acids aspartate and glutamate, reaching LODs in the sub-pM range [41] (Figure 3). This is
573
indeed the lowest LOD reported for chiral separations from last five years, together with
574
Pietansky et al [51]. Thanks to the high sensitivity of this approach it has been possible to
575
determine levels of D-aspartic and D-glutamic acids in single neurons isolated from the
576
neuronal model Aplysia californica. Differences in concentrations of these excitatory amino
577
acids were found depending on the clusters where the neurons were isolated from.
578
4.3. Food analysis
579
Chirality of a great variety of food components makes that their enantioselective
580
analysis has a significant role in food science and technology since it enables to obtain
581
information related to food quality, food processing, storage, or adulterations, among
582
others. Table 4 summarizes the characteristics of the most relevant and recent chiral CE
583
methodologies developed for food analysis in the last years. As this table shows, most of
584
these works are based on the use of CDs as chiral selectors and sometimes SDS is also
585
present in the BGE. As in pharmaceutical analysis, UV is the preferred detection system. 26
586
Different EKC methodologies have demonstrated their potential for the quality
587
control of food supplements [84, 85]. For instance, the enantioselective determination of
588
non-protein amino acids can provide information related to adulterations (the use of D-
589
enantiomers in the elaboration of dietary supplements is not allowed by legal regulations)
590
or food processing (fermentation, storage, etc). In this sense, different EKC methods based
591
on the use of anionic CDs (sulfated-α-CD (S-α-CD) or sulfated-γ-CD (S-γ-CD) depending
592
on the amino acid) were developed to carry out the enantiomeric separation of eight non-
593
protein amino acids previously labeled with FMOC [84]. After studying the effect of
594
different parameters, an optimized methodology using S-γ-CD as chiral selector was
595
applied to the enantiomeric analysis of citrulline in six food supplements. This method
596
enabled to reach LODs in the 10-7 M range. Data obtained demonstrated that the storage
597
time gave rise to a decrease in the amount of the L-enantiomer with respect to the labeled
598
content, but this effect could not be attributed to a racemization process since the D-
599
enantiomer was not detected in any of the samples analyzed [84]. Food supplements quality
600
can also be determined analyzing the origin of their constituents. A clear example of this
601
perspective is the enantioselective separation of 1,3-dimethylamylamine (DMAA) [85].
602
Using the combination of S-α-CD and S-β-CD in a chiral dual system and
603
benzyltriethylammonium chloride as chromophoric additive (to carry out an indirect UV
604
detection) it was possible to achieve the separation of the four DMMA diastereoisomers.
605
The application of this chiral method to the analysis of DMMA in dietary supplements
606
enabled to assume that DMMA probably was not of natural origin because its
607
diastereoisomeric ratios were identical to synthetic DMMA.
27
608
Geographical origin is also an important factor to determine the quality of
609
commercial tea products. One way to do that is using the content of cathechins and
610
methylxanthines as indicators. To do that, a MEKC method with HP-β-CD combined with
611
chemometric analysis was developed [86]. The presence of HP-β-CD in the BGE allowed
612
the enantioselective separation of some of the catechins ((+)-catechin, (‒)-catechin and (‒)-
613
epicatechin). Data obtained were evaluated using principal component and hierarchical
614
cluster analyses as exploratory techniques and by using discriminant models built using
615
linear and quadratic discriminant analyses. The results obtained demonstrated that the
616
developed methodology has a high potential to discriminate green tea samples according to
617
their geographical origin using catechines and methylxanthines as phytomarkers [86]. On
618
the other hand, the presence of (‒)-catechin and D-theanine (both considered as non-native
619
enantiomers) provides information of tea leaves treatment (fermentation, thermal treatment,
620
etc) which also enables to establish a tea classification. In this research line, Fiori et al.
621
developed a MEKC-UV methodology based on the use of SDS and 2,6-di-O-methyl-β-CD
622
(DM-β-CD) for the simultaneous enantioseparation of six major catechins and D,L-
623
theanine (previously derivatized with o-phtaldialdehyde in the presence of N-acetyl-L-
624
cystein) in green tea samples [87]. Once a set of different tea samples were analyzed, it was
625
possible to assign the presence of (‒)-catechin as indicator of a thermal degradation, and D-
626
theanine as marker of microbial or enzymatic processes.
627
The enantiomeric determination of protein amino acid in rice wine is an interesting
628
topic to obtain information about the wine age. For this reason, Miao et al. developed a
629
MEKC method for the determination of D-glutamic acid and D-aspartic acid (previously
630
derivatized with FMOC) in rice wine [88]. The method was based on the use of a dual
28
631
chiral system composed of β-CD and HP-β-CD, SDS, D-fructose (as additive) and
632
isopropanol (as organic modifier). Certain amounts of both D-amino acids were found in
633
the analyzed samples, but their percentage did not show a significant correlation with wine
634
age.
635
The potential of CE for the chiral analysis of lipids has also been demonstrated.
636
Analysis of hydroxyeicosatetraenoic acids requires separating both regioisomers and
637
enantiomers which was achieved in 35 min using a BGE containing HP-γ-CD as chiral
638
selector and SDS [89]. To demonstrate the viability of this MEKC methodology, the chiral
639
analysis of 8-, and 12-hydroxyeicosatetraenoic acids in two species of red algae was carried
640
out.
641
It is interesting to highlight that even though CDs are the most employed chiral
642
selectors and UV is the preferred detection system, an EKC methodology based on the use
643
of a (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) as selector and MS as
644
detection system has also been reported for the enantiomeric determination of underivatized
645
D,L-amino acids in three varieties of vinegar samples [50]. To avoid the contamination of
646
the ionization source due to the presence of the crown ether and make possible EKC-MS
647
hyphenation, the partial filling technique was employed. This provided LODs in the µg/mL
648
level and enabled the analysis of L and D enantiomers which gave information on the
649
fermentation degrees during the manufacturing process. The applicability of this
650
methodology could be extended to the sensitive determination of D,L-amino acids in other
651
samples.
652
The possibilities of ligand exchange, both in CE and in CEC (LE-CE and LE-CEC),
653
for the chiral analysis of short chain organic acids have also been demonstrated in the last 29
654
years. Aydogan et al., applied the ligand exchange principle in an open tubular CEC
655
column using copper (II) as central ion and L-histidine as chiral ligand. Using this
656
approach, it was possible to carry out the determination of D- and L-malic acid enantiomers
657
in apple juice with different dilutions, as Figure 4 shows [90]. On the other hand,
658
Kamencev et al. developed an alternative LE-CE method based on the combination of
659
copper (II)/aluminum (III)/D-quinic acid system and hexadecyltrimethylammonium
660
hydroxide (CTA-OH) to revert the EOF for the simultaneous enantiomeric separation of
661
DL-tartaric and DL-malic acids in wines [91]. While D-malic acid was determined in a
662
broad range of concentrations in different wines, D-tartaric acid was detected just in two of
663
the samples analyzed (at concentrations of 90 and 200 mg/L). It is worth noting that
664
whereas the use of DL-malic acid is allowed for wine acidification, the use of DL-tartaric
665
acid is not allowed. For this reason, the developed methodology acquires a high relevance,
666
especially from the winemaking point of view, since it enables the wine quality control
667
through the enantioselective determination of these short chain organic acids.
668
Until now, all the mentioned methodologies are based on direct approaches, which
669
as highlighted in the section 2, are the most commonly used in chiral separations. However,
670
two indirect strategies have also been described in the last five years. In one of them, D,L-
671
aldoses
672
dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole, were separated
673
in phenylboronate buffer containing SDS [92]. Under these conditions, and using LIF as
674
detection system, the enantiomeric separation of D,L-galactose in the hydrolysate of
675
commercial red seaweed samples was performed. The other work was focused on the
676
derivatization of selenomethionine with FLEC and their enantioselective separation
diastereomers,
formed
by
30
derivatization
with
(S)-(+)-4-(N,N-
677
employing APFO as pseudostationary phase [93]. The developed MEKC-UV methodology
678
was subsequently applied to the determination of L-selenomethionine in food supplements.
679
These two works demonstrate the usefulness of MEKC indirect approaches to the quality
680
control of edible marina algae and food supplements.
681
4.4. Environmental and forensic analysis
682
Chiral CE has also demonstrated its potential in the development of analytical
683
methodologies to be applied in other fields such as environmental and forensic analysis.
684
Table 5 groups some of the most relevant works published in the last years in these fields.
685
In the environmental field, different types of environmental waters (lake, river, ground,
686
wastewater) were the samples most frequently analyzed. Amino acids, drugs and herbicides
687
were enantiomerically determined. Native and derivatized CDs were the preferred chiral
688
selectors in these works to be used as the sole selectors or in dual systems based on
689
mixtures with other CDs or bile salts. In general, UV detection was employed although the
690
use of LIF detection was also reported. Due to the limited concentration sensitivity of UV
691
detection in CE and the low levels of these compounds in the analyzed samples,
692
preconcentration methods were employed to achieve the required sensitivity. With this aim,
693
new periodic mesoporous organosilica materials were synthesized and evaluated as
694
sorbents to carry out the solid-phase extraction of drugs and herbicides from water samples.
695
These novel mesoporous materials were synthesized with neutral ligands to be applied to
696
the preconcentration of a mixture of seven drugs of different characteristics [94] considered
697
as emergent pollutants and with cationic ligands to achieve the preconcentration of a group
698
of six herbicides [95]. The simultaneous separation of the fourteen enantiomers of these
699
drugs and the twelve enantiomers of the herbicides by CE enabled the application of the 31
700
developed methodologies to the determination of these compounds in different water
701
samples at µg/L levels. S-β-CD was employed as chiral selector for the simultaneous
702
enantiomeric determination by EKC of the seven studied drugs in 16 min and this method
703
was further optimized and applied to the evaluation of the enantiomer stability and the
704
toxicity of duloxetine and econazole on Daphnia magna [96]. The stability of these drugs
705
was studied under abiotic and biotic conditions. Figure 5 represents the analysis of
706
mixtures of both drugs. Figure 5A corresponds to an aqueous standard solution and Figure
707
5B to culture medium at zero time. In Figure 5C it can be observed that econazole
708
concentration was unstable and disappeared at 72 h of culture media incubation, the same
709
as in presence of Dapnhnia magna as shown in Figure 5D. Duloxetine concentration
710
decreased in presence of Dapnhnia magna (Figure 5D) [96]. On the other hand, the
711
simultaneous enantiomeric separation by EKC of the six herbicides studied in 11 min
712
required the use of a dual chiral system based on a mixture of TM-β-CD and HP-β-CD.
713
Other mesoporous silica materials were also developed to make possible the chiral analysis
714
of β-blockers by CE in water samples after their preconcentration by solid-phase extraction
715
[97, 98]. M-β-CD was employed as chiral selector enabling the simultaneous determination
716
of propranolol, atenolol, metoprolol and pindolol enantiomers in spiked water samples at
717
µg/l levels.
718
LIF detection was employed in the analysis of different amino acids in water
719
samples from the Mono Lake in USA that can be considered as an analogue for
720
astrobiologically revelant targets [42]. Two different methods were developed. One of them
721
was a MEKC method based on the use of a mixture of γ-CD and sodium taurocholate as
722
chiral system to study neutral amino acids and the other one was an EKC methodology
32
723
consisting of the use of γ-CD to analyze acidic amino acids. Sample preparation was easy
724
since no desalting or preconcentration procedures were necessary due to a derivatization
725
step with 5-carboxyfluorescein succinimidyl ester (CFSE). The use of CFSE increased the
726
sensitivity 2 orders of magnitude compared to fluorescein isothiocyanate (FITC)
727
derivatization.
728
Other interesting application of chiral CE is forensic analysis. CE can provide
729
relevant information related to the consumption of legal or illicit drugs and their
730
toxicological effects or about their origin and synthesis, among others [13]. Table 5 groups
731
detailed information of some of the most recent works published in relation with this type
732
of analysis. Seized samples of stimulant drugs, human hair and human blood are the
733
samples attracting most interest. CDs were the preferred chiral selectors and UV and MS
734
detection systems were employed. An EKC-MS method using S-γ-CD as chiral selector
735
was developed to carry out the analysis of eight amphetamine-like stimulants [99]. A
736
chemically modified capillary containing sulfonated groups was employed to improve the
737
migration times repeatability. This method was applied to two types of seized samples of
738
methamphetamine in order to detect impurities. (-)-ephedrine and (+)-ephedrine were
739
detected as impurities in both samples. The development of other EKC-MS method also
740
based on the use of S-γ-CD as chiral selector was reported and applied to the analysis of
741
twenty seized methamphetamine samples [45]. Results enabled to conclude that three of the
742
twenty samples were a mixture of R-methamphetamine and S-methamphetamine while the
743
rest of the samples contained only the S-enantiomer. This method could be used in the
744
analysis
745
methylenedioxyamphetamine, among others, thanks to the use of TOF-MS which provides
of
other
chiral
abuse
drugs
33
such
as
amphetamine
and
3,4-
746
unequivocal analyte identification. Seized samples were also analyzed by EKC-UV using
747
HP-β-CD as chiral selector. The regioisomeric and enantiomeric analysis of 24 design
748
cathinones and phenethylamines in these samples was reported [100]. The method
749
developed in this work was compared to a LC methodology with UV detection, using a
750
BEH Phenyl column as chiral stationary phase. The CE method was more advantageous, as
751
it resolved all 24 regioisomers while the LC method resolved 18 out of 24.
752
Regarding human hair and blood samples, an EKC-UV method using S-γ-CD as
753
chiral selector was proposed for the chiral separation of ketamine and norketamine which is
754
its principal metabolite, in hair samples from twelve ketamine abusers [101]. Ketamine is a
755
dissociative drug with analgesic, anesthetic and sedative properties. In order to carry out the
756
analysis of these compounds, LLE was used prior to CE. This work provides a fast method
757
for the study of the enantioselective metabolism of ketamine (see Figure 6). Moreover, an
758
EKC-UV method was developed for the determination of methorphan and its most
759
important metabolites in post-mortem blood samples from ten subjects who died due to
760
heroin overdose [102]. Dextromethorphan which is the D-form, can be used as anti-cough
761
medications, the L-enantiomer, levomethorphan, is an opiate agonist and unlike the D-
762
enantiomer, it has narcotic activity. Firstly, a LLE was necessary to extract the compounds
763
for the analysis. HP-β-CD was used as chiral selector which allowed baseline
764
enantioseparation of methorphan enantiomers in 20 min.
765
5. Conclusions and future trends
766
The attractive features of CE make this analytical technique one of the best options
767
to carry out a chiral separation. The research works published demonstrate the relevant role
768
of chiral CE in different fields such as the pharmaceutical, food analysis, biomedical, 34
769
environmental or forensic. This fact is possible thanks to the use of different CE modes
770
such as EKC, MEKC or NACE, and a wide variety of commercially available chiral
771
selectors. In the last years, EKC using a single CD or a combination of CDs has been the
772
most popular choice to develop chiral methodologies although other chiral selectors such as
773
maltodextrins, crown ethers or CILs have also been used to a lesser extent. Even though the
774
search of novel chiral selectors has received a significant attention in the last years and a
775
high number of research articles have been published in this area, until now the applications
776
of novel chiral selectors in the enantiomeric analysis of real samples are scarce. New
777
developments mainly related to the use of novel CILs and NPs as chiral selectors will be
778
expected in the near future.
779
UV detection being the most common system in chiral CE is followed by the
780
coupling with other detectors such as LIF or MS. The combination of these two detection
781
systems with preconcentration strategies has enabled the development of the most sensitive
782
chiral methodologies by CE with LODs in the pg/mL and sub-nM level. It is not surprising
783
that the lowest LODs were reached in the bioanalysis field as it is in this case where the
784
highest sensitivity is usually required. Even though CE-MS is a very powerful tool, its
785
application in chiral separations is limited by the incompatibility of nonvolatile chiral
786
selectors with the MS source. The use of the partial filling and countercurrent techniques as
787
well as of volatile selectors enables to increase the applicability of CE-MS to perform a
788
chiral analysis. Future trends should be redirected to the development of MS compatible
789
chiral selectors. In this way and using more versatile and efficient preconcentration
790
strategies, it would be possible to determine a higher number of chiral compounds at trace
791
levels in a big variety of matrices.
35
792
One of the main applications in pharmaceutical analysis has been the determination
793
of the enantiomeric purity of different drugs in pharmaceutical formulations with LODs at
794
the µg/mL level or relative LODs as low as 0.01%. To obtain low relative LODs in
795
pharmaceutical analysis is essential to ensure proper quality control of the enantiomeric
796
impurity to minimize detrimental effects of non-active substances. Bioanalysis is the field
797
in which more researchers have developed CE methods using MS detection. Amino acids
798
and drugs, among other compounds, are the most analyzed ones, being plasma and urine
799
from human or animal models the most analyzed samples. The low LODs achieved by
800
EKC-MS to study the enantioselective metabolism of pheniramine in urine (LOD of 80
801
pg/mL) or by EKC-LIF to determine D-glutamate and D-aspartate in single neurons (LODs
802
in the sub-pM range) can be highlighted. Although biofluids, EBC samples, and even single
803
neurons were analyzed in the last years, the analysis of tissues has not been reported. This
804
could be due to a more difficult sample preparation in the metabolite extraction from
805
tissues. This is something to keep in mind and hopefully future trends will also focus on
806
tissue analysis. This will definitely give insight on the chiral composition of the different
807
organs. Amino and organic acids were the compounds most frequently determined in food
808
samples. The lowest LODs achieved in this case were at the µg/mL and µM level. Several
809
types of water were analyzed in the environmental field to study the chiral separation of
810
different drugs and amino acids. LODs in the nM order were reached using MEKC or EKC
811
with LIF detection. Researchers working in the forensic field mainly focused their work on
812
the study of different drugs in seized samples as well as in human biological samples, being
813
ng/mL the lowest LODs achieved.
36
814
A notable number of research works published in the last years were focused on the
815
combined use of CE with NMR and molecular modeling studies in order to provide a
816
deeper knowledge on the enantioselective noncovalent intermolecular interactions taking
817
place in the chiral recognition mechanism. This is a very interesting area in which the full
818
potential of CE has not been demonstrated yet so new developments are expected in next
819
years.
820
821
Acknowledgements
822
Authors thank financial support from the Spanish Ministry of Economy and
823
Competitiveness (project CTQ2016-76368-P) and the Comunidad of Madrid and European
824
funding from FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL-II-
825
CM). S.B.B and M.C.P. also thank the Spanish Ministry of Economy and Competitiveness
826
for their predoctoral (BES-2017-082458) and “Ramón y Cajal” (RYC-2013-12688)
827
research contracts, respectively. E.S.L. thanks the University of Alcalá for her postdoctoral
828
contract.
829 830
37
831
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913
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929
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933
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973
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974
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975
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976
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985
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986
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989
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995
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1025
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1030
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1036
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1037
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1044
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1045
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1046
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1047
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1048
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1049
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1050
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1053
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1055
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1056
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1057
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1058
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1059
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1062
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1063
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1064
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1065
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1066
https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3B_R
1067
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1068
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1069
Chiral separation of methadone in exhaled breath condensate using capillary
1070
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1071
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1072
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1073
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1074
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1075
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1076
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1077
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1078
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1079
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1080
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1081
226-232.
dogs
of
under
pharmaceuticals
sevoflurane
vs.
for
medetomidine
49
human
comedication
use.
assessed
by
1082
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1083
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1084
capillary electrophoresis–mass spectrometry, Electrophoresis 37 (2016) 2410-2419.
1085
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1086
Enantioseparation of the constituents involved in the phenylalanine-tyrosine metabolic
1087
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1088
(2016) 372-382.
1089
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1090
Enantioseparation of D,L-2-hydroxyglutaric acid by capillary electrophoresis with tandem
1091
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1092
J. Chromatogr. A 1467 (2016) 383-390.
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1094
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1097
dimethylamylamine by capillary electrophoresis with indirect UV detection using a dual-
1098
selector system. Electrophoresis 36 (2015) 2866-2873.
1099
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1100
cyclodextrin-modified micellar electrokinetic chromatography and chemometric techniques
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1102
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1104
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1105
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1106
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1108
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1109
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1110
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1111
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1112
modified micellar electrokinetic chromatography. Electrophoresis 37 (2016) 23-24.
1113
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1114
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1115
electrochromatography. Food Chem. 187 (2015) 130-134
1116
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1117
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1118
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1119
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1120
D/L-aldoses by micellar electrokinetic chromatography using a chiral derivatization reagent
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1122
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1123
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1124
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51
liquid
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1125
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1126
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1127
phase extraction of drugs prior to simultaneous enantiomericseparation by capillary
1128
electrophoresis, J. Chromatogr. A 1566 (2018) 135-145.
1129
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1130
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1131
off-line solid-phase extraction of phenoxy acid herbicides from water samples prior to their
1132
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1133
1566 (2018) 146-157.
1134
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1135
Enantiomer stability and combined toxicity of duloxetine and econazole on Daphnia magna
1136
using real concentrations determined by capillary electrophoresis, Sci. Total Environ. 670
1137
(2019) 770-778.
1138
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1139
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1140
for SPE and their determination in waters by chiral CE, Electrophoresis 38 (2017) 1905-
1141
1912.
1142
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1143
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1144
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1145
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52
1146
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1147
Inouse, The use of a sulfonated capillary on chiral capillary electrophoresis/mass
1148
spectrometry of amphetamine-type stimulants for methamphetamine impurity profiling,
1149
Forensic Sci. Int. 249 (2015) 59-65.
1150
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1151
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1152
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1153
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1154
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1155
Forensic Sci. Int. 266 (2016) 304-310.
1156
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1157
methorphan in opiate-overdose related deaths by using capillary electrophoresis, J
1158
Chromatogr. B 1000 (2015) 130-135.
1159
1160
1161
53
1162
Figure Captions
1163 1164
Figure 1. Percentage of the number of research papers published from 2015 to 2019
1165
dealing with chiral analysis using different separation techniques obtained from Web of
1166
Science Thomson Reuters database (Accessed August 2019). The keywords employed were
1167
“chiral analysis” and “liquid chromatography”, “capillary electrophoresis”, “gas
1168
chromatography”, or “supercritical fluid chromatography”.
1169
Figure 2. Electropherogram corresponding to enantiomeric excess measurement of
1170
levamlodipine besylate tablets (4.312 mg/mL) obtained using 3.0 % (w/v) α-CD as chiral
1171
selector in velocity gap mode of CE. Reproduced with permission from Ref. [72].
1172
Figure 3. Comparison of EKC-LIF and LVSS-EKC-LIF in the analysis of D,L-aspartate
1173
(Asp) and D,L-glutamate (Glu). CE conditions: 110 mg/mL QA-β-CD in 60 mM 2-(N-
1174
morpholino)ethanesulfonic acid (pH 6.0) plus 10 mM KBr; applied voltage, -26 kV;
1175
temperature, 18°C; LVSS injection, 20 psi × 1 min; detection, emission wavelength at 440
1176
± 8 nm. Peaks in the electropherograms: (1) L-Asp, (2) D-Asp, (3) D-Glu, and (4) L-Glu.
1177
Adapted with permission from Ref. [41].
1178
Figure 4. Electropherograms corresponding to the chiral analysis of malic acid by OT-
1179
CEC. (A) Enantiomeric mixture of malic acid, (B, C, and D) the standard solution from
1180
apple juice diluted 10, 20 and 40-fold, respectively. CEC conditions: ACN/5.0 mM CuSO4,
1181
20.0 mM (NH4)2SO4 (60/40%); applied voltage, 20 kV; injection: -12 kV during 0.05 min;
1182
detection, 214 nm. Reproduced with permission from Ref. [90]
1183
Figure 5. Analysis of duloxetine and econazole racemates (both at a racemic concentration
1184
of 20 mg/L) in (A) an aqueous standard solution; (B) culture medium at zero time; (C) 54
1185
culture medium at 72 h incubation (abiotic conditions); (D) culture medium in presence of
1186
Daphnia magna at 72 h incubation. EKC conditions: 1.5% S-β-CD in 25 mM phosphate
1187
buffer (pH 3.0); applied voltage, -20 kV; temperature, 30°C; injection, 50 mbar × 10 s,
1188
detection, 210 nm. Reproduced with permission from Ref. [96].
1189
Figure 6. Analysis of ketamine and norketamine in three different hair sample from
1190
ketamine abusers. EKC conditions: 0.1% S-γ-CD in 15 mM Tris phosphate (pH 2.5);
1191
applied voltage, 20 kV; temperature, 20°C; injection, 7 kV × 20 s, detection, 200 nm.
1192
Reproduced with permission from Ref. [101].
55
Table 1. Some of the most representative and recent review articles on general aspects of enantiomeric separations by CE. Year
Title
Ref.
2019
Some thoughts about enantioseparations in capillary electrophoresis
[8]
2019
Chiral selectors in capillary electrophoresis: trends during 2017–2018
[6]
2018
Contemporary theory of enantioseparations in capillary electrophoresis
[9]
2018 2018 2017
Enantioseparation by capillary electrophoresis using ionic liquids as chiral selectors Ionic liquids in capillary electrophoresis for enantioseparation Chiral selectors in CE: recent development and applications (mid-2014 to mid2016)
[10] [11] [12]
]Table 2. Characteristics of the most representative CE methodologies developed for pharmaceutical analysis. Analyte
Sample
Montelukast enantiomeric and diastereoisomeric forms
Bulk product, chewable tablets and oral granules (pouches)
Tramadol
Pharmaceutical formulations (capsules and tablets)
CE modedetection
MEKC-UV
EKC-UV
BGE
LOD
Application
Ref.
CS: 10 mM SBE-β-CD +10 mM TM-β-CD B: 10 mM SDS + 20 mM borate (pH 9.0)
0.30 µg/mL (RLOD 0.02%)
Determination of montelukast enantiomeric and diastereoisomeric forms and its main degradation product in bulk product, chewable tablets and oral granules
[54]
CS: 5 mM CM-β-CD B:25 mM Borate pH (11.0)
0.02 mg/mL ((S,S)tramado) 0.024 mg/mL ((R,R)tramadol)
Enantioseparation of two tramadol enantiomers
[55]
Ambrisentan
A chemical supplier sample
EKC-UV
CS: 30 mM γ-CD B: 50 mM acetate (pH 4.0)
0.2 µg/mL
Levomepromazine
Injection solution and a reference substance of the European Pharmacopoeia
EKC-UV
CS: 3.6 mg/mL HP-γ-CD B: 100 mM citric acid (pH 2.85)
0.03% (dextromepromazine)
Dextromethorphan
Capsules
EKC-UV
Dapoxetine
Tablets
EKC-UV
CS: 20 mg/mL S-β-CD + 10 mg/mL M-α-CD B: 50 mM phosphate (pH 7.0)
0.3 µg/mL (0.02%) (levomethorphan)
CS: 45 mg/mL S-γ-CD and
0.66 µg/mL ((R)-
Evaluation of the enantiomeric purity of (S)-ambrisentan in a chemical supplier sample Determination of dextromepromazine and the oxidation product levomepromazine sulfoxide in levomepromazine Determination of the enantiomeric purity of dextromethorphan in capsules Determination of the
[56]
[57]
[58] [59]
40.2 mg/mL DM-β-CD (50%) B: 50 mM phosphate (pH 6.3) CS: 10 mM S-β-CD + 34 mM M-β-CD B: 5 mM Britton Robinson (pH 3.45)
dapoxetine) 1.2 µg/mL for enantiomeric impurity (R-levosulpiride)
Levosulpiride
Pharmaceutical formulations
EKC-UV
Valsartan
Tablets
EKC-UV
CS: 10 mM A-β-CD B: 25 mM phosphate (pH 8)
0.01% (R-valsartan)
Cinacalcet
Tablets
EKC-UV
CS: 0.5% (w/v) HP-γ-CD B: 150 mM phosphate (pH 2.5) + 20% (v/v) of methanol
0.1% ((S)-cinacalcet)
Colchicine
Pharmaceutical formulations
EKC-UV
CS: 7 mM Succ-γ-CD B: 50 mM borate (pH 9.0)
0.3 mg/L ((R)colchicine)
Rasagiline
Tablets
EKC-UV
2 µg/mL ((S)rasagiline)
Mn-L-Asp
Batch samples and drug products
EKC-LIF
CS: 30 mM SBE-β-CD B: 50 mM glycine-HCl (pH 2) CS: 18 mM HP-β-CD B: 50 mM phosphate (pH 7.0) + 18% (v/v) DMSO
Amlodipine
Tablets
EKC-UV
CS: 20 mM M-β-CD B: 50 mM phosphate (pH 3.0)
Carvedilol
Tablets
EKC-UV
CS: 10 mM β-CD B: 25 mM phosphate (pH 2.5)
Lercanidipine
Tablets
EKC-UV
CS: 10 mM of TM-β-CD B: 200 mM acetate (pH 4.0)
LOQ: 0.03 % (Mn-DAsp) 2.31 µg/mL ((R)amlodipine) 2.43 µg/mL ((S)amlodipine) 1.13 µg/mL ((R)carvedilol) 1.18 µg/mL ((S)carvedilol) 0.8 µg/mL ((S)lercanidipine)
purity of dapoxetine in tablets Quantification of levosulpiride in pharmaceutical formulations Determination of the (R)-enantiomer of valsartan in tablets Quantification of cinacalcet in tablets Quantification of colchicine in pharmaceutical formulations Determination of (S)rasagiline in tablets Quantification of Mn-LAsp in batch samples and drug products
[60]
[61]
[62]
[63]
[64] [65]
Determination of amlodipine enantiomers in tablets
[66]
Quantification of carvedilol in tablets
[67]
Quantification of lercanidipine in tablets
[68]
Pindolol
Pills
EKC-UV
CS: 6 mM OS-γ-CD B: 40/80 mM Sodium/MOPS pH (7.12)
Praziquantel
Tablets
EKC-UV
CS: 15 mM S-β-CD B: 50 mM phosphate (pH 2.0)
Tramadol and methadone
Tablets
EKC-UV
Levamlodipine besylate
Tablets
EKC-UV
Naproxene
Bulk sample
LEKC-UV
Propranolol
Tablets
NACE-UV
Clenbuterol
Oral solution
NACE-UV
0.6 µg/mL ((R)lercanidipine) 0.6 µg/mL ((R)pindolol) 0.6 µg/mL ((S)pindolol)
Enantioseparation of pindolol enantiomers in pills
[69]
0.75 µg/mL
Separation of the Praziquantel enantiomers in tablets
[70]
CS: 20% (w/v) Maltodextrin (DE=4-7) B: 100mM phosphate (pH 8.0)
2 µg/mL (tramadol) 1.5 µg/mL (methadone)
Determination of tramadol and methadone in different samples
[71]
CS: 3.0% (w/v) α-CD B: not stated
1 % ((R)levamlodipine)
Quantification of levamlodipine besylate in tablets
[72]
4.5 µg/mL
Enantioseparation of Naproxene in bulk sample
[73]
0.25 µg/mL (((R)propranolol) 0.50 µg/mL ((S)propranolol)
Quantification of propranolol in tablets
[74]
0.25 µg/mL
Separation of clenbuterol agonists in oral solution
[75]
CS: 2% SBE-β-CD (w/v) + 1.2% (w/v) nanoliposome B: 20 mM phosphate buffer (pH 5.2) CS: 8 mM lactobionic acid, 100 mM boric acid B: 14.4 mM triethylamine in methanol CS:80 mM diacetone-Dmannitol B: 100 mM boric acid + 50.4 mM triethylamine in methanol
Abbreviations: A-β-CD, acetyl-β-CD; B, buffer; CM-β-CD, carboxymethyl-β-CD; CS, chiral selector; DE, dextrose equivalent; DM-β-CD, 2,6-dimethyl-βCD; HP-γ-CD, 2-hydroxypropyl-γ- CD; γ-CD,γ -CD; M-α-CD, methyl-α-CD; M-β-CD, methyl-β- CD; MOPS, 3-(N-morpholino) propanesulphonic acid;
OS-γ-CD, octa (6-O-sulfo) γ- CD; Succ-γ-CD, succinyl-γ- CD; S-β-CD: sulfated β- CD; S-γ-CD, sulfated γ- CD; SBE-β-CD, sulfobutyl ether-β-CD; TM-βCD, 2,3,6-tri-O-methyl-β-CD; SDS, sodium dodecyl sulphate.
Table 3. Characteristics of the most representative methodologies developed by CE for bioanalysis. Analyte
Sample
CE modedetection
BGE
LOD
Methadone
EBC
EKC-UV
CS: (0.8%) CM-β-CD B: 100 mM phosphoric acidTEA (pH 2.5)
LLOQ: 0.15 µg/mL
API derived from pyroglutamic acid
Rat plasma
EKC-UV
CS: 2.5% (w/v) S-β-CD B: 50 mM phosphate (pH 2.5)
0.15-0.17 µg/mL
CS: 0.66 % (w/v) S-γ-CD B: disodium hydrogenphosphate buffer (pH 3.0)
3 ng/mL
Ketamine and metabolites
Dog plasma
EKC/UV
Pheniramine
Rat plasma
EKC/UV
Pheniramine and metabolites
Human urine
EKC/MS
3-hydroxyaspartate
Rat CSF
EKC/MS
CS: 6 mM β-CD B: 49 mM NH4Ac + 15% (v/v) isopropanol
87 nM
Proteinogenic amino acids
Human CSF
EKC/MS
CS: 10 mM β-CD B: 50 mM ammonium bicarbonate (pH 8) + 15%
0.5-84.3 µM
CS: 30 mg/mL S-β-CD B: 30 mM phosphate buffer (pH 3.0) CS: 5 mg/mL CM-β-CD B: 25 mM ε-aminocaproic acid + 25 mM acetic acid (pH 4.5) + 0.05 % (w/v) methylhydroxyethylcellulose
LLOQ: 10 ng/mL
80 pg/mL
Application Quantification of methadone enantiomers in EBC Study on drug enantiomeric inversion in vivo Determination of ketamine and metabolites in dog plasma under sevoflurane or medetomidine and ketamine intake Pharmacokinetic study on rats treated with racemic pheniramine Determination of pheniramine and its metabolites in urine of healthy patients after pheniramine intake Simultaneous determination of four isomers of 3hydroxyaspartate in rat CSF Enantioselective analysis of proteinogenic amino
Ref. [77]
[78]
[79]
[80]
[51]
[46]
[81]
Phenylalanine, tyrosine, DOPA, dopamine, norepinephrine, epinephrine
2-hydroxyglutaric acid
Rat plasma
Human urine
EKC-MS/MS
(v/v) isopropanol
acids in CSF
CS: 180 mM M-β-CD + 40 mM HP-β-CD B: 2 M formic acid (pH 1.2)
Chiral separation of the constituents of the PheTyr metabolic pathway in rat plasma
40-150 nM
EKC/MS
CS: 25 mM vancomycin B: 50 mM ammonium acetate (pH 4.5)
31-38 nM
10.5-15 ng/mL 15 ng/mL
Sub-pM
Venlafaxine and metabolites
Human plasma
MEKC/MS
CS: 15 mM poly-L,L-SULA B: 20 mM ammonium acetate + 25 mM TEA (pH 8.5)
Aspartate and glutamate
Single neurons from Aplysia californica
EKC/LIF
CS: 110 mg/mL QA-β-CD B:60 mM MES (pH 6.0) + 10 mM KBr
Determination of 2hydroxyglutaric acid in urine of healthy patients and children with abnormal excretion of 2hydroxyglutaric acid Determination of venlafaxine and metabolites in plasma of healthy patients after venlafaxine intake with or without indinavir Study on excitatory amino acids in neurons isolated from different neurological clusters of sea slug
[82]
[83]
[49]
[41]
Abbreviations: API, active pharmaceutical ingredient; B, buffer; CM-β-CD, carboxymethyl-β-CD; CS: chiral selector; CSF, cerebrospinal fluid; EBC, exhaled breath condensate; HP-β-CD, hydroxypropyl-β-CD; S-β-CD: sulfated-β-CD; S-γ-CD, highly sulfated-γ-CD; LLOQ, lower limit of quantification; MES, 2-(N-morpholino)ethanesulfonic acid; M-β-CD, methyl-β-CD, poly-L,L-SULA, poly-sodium N-undecenoyl-l,l-leucylalaninate; QA-β-CD, quaternary ammonium β-cyclodextrin; S-β-CD, sulfated-β-CD; TEA, triethylamine.
Table 4. Characteristics of the most representative methodologies developed by CE for food analysis. Analyte
Sample
CE modedetection
BGE
LOD
D,L-Citrulline
Food supplements
EKC-UV
CS: 10 mM S-γ-CD B: 100 mM formate (pH 3.0)
0.21 µM (D-Cit) 0.18 µM (L-Cit)
7.82-9.24 µg/mL
Application Analysis of D- and Lcitrulline in food supplements Determination of the stereoisomeric composition of DMAA in food supplements to verify their potential natural origin Analysis of the principal catechins and methylxanthines in green tea samples Chiral analysis of theanine and catechin in the characterization of green tea samples
Ref. [84]
1,3Dimethylamylamine
Food supplements
EKC-UV
CS: 1.1% (w/v) S-α-CD + 0.2 % (w/v) S-β-CD B: 5 mM phosphate/ Tris (pH 3.0) and 10 mM BTEAC
Catechins
Green tea
MEKC-UV
CS: 25 mM HP-β-CD B: 90 mM SDS + 25 mM borate-phosphate (pH 2.5)
-
Theanine and catechins
Green tea
MEKC-UV
CS: 28 mM DM-β-CD B: 65 mM SDS + 25 mM borate-phosphate (pH 2.5)
0.1-02 µg/mL
MEKC-UV
CS: 35 mM β-CD + 6 mM HPβ-CD + 25 mM D-fructose B: 30 mM SDS + 100 mM borate (pH 9.5) + 15% IPA (v/v)
2.5 µM
Determination of D-Glu and D-Asp in rice wine
[88]
MEKC-UV
CS: 30 mM HP-γ-CD B: 75 mM SDS + 30 mM phosphate-15 mM borate (pH 9.0)
0.95-0.99 µg/mL
Determination of the enantiomeric composition of the hydroxyeicosatetraenoic acids found in red algae
[89]
EKC-MS
CS: 30 mM (18C6H4)
0.07-1.03 µg/mL
Determination of D- and
[50]
Glutamic acid and aspartic acid
8- and 12Hydroxyeicosatetrae noic acids Proteinogenic amino
Rice wine
Marine Red Algae (Gracilaria vermiculophylla and Gracilaria arcuata) Vinegars
[85]
[86]
[87]
acids
B: 1M formate
D- and L-Malic acid
Apple juice
LE-CEC-UV
D- and L-Tartaric acid and D- and Lmalic acid
Wine
LECE-UV
CS: 5 mM Cu (II) sulfate B: 20 mM ammonium sulfate (60:40 v/v) (pH 3.0) CS: 100 mM D-quinic acid, 10 mM Cu (II), 0.5 mM Al (III) and 0.5 mM CTA-OH B: 20 mM acetate
1.5 mg/L (D-tartaric acid) 3 mg/L (D-malic acid)
L-amino acids in vinegars Determination of malic acid enantiomers in apple juice Determination of Denantiomers of both acids in wines.
[90]
[91]
Abbreviations: B, buffer, BTEAC: benzyltriethylammonium chloride, CS: chiral selector, Cit: citrulline, CTA-OH, hexadecyltrimethylammonium hydroxide; 18C6H4, (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid; DM-β-CD, 2,6-di-O-methyl-β-cyclodextrin; HP-β-CD, 2-hydroxypropyl-βcyclodextrin; IPA, isopropanol; LECE, ligand exchange capillary electrophoresis; LE-CEC, ligand exchange capillary electrochromatography;, SDS, sodium dodecyl sulfate; S-α-CD, sulfated-α- CD; S-β-CD, sulfated-β-CD.
Table 5. Characteristics of the most representative methodologies by CE in other fields. Analyte Duloxetine, terbutaline, econazole, propranolol, verapamil, metoprolol and betaxolol Fenoprop, mecoprop, dichloroprop, 2-(4chlorophenoxy) propionic acid, 2-(3chlorophenoxy)propi onic acid and 2phenoxypropionic acid Duloxetine and econazole Pindolol, atenolol, propranolol and metoprolol Pindolol, atenolol, propranolol and metoprolol
Sample
Wastewater of different treatment plants
River water and wastewater of effluent treatment plants from different Spanish regions
CE modedetection
BGE
LOD
Application
Ref.
EKC-UV
CS: 2% (w/v) S-β-CD B: 25 mM phosphate (pH 3.0)
0.4-1.5 mg/L
Enantiomeric determination of drugs in waste water
[94]
EKC-UV
CS: 20 mM of TM-β-CD + 7 mM of HP-β-CD B: 50 mM phosphate (pH 7.0)
0.1-4.3 µg/L
Enantiomeric determination of phenoxy acid herbicides in water samples
[95]
Daphnia magna
EKC-UV
CS: 15% (w/v) S-β-CD B: 25 mM phosphate (pH 3.0)
0.3-1,1 mg/L
River and ground water
EKC-UV
CS: 1.25% (w/v) M-β-CD B: 50 mM phosphate (pH 2.5)
1-1.6 µg/L
EKC-UV
CS: 1.25% (w/v) M-β-CD B: 50 mM phosphate (pH 2.5)
0.4- 0.6 µg/L
River water and sewage water of the waste water treatment plant (effluent water)
Enantiomer stability and combined toxicity of duloxetine and econazole on Daphnia magna Determination of βblockers in environmental waters Chiral analysis of β blockers in river and sewage water
[96]
[97]
[98]
Neutral amino acids CS: 30 mM γ-CD and 30 mM sodium taurocholate B: 80 mM tetraborate (pH 9.2) and 5% (v/v) ACN Acidic amino acids CS: 30 mM γ-CD B: 80 mM tetraborate (pH 9.2) CS: 20 mM S-γ-CD B: 10 mM formic acid (pH 2.5)
Amino acids
Lake water
Neutral amino acids: MEKCLIF Acidic amino acids: EKC-LIF
8 Amphetaminerelated stimulants
Seized samples
EKC-MS
Amphetamine‐type stimulants and ephedrine
Seized samples
EKC-MS
CS: 0.26% (w/v) S-γ-CD B: 50 mM ammonium formate (pH 2.2)
3.24-8.57 µg/mL
EKC-UV
CS: 80 mM HP-β-CD B: Celixir initiator solution (pH 2.5)
5 µg/mL
24 Design cathinones and phenethylamines
Ketamine and norketamine
Methorphan
Seized samples
Human hair
Human blood
EKC-UV
EKC-UV
CS: 0.1% (w/v) S-γ-CD B: 15 mM Tris-phosphate (pH 2.5) CS: 5 mM HP-β-CD B: 150 mM phosphate (pH 4.4) containing 20% (v/v) of MeOH
Neutral amino acids: 5-100 nM Acidic amino acids: 500-750 nM
2 µg/mL (for (-)ephedrine and (+)pseudoephedrine)
Chiral separation of DLamino acids in lake water
Analysis of seized samples of methamphetamine Analysis of seized samples of amphetamine‐type stimulants and ephedrine Regioisomeric and enantiomeric analysys of cathinones and phenethylamines in seized samples
[42]
[99]
[45]
[100]
0.08 ng/mL
Determination of ketamine in hair from ketamine abusers
[101]
8 ng/mL
Analysis of post-mortem blood samples of corpses overdosed by heroin
[102]
Abbreviations: ACN, acetonitrile; B, buffer; CS, chiral selector; S-γ-CD, sulfated-γ- CD; HP-β-CD, (2-hydroxypropyl)-β-cyclodextrin; M-β-CD, methylated-β- CD; LIF, laser-induced fluorescence; S-β-CD: sulfated-β-CD; TM-β-CD, (2,3,6-tri-O-methyl)-β-CD.
Figure 1.
73
Figure 2.
74
Figure 3.
75
Figure 4.
76
Figure 5.
77
Figure 6.
78
Highlights • Most relevant and recent contributions of Chiral CE were reviewed • Fundamentals and characteristics of Chiral CE were described • Most recent technological and methodological developments were presented • Applications in the pharmaceutical, food, biomedical or other fields were included
Conflicts of Interest: The authors declare no conflict of interest.