Accepted Manuscript Title: Cationic permethylated 6-monoamino-6-monodeoxy--cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis Author: Krisztina N´emeth Celesztina Domonkos Vir´ag Sarnyai Julianna Szem´an L´aszl´o Jicsinszky Lajos Szente J´ulia Visy PII: DOI: Reference:
S0731-7085(14)00316-1 http://dx.doi.org/doi:10.1016/j.jpba.2014.06.028 PBA 9631
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
18-4-2014 17-6-2014 18-6-2014
Please cite this article as: K. N´emeth, C. Domonkos, V. Sarnyai, J. Szem´an, L. Jicsinszky, L. Szente, J. Visy, Cationic permethylated 6-monoamino-6-monodeoxyrmbeta-cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.06.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
1
Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of
2
dansylated amino acids in capillary electrophoresis
3 Krisztina Németh1*, Celesztina Domonkos1, Virág Sarnyai1, Julianna Szemán2, László
5
Jicsinszky2, Lajos Szente2, Júlia Visy1
6
1
7
of Sciences, H-1519 Budapest P.O.B.:286, Hungary
8
2
ip t
4
us
CycloLab R&D Ltd., H-1097 Budapest, Illatos út 7, Hungary
cr
Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy
an
9 *Corresponding author: K. Németh
11
Postal address: 1Institute of Organic Chemistry, Research Centre for Natural Sciences,
12
Hungarian Academy of Sciences, H-1519 Budapest P.O.B.:286, Hungary
13
Tel.: +36 1 382 6659; E-mail address: [email protected]
d
M
10
te
14 15
24
Highlights • • • • •
Ac ce p
16 17 18 19 20 21 22 23
Permethylated 6-monoamino-6-monodeoxy-βCD resolves dansylated amino acids. Maximal chiral resolutions could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of the selector. Low CD concentration was enough for the separation of the most apolar Dns-AAs. Resolution power of PMMABCD was proved by the separation of Dns-AAs’ mixture.
25 26
Abstract
27 28 29
The
resolution
power
of
permethylated
6-monoamino-6-monodeoxy-βCD
(PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral
1
Page 1 of 25
analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-
31
AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-
32
AAs by varying pH and selector concentrations were investigated and correlated with their
33
structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions
34
could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of
35
the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be
36
achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs.
37
Chiral discrimination ability of PMMABCD was demonstrated by the separation of an
38
artificial mixture of 8 Dns-AA pairs.
an
us
cr
ip t
30
39
Keywords: cationic CD derivative, dansylated amino acid, enantioseparation, isoelectric
41
point, lipophilicity
M
40
d
42 Abbreviations:
44
Dns-AA: dansylated amino acid; Dns-Abu: dansylated α-amino butyric acid; Dns-Asp:
45
dansylated aspartic acid; Dns-Glu: dansylated glutamic acid; Dns-Leu: dansylated leucine;
46
Dns-Met: dansylated methionine; Dns-Nle: dansylated norleucine; Dns-Nva: dansylated
47
norvaline; Dns-Phe: dansylated phenylalanine; Dns-Thr: dansylated threonine; Dns-Trp:
48
dansylated tryptophan; Dns-Val: dansylated valine; PMMABCD: permethylated 6A-
49
monoamino-6A-monodeoxy- β-cyclodextrin;
Ac ce p
50
te
43
51
2
Page 2 of 25
51
1. Introduction
52 Analytical methods particularly capillary electrophoresis (CE) using chiral selectors
54
are appropriate for stereoselective separations [1-6]. Cyclodextrin (CD) derivatives are one of
55
the most favorable selectors to be applied in CE [7]. Development of new chiral selectors,
56
including charged cyclodextrins is important due to the increasing demand to analyze a great
57
number of pharmaceutical, environmental, and nutritional compounds. The advantage of the
58
charged selectors is that they provide relatively large separation window in the case of the
59
oppositely charged analytes [8]. Great numbers of anionic and even cationic CD derivatives
60
have been synthesized and released in the last decades [9-21]. Amino substituted CDs are
61
among the most widely investigated new selectors. Application of cationic –principally
62
amino- CD derivatives for chiral separation of several underivatized amino acids (AA) or
63
their fluorescent derivatives - for more sensitive detection –has been demonstrated [8,13,14-
64
16,18-25]. Iványi et al. [26] reported that permethylated 6-monoamino-6-monodeoxy-βCD
65
(PMMABCD) developed previously as stereoselective agent for CE [27] was a good chiral
66
selector for dansyl-phenylalanine and dansyl-leucine as well as for many other – acidic -
67
chiral substances [26,28-32]. This cationic (pKa = 9.05 [28]), single-isomer βCD derivative is
68
completely O-methylated and substituted by an amino group in C6 position of one of the
69
glucopyranoside units (Degree of substitution = 20+1). The advantage of single isomer
70
selectors over randomly substituted ones is their outstanding batch-to-batch reproducibility.
cr
us
an
M
d
te
Ac ce p
71
ip t
53
Our study aimed to demonstrate the applicability of PMMABCD in chiral separation
72
of further dansylated amino acids (Dns-AAs) as amphoteric model compounds. Using Dns-
73
AAs is explained by the increasing importance of the evaluation of the enantiomer ratio of
74
amino acids in biological samples and in food analysis [33-38] since the biological activity,
75
pharmacology and toxicology of D-amino acids are widely studied [39-42]. The pH and
3
Page 3 of 25
selector concentration dependencies of chiral resolutions were characterized and correlated
77
with the charged states of the analytes and selector and with the structure and lipophilicity of
78
the analytes. The chiral resolution power of PMMABCD was further examined with an
79
artificial mixture of Dns-AAs.
ip t
76
80 2. Materials and methods
cr
81 82
2.1. Materials
us
83
an
84
Background electrolyte (BGE) components phosphoric acid, sodium hydroxide,
86
glacial acetic acid, boric acid and ethanol were purchased from Merck GmbH (Darmstadt,
87
Germany), eleven racemic (Table 1) and nine L-Dns-AA were obtained from Sigma-Aldrich
88
(St.Louis, USA). PMMABCD was the product of CycloLab Ltd. (Budapest, Hungary). Its
89
synthesis route has been described in details [27].
te
d
M
85
90
2.2. Capillary electrophoresis
92 93
Ac ce p
91
Capillary electrophoresis was performed with an Agilent Capillary Electrophoresis
94
3D
95
capillary having a 64.5 cm total and 56 cm effective length with 50 μm I.D. (Agilent
96
Technologies, Santa Clara, CA, USA). On-line absorption at 220 nm was monitored by DAD
97
UV-Vis detector. The capillary was thermostated at 25°C. Between measurements, the
98
capillary was rinsed subsequently with 0.1 M HCl, 1.0 M NaOH, 0.1 M NaOH and distilled
99
water for 3 min each and with BGE for 5 min. Various BGE buffers were prepared either
100
from 82 mM boric acid (pH 9); or from 16 mM phosphoric acid (pH 8); or from 22 mM
CE system (Agilent Technologies, Waldbronn, Germany) applying bare fused silica
4
Page 4 of 25
phosphoric acid (pH 7); or from 35 mM phosphoric acid (pH 6); or from 65 mM acetic acid
102
(pH 5); or from 255 mM acetic acid (pH 4); or from 50 mM phosphoric acid (pH 3) with
103
sodium hydroxide to attain the desired pHs as indicated and the same ionic strength (I= 44
104
mM). The appropriate amounts of PMMABCD were dissolved in the running buffers. Dns-
105
AA samples were dissolved in 50% ethanol and further diluted with distilled water, final
106
concentrations were 0.1 mg/ml. The racemic Dns-AAs were spiked by the pure L-enantiomers
107
(cL-Dns-AA/cD-Dns-AA= 7/3) – with the exception of Dns-Nle and Dns-Abu, their pure ‘L’
108
enantiomers were lacking. The artificial mixture was prepared by mixing the stock solutions
109
of the Dns-AA samples and further diluted with distilled water. The ethanol content of the
110
mixture was higher, the concentrations of Dns-AAs were 0.03 mg/ml and enantiomer ratios
111
were similar with the individual samples. Samples were injected by 5×103 Pa pressure for 3
112
sec. Runs were performed in the positive-polarity mode with 30 kV. Enantiomer separations
113
were carried out with 2.5, 5, 10, 20 mM concentrations of PMMABCD. The resolution (Rs) of
114
the enantiomers is given by the following equation [43]:
cr
us
an
M
d
1.18 × (t 2 − t1 ) w (0.5 )1 + w (0.5 )2
te
Rs =
Eq. 1
Ac ce p
115
ip t
101
116
where t is the migration time of the enantiomers (1, 2 in lower index), w(0.5) is the peak width
117
at half height.
118
Selectivity values are given as αt,migr = t migr,2/tmigr,1 , where t migr,1 and t migr,2 are the migration
119
times of the Dns-AA enantiomers, and are also given as αµ,eff = µeff,2/µeff,1 , where µeff,1 and µeff,2
120
are the mobilities of the Dns-AA enantiomers, respectively.
121 122
2.3. Estimation of pI values of Dns-AAs
123 124
pIs of the Dns-AAs were determined from the effective mobilities (μeff) measured at
125
several pHs. The effective mobilities were plotted against the applied pHs and the intercept of 5
Page 5 of 25
126
the fitted curve on the abscissa gave the pI of the given analyte (Fig. S1, Table S1) [44,45]. Predicted logP values were calculated by ChemAxon’s Marvin Calculator Plugin
128
software (version 5.9.2; URL: http://www.chemaxon.com/marvin) called as “Marvin” in the
129
followings.
ip t
127
130 3. Results and discussion
cr
131 132
Chiral separations of 11 Dns-AA enantiomer pairs (Table 1) were studied by the
134
cationic PMMABCD. Separations were carried out in the pH 3-9 range and by applying the
135
selector in the 2.5 mM to 20 mM concentration range. Results are summarized in (Table 2,
136
Table S2). Using these conditions eight enantiomer pairs can be resolved at least at baseline
137
or better and additional two pairs can be separated partially. Separations can be carried out in
138
reasonable running times. Changes in resolution of Dns-AAs as a function of pH and
139
PMMABCD concentration is discussed in details as follows.
142 143 144 145
an
M
d
te
141
Ac ce p
140
us
133
[Table 1] [Table 2]
3.1. pH dependence of resolution of Dns-AA enantiomers
Changes in the chiral separations of the enantiomers of Dns-AAs were investigated by
146
varying the pH between pH 3 and pH 9. In order to evaluate charge relationships between the
147
host and guest molecules in this pH range the pI values of the analytes were measured by CE.
148
The pI values of the Dns-AAs are between 3.46-3.79 (Table 1) being in good agreement with
149
literature data (Table S1) [46]. Consequently, the Dns-AAs are positively charged below and
150
negatively charged above these values while the PMMABCD is positively charged
6
Page 6 of 25
151
permanently below pH 9. The effects of the pH on chiral separations of Dns-AAs were
152
studied by investigating several combinations of different charge states of the selector and
153
selectands (at pH 3, 4, 6 and 9). Migrations of the Dns-AAs were accelerated (μcompl>μfree) by PMMABCD at pHs
155
above their pI values (Table S2). The negatively charged Dns-AAs if complexed with
156
PMMABCD migrate faster than the free analytes alone due to the decrease in their negative
157
charges and increase in their mass (i.e. hydrodynamic radius). Using this cationic selector
158
below the pIs of the Dns-AAs the complexes gain positive charges not only from the analyte
159
but also from the selector, in addition their sizes are increased. Therefore their mobility
160
changes are not substantial and could not be predicted well due to those counter-effects.
an
us
cr
ip t
154
Resolutions show maxima at pH 4 or at pH 6 (Table 2, Fig. 1) where PMMABCD is
162
cationic and the Dns-AAs are at least partially negatively charged. The highest selectivities
163
can be observed for Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp and Dns-Thr
164
at pH 4 and for Dns-Met, Dns-Val and Dns-Asp at pH 6. Resolutions are decreased at pH 9
165
where PMMABCD is partially in uncharged form and the Dns-AAs are negatively charged.
166
Resolutions are weaker too at pH 3 where the selector is fully positively charged and the
167
analytes are partially cationic.
d
te
Ac ce p
168
M
161
In general, the amount of the D-amino acids is substantially lower than that of their L
169
pairs in nature. In most of our cases the migration order of the enantiomers (EMO) is D,L in
170
the entire pH range investigated. So that EMO is advantageous because the evaluation of
171
peaks is more accurate when the smaller component migrates before the major one.
172 173
[Fig. 1]
174 175
3.2. CD concentration dependence of resolution of Dns-AA enantiomers
7
Page 7 of 25
176 Selectivities increase with increasing selector concentrations in most cases of the Dns-
178
AAs investigated. Our finding that the maximal selectivities are in the high concentration
179
range of the selector corresponds [2,47,48] to the weak interactions between PMMABCD and
180
Dns-AAs (Kassoc ≤ 10 M-1) estimated by the well known non-linear regression of the
181
electrophoretic mobility shifts [49-53].
cr
ip t
177
Whereas, increasing the selector concentration results in the improvement of
183
separations usage of PMMABCD in concentrations substantially higher than 20 mM is not
184
recommended because of longer analysis times and peak distortions. In addition, due to the
185
elevated current other counterproductive effects of Joule heating can also occur. Furthermore,
186
it is noteworthy that the pH and selector concentration dependencies for Dns-Nle and Dns-
187
Phe deviate from the others (Fig. 1) because in these two cases the migration time of the
188
peaks of the enantiomers overlaps with the time of the electroosmotic flow at high (15-20
189
mM) concentrations of PMMABCD at pH 4.
te
d
M
an
us
182
Baseline or better separation can be achieved for some of the enantiomer pairs of these
191
Dns-AAs even with low concentrations of PMMABCD. In the presence of only 2.5 mM-10
192
mM concentrations of the selector Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp
193
and Dns-Met exhibited good resolution with further improvements with elevated CD
194
concentrations. In the group of Dns-AAs with nonpolar aliphatic sidechains the higher is the
195
lipophilicity the better is the resolution. 15 mM - 20 mM concentrations of PMMABCD are
196
needed for the resolution of the more polar (cf. the logP values in Table 1) Dns-AAs (namely:
197
Dns-Val, Dns-Thr and Dns-Asp). Unfortunately, enantiomers of the polar Dns-Glu can not be
198
separated at all with PMMABCD.
Ac ce p
190
199
Limit of detection (LOD at signal to noise ratio S/N = 3) and the limit of quantitation
200
(LOQ at S/N = 10) were determined for several dansylated D- and L-amino acids (Table 3).
8
Page 8 of 25
The LOD and LOQ values were between 0.2 - 3 µg/ml and 6 - 10 µg/ml, respectively. Most
202
biological samples contain D- and L-amino acid enantiomers approximately in a ratio of
203
1:100 [54]. Accordingly, the sensibility of our system for the detection of the minor (D-Dns-
204
AA) component followed by the major ‘L’ enantiomer is demonstrated here with some
205
examples (Dns-Phe, Dns-Trp, Dns-Leu, Dns-Val, Dns-Nva and Dns-Met) where the
206
enantiomer ratios is also 1 to 100. In these setups (20 mM PMMABCD at pH 6.0) the
207
concentrations of D-Dns-AAs is around at their LOQ values (10 µg/ml) and those of their ‘L’
208
pairs were 1 mg/ml (Fig. 2).
us
cr
ip t
201
an
209 210
[Table 3] [Fig. 2]
212
M
211
3.4. Resolution of an artificial mixture of Dns-AAs
d
213
The resolution power of PMMABCD is further studied by mixture of Dns-AAs. From
215
our enantiomer pairs we chose those eight Dns-AAs which were separated at baseline at least.
216
Migration times of the enantiomers were taken into account too in selecting optimal running
217
parameters (pH and CD concentration) in order to obtain separated peaks for each constituent
218
of the mixture. Despite that in some cases pH 4 can provide more improved resolution for the
219
individual Dns-AAs than pH 6 the number of the enantiomer pairs resolved is greater at pH 6
220
than at pH 4. Consequently, we have chosen 20 mM PMMABCD at pH 6.0 and 7 enantiomer
221
pairs separated well and two further peaks partially of that mixture containing 8 pairs (Fig. 3).
222
[Fig. 3]
Ac ce p
te
214
223 224 225
9
Page 9 of 25
226
4. Conclusions
227 The amino and methyl substituted βCD derivative PMMABCD is introduced as an
229
excellent cationic selector for Dns-AAs having nonpolar aliphatic and aromatic side chains
230
and a usable selector for some Dns-AAs with polar uncharged or charged groups. The
231
advantage of using PMMABCD is that it consumes a small amount of CD derivative, the
232
separations can be carried out in reasonable running times, and it provides the preferable ‘DL’
233
migration order for the resolution of Dns-AAs at pHs above their pI values. Furthermore, the
234
chiral recognition ability of PMMABCD is proved by the separation of a mixture of Dns-
235
AAs.
an
us
cr
ip t
228
238
M
236
239
We gratefully acknowledge Dr. Róbert Iványi, Katalin Tuza (Cyclolab R&D Ltd.) as well as
240
Prof. Miklós Simonyi, Dr. Gábor Tárkányi, Mrs. Ilona Kawka (RCNS, HAS) and Dr.
241
Zsuzsanna Lakatos for their helpful contributions. The authors thank for the financial support
242
from the following grants: NKFP_A3-2008-0211 NATURSEP, TECH-09-AI-2009-0117
243
NanoSEN9, the Hungarian Scientific Research Fund (OTKA, grant numbers K-100134 and
244
NN-110214) and the “Lendület” Program of the Hungarian Academy of Sciences (LP2013-
245
55/2013).
te
d
Acknowledgements
246
Ac ce p
237
247
Appendix A. Supplementary data
248
Supplementary data associated with this article can be found, in the online version, at doi:
249 250
The authors have declared no conflict of interest.
10
Page 10 of 25
251 5. References
253
[1] G. Gübitz, M.G. Schmid, Chiral separation principles in capillary electrophoresis, J.
254
Chromatogr. A 792 (1997) 179-225.
255
[2] S. Fanali, Enantioselective determination by capillary electrophoresis with cyclodextrins
256
as chiral selectors, J. Chromatogr. A 875 (2000) 89-122.
257
[3] G. Blaschke, B. Chankvetadze, Enantiomer separation of drugs by capillary
258
electromigration techniques , J. Chromatogr. A 875 (2000) 3-25.
259
[4] B. Chankvetadze, Enantioseparations by using capillary electrophoretic techniques. The
260
story of 20 and a few more years, J. Chromatogr. A 1168 (2007) 45-70.
261
[5] P. Jac, G.K.E. Scriba, Recent advances in electrodriven enantioseparations, J. Sep Sci. 36
262
(2013) 52-74.
263
[6] L. Qi, Y. Chen, M. Xie, Z. Guo, X. Wang, Separation of dansylated amino acid
264
enantiomers by chiral ligand-exchange CE with a zinc(II) L-arginine complex as the selecting
265
system Electrophoresis 29 (2008) 4277-4283.
266
[7] D.A. Tsioupi R-I. Stefan-van-Staden, C.P. Kapnissi-Christodoulou, Chiral selectors in CE:
267
Recent developments and applications, Electrophoresis 34 (2013) 178-204.
268
[8] B. Chankvetadze, G. Endresz, G. Blaschke, Charged cyclodextrin derivatives as chiral
269
selectors in capillary electrophoresis, Chem. Soc. Review, 25 (1996) 141-148.
270
[9] V. Cucinotta, A. Contino, A. Giuffrida, G. Maccarrone, M. Messina, Application of
271
charged single isomer derivatives of cyclodextrins in capillary electrophoresis for chiral
272
analysis, J. Chromatogr. A 1217 (2010) 953-967.
273
[10] Z. Juvancz, R. Bodáné-Kendrovics, R. Iványi, L. Szente, The role of cyclodextrins in
274
chiral capillary electrophoresis, Electrophoresis 29 (2008) 1701-1712.
Ac ce p
te
d
M
an
us
cr
ip t
252
11
Page 11 of 25
[11] Y. Tanaka, S. Terabe, Enantiomer separation of acidic racemates by capillary
276
electrophoresis using cationic and amphoteric beta-cyclodextrins as chiral selectors, J.
277
Chromatogr. A 781 (1997) 151-160.
278
[12] H. Yamamura, Y. Yamada, R. Miyagi, K. Kano, S. Araki, M. Kawai, Preparation of β-
279
Cyclodextrin Derivatives Possessing Two Trimethylammonio Groups on Their Primary
280
Hydroxy Sides as Chiral Guest Selectors, J. Incl. Phenom. Macrocycl. Chem. 45 (2003) 211–
281
216.
282
[13] W. Tang, I. W. Muderawan, S.-C. Ng, H. S. O. Chan, Enantiomeric separation of 8
283
hydroxy, 10 carboxylic and 6 dansyl amino acids by mono(6-amino-6-deoxy)-beta-
284
cyclodextrin in capillary electrophoresis, Anal. Chim. Acta 554 (2005) 156-162.
285
[14] W. Tang, I. W. Muderawan, T.-T. Ong, S.-C. Ng, Enantio separation of dansyl amino
286
acids by a novel permanently positively charged single-isomer cyclodextrin: Mono-6-N-
287
allylammonium-6-deoxy-beta-cyclodextrin chloride by capillary electrophoresis, Anal. Chim.
288
Acta 546 (2005) 119-125.
289
[15] Sz. Béni, T. Sohajda, G. Neumajer, R. Iványi, L. Szente, B. Noszál, Separation and
290
characterization of modified pregabalins in terms of cyclodextrin complexation, using
291
capillary electrophoresis and nuclear magnetic resonance, J. Pharm. Biomed.Anal. 51
292
(2010) 842-852.
293
[16] A. Giuffrida, A. Contino, G. Maccarrone, M. Messina, V. Cucinotta The 3-amino-
294
derivative of gamma-cyclodextrin as chiral selector of Dns-amino acids in electrokinetic
295
chromatography, J. Chromatogr. A 1216 (2009) 3678-3686.
296
[17] P. Liu, W. He, X-Y.,Qin, X-L. Sun, H. Chen, S-Y. Zhang, Synthesis and application of a
297
novel
298
tetramethylenediamine)-beta-cyclodextrin as chiral selector in capillary electrophoresis
299
Chirality 22 (2010) 914-921.
Ac ce p
te
d
M
an
us
cr
ip t
275
single-isomer
mono-6-deoxy-6-((2S,3S)-(+)-2,3-O-isopropylidene-1,4-
12
Page 12 of 25
[18] A. Giuffrida, R. Caruso, M. Messina, G. Maccarrone, A. Contino, A. Cifuentes, V.
301
Cucinotta, Chiral separation of amino acids derivatised with fluorescein isothiocyanate by
302
single isomer derivatives 3-monodeoxy-3-monoamino-beta- and gamma-cyclodextrins: the
303
effect of the cavity size J. Chromatogr. A 1269 (2012) 360-365.
304
[19] Y. Dai, S. Wang, J. Zhou, Y. Liu, D. Sun, J. Tang, W. Tang, Cationic cyclodextrin as
305
versatile chiral selector for enantiomeric separation in capillary electrophoresis, J.
306
Chromatogr. A, 1246 (2012) 98-102.
307
[20] S. Wang, Y. Dai, J. Wu, J. Zhou, J. Tang, W. Tang, Methoxyethylammonium
308
monosubstituted β-cyclodextrin as the chiral selector for enantioseparation in capillary
309
electrophoresis, J. Chromatogr. A 1277 (2013) 84-92.
310
[21] J. Zhou, F. Ai, B. Zhou, J. Tang, S.-C. Ng, W. Tang, Hydroxyethylammonium
311
monosubstituted cyclodextrin as chiral selector for capillary electrophoresis, Anal. Chim.
312
Acta 800 (2013) 95-102.
313
[22] H. Wan, L.G. Blomberg, Chiral separation of amino acids and peptides by capillary
314
electrophoresis, J. Chromatogr. A 875 (2000) 43-88.
315
[23] V. Poinsot, M.-A. Carpene, J. Bouajila, P. Gavard, B. Feurer, F. Couderc, Recent
316
advances in amino acid analysis by capillary electrophoresis, Electrophoresis 33 (2012) 14-
317
35.
318
[24] V. Poinsot, C. Bayle, F. Couderc, Recent advances in amino acid analysis by capillary
319
electrophoresis, Electrophoresis 24 (2003) 4047-4067.
320
[25] M. Chiari, M. Cretich, G. Crini, L. Janus, M. Morcellet, Recent advances in amino acid
321
analysis by capillary electrophoresis, J. Chromatogr. A 894 (2000) 95-103.
322
[26] R. Iványi, L. Jicsinszky, Z. Juvancz, Permethyl monoamino beta-cyclodextrin a new
323
chiral selective agent for capillary electrophoresis, Chromatographia 53 (2001) 166-172.
Ac ce p
te
d
M
an
us
cr
ip t
300
13
Page 13 of 25
[27] L. Jicsinszky, R. Iványi, Catalytic transfer hydrogenation of sugar derivatives,
325
Carbohydr. Polym. 45 (2001) 139-145.
326
[28] R. Iványi, L. Jicsinszky, Z. Juvancz, Chiral separation of pyrethroic acids with single
327
isomer permethyl monoamino beta-cyclodextrin selector, Electrophoresis 22 (2001) 3232-
328
3236.
329
[29] A.M. Abushoffa, M. Fillet, A.C. Servais, P. Hubert, J. Crommen, Enhancement of
330
selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of
331
oppositely charged cyclodextrins in capillary electrophoresis, Electrophoresis 24 (2003) 343-
332
350.
333
[30] B. Tőkés, L. Ferencz, P. Buchwald, G. Donázh-Nagy, Sz. Vancea, N. Sántha, E. L. Kis,
334
Structural studies on the chiral selector capacity of cyclodextrin derivatives, J. Biochem.
335
Biophys. Methods 70 (2008) 1276–1282.
336
[31] A.G. Cârje; Z. Juvancz, R. Iványi, V. Schurig, B. Tőkés, Comparative study on chiral
337
separation of pyrethroic acids with amino and neutral cyclodextrine derivatives, Acta
338
Medica Marisiensis 57 (2011) 52-54.
339
[32] G. Tárkányi, K. Németh, R. Mizsei, O. Tőke, J. Visy, M. Simonyi, L. Jicsinszky, J.
340
Szemán, L. Szente, Structure and stability of warfarin-sodium inclusion complexes formed
341
with permethylated monoamino-beta-cyclodextrin, J. Pharm. Biomed.Anal. 72 (2013) 292-
342
298.
343
[33] R.M. Callejón, A.M. Troncoso, M.L. Morales, Determination of amino acids in grape-
344
derived products: A review, Talanta 81 (2010) 1143-1152.
345
[34] M. Wcisło, D. Compagnone, M. Trojanowicz, Enantio selective screen-printed
346
amperometric biosensor for the determination of D-amino acids, Bioelectrochemistry 71
347
(2007) 91-98.
Ac ce p
te
d
M
an
us
cr
ip t
324
14
Page 14 of 25
[35] X. He, X. Cui, M. Li, L. Lin, X. Liu, X. Feng, Highly enantioselective fluorescent sensor
349
for chiral recognition of amino acid derivatives, Tetrahedron Lett. 50 (2009) 5853-5856.
350
[36] H. Zahradníčková, P. Hušek, P. Šimek, P. Hartvich, B. Maršálek, I. Holoubek,
351
Determination of D- and L-amino acids produced by cyanobacteria using gas chromatography
352
on Chirasil-Val after derivatization with pentafluoropropyl chloroformate, Anal. Bioanal.
353
Chem. 388 (2007) 1815-1822.
354
[37] Y. Miyoshi, R. Koga, T. Oyama, H. Han, K. Ueno, K. Masuyama, Y. Itoh, K. Hamase,
355
HPLC analysis of naturally occurring free D-amino acids in mammals, J. Pharm. Biomed.
356
Anal. 69 (2012) 42-49.
357
[38] M. Herrero, C., Simo, V. Garcia-Canas, S. Fanali, A. Cifuentes, Electrophoresis 31
358
(2010) 2106-2114.
359
[39] K. Hamase, Sensitive two-dimensional determination of small amounts of D-amino acids
360
in mammals and the study on their functions, Chem. Pharm. Bull. 55 (2007) 503-510.
361
[40] M. Friedman, Origin, Microbiology, Nutrition, and Pharmacology of D-Amino Acids,
362
Chem. Biodiversity 7 (2010) 1491-1530.
363
[41] K. Hamase, Analysis and biological relevance of D-amino acids and relating compounds,
364
J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3077-3077.
365
[42] H. Ohide, Y. Miyoshi, R. Maruyama, K. Hamase, K. R. Konno, D-Amino acid
366
metabolism in mammals: Biosynthesis, degradation and analytical aspects of the metabolic
367
study, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3162-3168.
368
[43] S. Fanali, Controlling enantioselectivity in chiral capillary electrophoresis with inclusion-
369
complexation, J. Chromatogr. A 792 (1997) 227-267.
370
[44] D. Koval, V. Kasicka, J. Jiracek, M. Collinsová, Determination of pK(a) values of
371
diastereomers of phosphinic pseudopeptides by CZE, Electrophoresis 27 (2006) 4648-4657.
Ac ce p
te
d
M
an
us
cr
ip t
348
15
Page 15 of 25
[45] S.K. Poole, S. Patel, K. Dehring, H. Workman, C.F. Poole, Determination of acid
373
dissociation constants by capillary electrophoresis, J. Chromatogr. A 1037 (2004) 445-454.
374
[46] A. Bianchi-Bosisio, P.G. Righetti, N.B. Egen, M. Bier, Isoelectric focusing of
375
dansylated amino-acids in immobilized pH gradients, Electrophoresis 7 (1986) 128-133.
376
[47] A. Rizzi, Fundamental aspects of chiral separations by capillary electrophoresis,
377
Electrophoresis 22 (2001) 3079-3106.
378
[48] S. Wren, The separation of enantiomers by capillary electrophoresis, Chromatographia
379
54 (2001) S1-S95.
380
[49] S. G. Penn, D. M. Goodall, J. S. Loran, Differential Binding of tioconazole enantiomers
381
to hydroxypropyl-beta cyclodextrin studied by capillary, J. Chromatogr. 636 (1993) 149-152.
382
[50] K. L. Rundlett, D. W. Armstrong, Examination of the origin, variation, and proper use of
383
expressions for the estimation of association constants by capillary electrophoresis, J.
384
Chromatogr. A 721 (1996) 173-186.
385
[51] S. A.C. Wren, Mobility measurements on dansylated amino acids, J. Chromatogr. A 768
386
(1997) 153-159.
387
[52] A. Salvador, E. Varesio, M. Dreux, J-L. Veuthey, Binding constant dependency of
388
amphetamines with various commercial methylated beta-cyclodextrins, Electrophoresis 20
389
(1999) 2670-2679.
390
[53] Y. Tanaka, S. Terabe, Estimation of binding constants by capillary electrophoresis, J.
391
Chromatogr. A 768 (2002) 81-92.
392
[54] Zs. Wagner, T. Tábi, T. Jakó, G. Zachar, A. Csillag, É. Szökő, Chiral separation and
393
determination of excitatory amino acids in brain samples by CE-LIF using dual
394
cyclodextrin system, Anal. Bioanal. Chem. 404 (2012) 2363-2368.
Ac ce p
te
d
M
an
us
cr
ip t
372
395
16
Page 16 of 25
395
Figure captions
396 Fig. 1. Changes in selectivities (αt,migr) of several Dns-AAs as a function of pH and selector
398
concentration
399
Fig. 2. Separations of D- and L-Dns-AA enantiomers in a concentration ratio of 1:100 (10
400
µg/ml and 1 mg/ml, respectively) in the presence of 20 mM PMMABCD at pH 6
401
Fig. 3. Separation of Dns-AA mixture by 20 mM PMMABCD at pH 6
cr
ip t
397
us
402
an
403
Ac ce p
te
d
M
404
17
Page 17 of 25
404 405
Table 1: Structure, isoelectric point (pI) and lipophilicity (logP) of dansyl-amino acids H3C N O S
R group
COOH NH
pI
R
O
Nonpolar, aliphatic R group, linear sidechain: Dns-DL-α-aminobutyric acid (Dns-Abu)
-CH2CH3
Dns-DL-norvalin (Dns-Nva)
-(CH2)2CH3
Dns-DL-norleucine (Dns-Nle)
-(CH2)3CH3
-2.54
3.67
-2.11
3.67
-1.52
-CH(CH3)2
3.70
-2.26
-CH2CH(CH3)2
3.63
-1.52
-CH2C6H5
3.67
-1.38
-CH2C8NH6
3.79
-1.05
-(CH2)2SCH3
3.55
-1.87
-CHCH3OH
3.66
-2.94
-CH2COOH
3.46
-3.89
-(CH2)2COOH
3.50
-3.69
cr
3.68
Nonpolar, aliphatic R group, branched sidechain:
us
Dns-DL-valine (Dns-Val) Dns-DL-leucine (Dns-Leu) Nonpolar, aromatic R group:
an
Dns-DL-phenylalanine (Dns-Phe) Dns-DL-tryptophan (Dns-Trp) Nonpolar, uncharged R group:
M
Dns-DL-methionine (Dns-Met) Polar, uncharged R group: Dns-DL-threonine (Dns-Thr) Dns-DL-aspartic acid (Dns-Asp)
Ac ce p
te
Dns-DL-glutamic acid (Dns-Glu)
d
Polar, negatively charged R group:
406 407 408
logP
*
ip t
H3C
18
Page 18 of 25
Table 2: Migration times (t1), selectivities (α) and resolutions (Rs) of Dns-AAs at pH 9-3 in the presence of 2.5-20 mM PMMABCD as chiral selector pH 2.5
DnsLeu
DnsPhe
DnsTrp
DnsThr DnsAsp
411 412 413 414 415 416 417 418 419 420
20
2.5
5
10
20
2.5
5
10
15
20
n.a.
4.63
4.79
5.23
n.a.
1.004
1.010
n.a.
n.a.
nr
0.5
1.5
5.90
6.54
6.83
7.33
7.79
1.000
1.012
1.025
1.042
1.073
nr
1.8
3.9
6.5
11.1
4
12.19
14.19
17.21
19.23
20.30
1.000
1.009
1.039
1.085
1.131
nr
3
n.a.
n.a.
14.67
15.49
19.16
n.a.
n.a. 1.000
1.008
1.000
n.a.
9
4.22
4.30
4.47
4.64
5.07
1.000
1.000
1.000
1.000
1.012
nr
6
5.59
5.94
6.32
6.51
7.58
1.000
1.013
1.029
1.042
1.092
nr
4
12.21
14.02
16.14
17.22
17.59
1.000
1.016
1.068
1.133
1.179
3
12.56
12.65
20.52
22.62
26.37
1.000
1.000
1.000
1.000
1.016
1.0
4.0
8.1
11.8
n.a.
nr
nr
nr
nr
nr
nr
1.7
2.1
4.6
8.0
12.9
nr
1.6
6.8
13.2
17.3
nr
nr
nr
nr
0.8
n.a.
n.a.
0.6
2.5
4.9
0.9
5.2
10.1
14.0
19.5
9
n.a.
n.a.
4.45
4.55
4.89
n.a.
1.018
1.032
6
5.49
5.68
5.92
6.05
6.40
1.006
1.032
1.068
1.088
1.160
4
12.14
13.20
13.42
14.84
17.52
1.015
1.065
1.197
1.149
n.a.
1.9
5.5
20.4
20.3
n.a.
3
n.a.
n.a.
15.91
17.24
21.66
n.a.
n.a. 1.022
1.067
1.060
n.a.
n.a.
1.8
2.8
3.6
9
n.a.
n.a.
4.56
4.73
5.19
n.a.
6
5.62
6.07
6.62
7.01
8.45
1.000
4
11.53
13.43
16.16
18.85
20.59
3
n.a.
n.a.
15.64
16.60
n.a.
9
4.17
4.24
4.41
4.58
5.08
1.003
1.005
1.007
1.006
6
5.85
6.66
6.91
7.27
7.91
1.004
1.000
1.000
1.013
4
11.99
13.96
16.89
18.46
19.17
1.000
1.010
1.059
1.137
3
12.91
13.19
24.46
27.14
34.52
1.000
1.000
1.012
1.023
9
4.15
4.21
4.23
4.25
4.58
1.142
1.000
1.019
6
5.63
5.95
5.66
5.77
6.07
1.016
1.071
1.115
4
11.43
12.26
12.70
16.23
17.24
1.021
1.085
1.216
n.a.
3
13.05
13.16
22.41
24.90
31.77
1.000
1.000
1.015
1.017
9
4.16
4.26
4.48
4.64
5.11
1.000
1.000
1.000
1.000
6
5.47
5.89
6.37
6.64
7.33
1.000
1.000
1.014
1.019
4
n.a. 12.21
12.49
12.69
n.a. 1.000
us
n.a. 1.005
ip t
n.a.
6
cr
n.a. 1.000
15
9
3
DnsMet
15
Rs
an
DnsVal
10
1.000
1.000
n.a.
n.a.
nr
nr
nr
1.000
1.000
1.016
nr
nr
nr
nr
2.3
1.000
n.a. 1.000
1.000
1.000
nr
n.a.
nr
nr
nr
n.a.
n.a. 1.000
1.000
n.a.
n.a.
n.a.
nr
nr
n.a.
1.004
0.5
0.9
1.2
1.5
0.6
1.035
0.6
nr
nr
2.0
4.4
1.220
nr
1.2
6.0
12.8
19.5
1.036
nr
nr
1.0
1.7
6.9
1.039
1.059
0.9
nr
3.4
5.9
6.7
1.148
1.168
2.5
10.5
16.3
21.2
21.2
n.a.
nr
7.4
27.1
n.a.
n.a.
1.000
nr
nr
1.0
1.4
nr
1.000
nr
nr
nr
nr
nr
1.045
nr
nr
2.1
1.4
7.4
1.000
M
DnsNle
5
d
DnsNva
d
Ac ce p
DnsAbu
αe
t1 (min)
te
408 409 410
14.33
n.a.
17.53
17.33
20.05
23.72
n.a. 1.006 1.000
9
n.a.
n.a.
4.59
4.70
5.14
n.a.
6
5.96
6.78
6.92
7.20
7.94
1.000
1.000
1.026
n.a.
1.092
n.a.
0.8
2.8
n.a.
8.7
1.000
1.000
1.006
nr
nr
nr
nr
nr
1.011
1.021
n.a.
n.a.
nr
1.3
3.2
1.021
n.a. 1.000 1.044
1.071
1.134
nr
3.3
6.8
10.9
17.0
n.a. 1.000
1.000
1.000
1.000
n.a.
nr
nr
nr
nr
n.a. 1.000
1.000
1.010
n.a.
n.a.
nr
nr
0.5
4
n.a.
n.a.
n.a.
n.a.
n.a.
3
n.a.
n.a.
18.25
20.60
24.35
n.a.
4
12.74
14.96
19.15
24.44
29.21
1.000
1.000
1.000
1.000
1.015
nr
nr
nr
nr
1.0
6
9.88
11.43
13.31
16.10
19.79
1.000
1.000
1.000
1.011
1.016
nr
nr
nr
1.0
1.4
a) n.a. not available b) nr not resolved; Rs < 0.5 c) t1 migration time of the faster enantiomer d) Selector concentrations applied is given in mM in the second row of the header e) Selectivity is given as follows: α = t migr,2/tmigr,1 f) The enantiomer migration order is D,L in the majority of the cases. The selectivity values are underlined where the enantiomer migration order is reversed (L,D).
19
Page 19 of 25
Table 3: Limit of detection (LOD) and quantitation (LOQ) of D- and L-Dns AAs 421 D-Dns-AA LOD (µg/ml)
L-Dns-AA LOQ (µg/ml)
LOD (µg/ml)
LOQ (µg/ml)
0.4
5.8
0.4
6.4
Dns-Nva
0.2
5.7
0.2
6.3
Dns-Nle
0.1
5.2
0.1
6.5
Dns-Val
1.7
7.9
1.7
7.7
Dns-Leu
1.5
7.4
1.5
Dns-Phe
1.3
6.2
1.3
Dns-Trp
1. 8
6.1
1.8
6.1
Dns-Met
3.0
10.0
2.9
10.2
ip t
Dns-Abu
cr
420
7.5
us
7.5
Ac ce p
te
d
M
an
422 423
20
Page 20 of 25
6.5 7.0 7.5 8.0
cr
8.5
Migration time (min) 9.0
Dns-L-Met
Dns-L-Nva Dns-L-Leu Dns-L-Trp Dns-D-Val Dns-Abu Dns-L-Val
i
(OCH3)6 and (NH2)1
Dns-Abu Dns-D-Trp Dns-D-Leu
Dns-Nle
us
C(2)
Dns-D-Met
M an
Dns-L-Phe
(OCH3)7
Dns-D-Nva
ed
ce pt
C(3)
Dns-Nle
Dns-D-Phe
(CH3O)7
Ac
EOF
Absorbance (220 nm)
*Graphical Abstract C(6)
9.5 Page 21 of 25
20
20
cr
20
i
Figure(s)
1.100 10
1.125 1.150 1.175
Dns-Abu
5
1.050 1.075 1.100
10
1.125
5
1.200
Dns-Nva
1.225 3
4
5
6 pH
7
8
9
3
1.250
20
1.050 1.075 1.125 1.150 1.175
Dns-Leu
5
1.200
6 pH
7
8
9
5
6 pH
7
8
9
1.250
Ac
4
20
3
4
1.100 10
1.125 1.150 1.175
Dns-Nle
1.200 1.225
3
4
5
6 pH
7
8
9
1.100 1.150 1.175 1.200
1.000 1.025 15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Trp
1.200 1.225
1.225 5
6 pH
7
8
9
1.250
20
1.075
Dns-Phe
1.050 1.075
1.250
1.125
5
1.025
15
1.225
1.050
10
1.000
5
1.200
1.025
1.225
3
1.175
1.000
15
ce pt
1.100
10
CD concentration (mM)
1.025 15
5
ed
20 1.000
CD concentration (mM)
4
1.150
CD concentration (mM)
1.075
15
CD concentration (mM)
1.050
1.025
us
15
1.000
M an
1.025
CD concentration (mM)
CD concentration (mM)
1.000
1.250
3
4
5
6 pH
7
8
9
1.250
CD concentration (mM)
1.000 1.025
15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Met
1.200 1.225
3
4
5
6 pH
7
8
9
1.250
Page 22 of 25
Fig. 1
20
20
cr
20
i
Figure(s)
1.100 10
1.125 1.150 1.175
5
Dns-Abu
1.050 1.075 1.100
10
1.125
5
1.200
Dns-Nva
1.225 3
4
5
6 pH
7
8
9
3
1.250 20
15
1.050 1.075 1.100
10
1.125 1.150
5
1.175
Dns-Leu
CD concentration (mM)
1.025
1.200
5
6 pH
7
8
9
1.250
Ac
4
20
7
8
9
4
1.050 1.075 1.100
10
1.125 1.150 1.175
Dns-Nle
1.200
1.225
1.225 3
1.250
4
5
6 pH
7
8
9
1.025 1.050 1.075 1.100
1.175
Dns-Phe 6 pH
7
8
1.200
1.000 1.025 15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Trp
1.225 9
1.250
20
1.150
5
1.025
15
5
1.200
1.125
5
3
1.175
1.000
1.000
10
1.225
3
6 pH
15
ce pt
CD concentration (mM)
1.000
5
ed
20
4
1.150
CD concentration (mM)
1.075
15
CD concentration (mM)
1.050
1.025
us
15
1.000
M an
1.025
CD concentration (mM)
CD concentration (mM)
1.000
1.250
3
4
5
6 pH
7
8
1.200 1.225 9
1.250
CD concentration (mM)
1.000 1.025
15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Met
1.200 1.225
3
4
5
6 pH
7
8
9
1.250
23 of 25 Fig. 1 Page in grey
7.5 8.0 8.5
8.5
Migration time (min) 9.0 8.5
8.5
9.0
7.5 8.0
Migration time (min)
9.0
Dns-L-Nva
Dns-D-Nva
Absorbance (220 nm)
8.0
Dns-L-Met
8.0
8.0
us
cr
Absorbance (220 nm)
9.0
8.5
9.5
Migration time (min)
Dns-L-Val
Dns-D-Val
M an
Dns-L-Phe
Dns-D-Phe
Absorbance (220 nm)
i
Fig. 2
Dns-D-Met
7.5 7.5
Dns-L-Trp
7.0
Absorbance (220 nm)
ed Dns-D-Trp
Migration time (min)
Dns-L-Leu
7.0
ce pt
6.5
Dns-D-Leu
Absorbance (220 nm)
6.0
Ac
Absorbance (220 nm)
Figure(s)
Migration time (min)
9.5 10.0
Migration time (min)
9.0 9.5
10.0
Page 24 of 25
6.5 7.0 7.5 8.0
cr
8.5
Migration time (min) 9.0
Dns-L-Met
Dns-L-Nva Dns-L-Leu Dns-L-Trp Dns-D-Val Dns-Abu Dns-L-Val
i
(OCH3)6 and (NH2)1
Dns-Abu Dns-D-Trp Dns-D-Leu
Dns-Nle
C(2)
us
(OCH3)7
Dns-D-Met
M an
Dns-L-Phe
C(6)
Dns-D-Nva
ed
ce pt
C(3)
Dns-Nle
Dns-D-Phe
(CH3O)7
Ac
EOF
Absorbance (220 nm)
Figure(s)
Fig. 3
9.5 Page 25 of 25
S0731-7085(14)00316-1 http://dx.doi.org/doi:10.1016/j.jpba.2014.06.028 PBA 9631
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
18-4-2014 17-6-2014 18-6-2014
Please cite this article as: K. N´emeth, C. Domonkos, V. Sarnyai, J. Szem´an, L. Jicsinszky, L. Szente, J. Visy, Cationic permethylated 6-monoamino-6-monodeoxyrmbeta-cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.06.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
1
Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of
2
dansylated amino acids in capillary electrophoresis
3 Krisztina Németh1*, Celesztina Domonkos1, Virág Sarnyai1, Julianna Szemán2, László
5
Jicsinszky2, Lajos Szente2, Júlia Visy1
6
1
7
of Sciences, H-1519 Budapest P.O.B.:286, Hungary
8
2
ip t
4
us
CycloLab R&D Ltd., H-1097 Budapest, Illatos út 7, Hungary
cr
Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy
an
9 *Corresponding author: K. Németh
11
Postal address: 1Institute of Organic Chemistry, Research Centre for Natural Sciences,
12
Hungarian Academy of Sciences, H-1519 Budapest P.O.B.:286, Hungary
13
Tel.: +36 1 382 6659; E-mail address: [email protected]
d
M
10
te
14 15
24
Highlights • • • • •
Ac ce p
16 17 18 19 20 21 22 23
Permethylated 6-monoamino-6-monodeoxy-βCD resolves dansylated amino acids. Maximal chiral resolutions could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of the selector. Low CD concentration was enough for the separation of the most apolar Dns-AAs. Resolution power of PMMABCD was proved by the separation of Dns-AAs’ mixture.
25 26
Abstract
27 28 29
The
resolution
power
of
permethylated
6-monoamino-6-monodeoxy-βCD
(PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral
1
Page 1 of 25
analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-
31
AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-
32
AAs by varying pH and selector concentrations were investigated and correlated with their
33
structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions
34
could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of
35
the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be
36
achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs.
37
Chiral discrimination ability of PMMABCD was demonstrated by the separation of an
38
artificial mixture of 8 Dns-AA pairs.
an
us
cr
ip t
30
39
Keywords: cationic CD derivative, dansylated amino acid, enantioseparation, isoelectric
41
point, lipophilicity
M
40
d
42 Abbreviations:
44
Dns-AA: dansylated amino acid; Dns-Abu: dansylated α-amino butyric acid; Dns-Asp:
45
dansylated aspartic acid; Dns-Glu: dansylated glutamic acid; Dns-Leu: dansylated leucine;
46
Dns-Met: dansylated methionine; Dns-Nle: dansylated norleucine; Dns-Nva: dansylated
47
norvaline; Dns-Phe: dansylated phenylalanine; Dns-Thr: dansylated threonine; Dns-Trp:
48
dansylated tryptophan; Dns-Val: dansylated valine; PMMABCD: permethylated 6A-
49
monoamino-6A-monodeoxy- β-cyclodextrin;
Ac ce p
50
te
43
51
2
Page 2 of 25
51
1. Introduction
52 Analytical methods particularly capillary electrophoresis (CE) using chiral selectors
54
are appropriate for stereoselective separations [1-6]. Cyclodextrin (CD) derivatives are one of
55
the most favorable selectors to be applied in CE [7]. Development of new chiral selectors,
56
including charged cyclodextrins is important due to the increasing demand to analyze a great
57
number of pharmaceutical, environmental, and nutritional compounds. The advantage of the
58
charged selectors is that they provide relatively large separation window in the case of the
59
oppositely charged analytes [8]. Great numbers of anionic and even cationic CD derivatives
60
have been synthesized and released in the last decades [9-21]. Amino substituted CDs are
61
among the most widely investigated new selectors. Application of cationic –principally
62
amino- CD derivatives for chiral separation of several underivatized amino acids (AA) or
63
their fluorescent derivatives - for more sensitive detection –has been demonstrated [8,13,14-
64
16,18-25]. Iványi et al. [26] reported that permethylated 6-monoamino-6-monodeoxy-βCD
65
(PMMABCD) developed previously as stereoselective agent for CE [27] was a good chiral
66
selector for dansyl-phenylalanine and dansyl-leucine as well as for many other – acidic -
67
chiral substances [26,28-32]. This cationic (pKa = 9.05 [28]), single-isomer βCD derivative is
68
completely O-methylated and substituted by an amino group in C6 position of one of the
69
glucopyranoside units (Degree of substitution = 20+1). The advantage of single isomer
70
selectors over randomly substituted ones is their outstanding batch-to-batch reproducibility.
cr
us
an
M
d
te
Ac ce p
71
ip t
53
Our study aimed to demonstrate the applicability of PMMABCD in chiral separation
72
of further dansylated amino acids (Dns-AAs) as amphoteric model compounds. Using Dns-
73
AAs is explained by the increasing importance of the evaluation of the enantiomer ratio of
74
amino acids in biological samples and in food analysis [33-38] since the biological activity,
75
pharmacology and toxicology of D-amino acids are widely studied [39-42]. The pH and
3
Page 3 of 25
selector concentration dependencies of chiral resolutions were characterized and correlated
77
with the charged states of the analytes and selector and with the structure and lipophilicity of
78
the analytes. The chiral resolution power of PMMABCD was further examined with an
79
artificial mixture of Dns-AAs.
ip t
76
80 2. Materials and methods
cr
81 82
2.1. Materials
us
83
an
84
Background electrolyte (BGE) components phosphoric acid, sodium hydroxide,
86
glacial acetic acid, boric acid and ethanol were purchased from Merck GmbH (Darmstadt,
87
Germany), eleven racemic (Table 1) and nine L-Dns-AA were obtained from Sigma-Aldrich
88
(St.Louis, USA). PMMABCD was the product of CycloLab Ltd. (Budapest, Hungary). Its
89
synthesis route has been described in details [27].
te
d
M
85
90
2.2. Capillary electrophoresis
92 93
Ac ce p
91
Capillary electrophoresis was performed with an Agilent Capillary Electrophoresis
94
3D
95
capillary having a 64.5 cm total and 56 cm effective length with 50 μm I.D. (Agilent
96
Technologies, Santa Clara, CA, USA). On-line absorption at 220 nm was monitored by DAD
97
UV-Vis detector. The capillary was thermostated at 25°C. Between measurements, the
98
capillary was rinsed subsequently with 0.1 M HCl, 1.0 M NaOH, 0.1 M NaOH and distilled
99
water for 3 min each and with BGE for 5 min. Various BGE buffers were prepared either
100
from 82 mM boric acid (pH 9); or from 16 mM phosphoric acid (pH 8); or from 22 mM
CE system (Agilent Technologies, Waldbronn, Germany) applying bare fused silica
4
Page 4 of 25
phosphoric acid (pH 7); or from 35 mM phosphoric acid (pH 6); or from 65 mM acetic acid
102
(pH 5); or from 255 mM acetic acid (pH 4); or from 50 mM phosphoric acid (pH 3) with
103
sodium hydroxide to attain the desired pHs as indicated and the same ionic strength (I= 44
104
mM). The appropriate amounts of PMMABCD were dissolved in the running buffers. Dns-
105
AA samples were dissolved in 50% ethanol and further diluted with distilled water, final
106
concentrations were 0.1 mg/ml. The racemic Dns-AAs were spiked by the pure L-enantiomers
107
(cL-Dns-AA/cD-Dns-AA= 7/3) – with the exception of Dns-Nle and Dns-Abu, their pure ‘L’
108
enantiomers were lacking. The artificial mixture was prepared by mixing the stock solutions
109
of the Dns-AA samples and further diluted with distilled water. The ethanol content of the
110
mixture was higher, the concentrations of Dns-AAs were 0.03 mg/ml and enantiomer ratios
111
were similar with the individual samples. Samples were injected by 5×103 Pa pressure for 3
112
sec. Runs were performed in the positive-polarity mode with 30 kV. Enantiomer separations
113
were carried out with 2.5, 5, 10, 20 mM concentrations of PMMABCD. The resolution (Rs) of
114
the enantiomers is given by the following equation [43]:
cr
us
an
M
d
1.18 × (t 2 − t1 ) w (0.5 )1 + w (0.5 )2
te
Rs =
Eq. 1
Ac ce p
115
ip t
101
116
where t is the migration time of the enantiomers (1, 2 in lower index), w(0.5) is the peak width
117
at half height.
118
Selectivity values are given as αt,migr = t migr,2/tmigr,1 , where t migr,1 and t migr,2 are the migration
119
times of the Dns-AA enantiomers, and are also given as αµ,eff = µeff,2/µeff,1 , where µeff,1 and µeff,2
120
are the mobilities of the Dns-AA enantiomers, respectively.
121 122
2.3. Estimation of pI values of Dns-AAs
123 124
pIs of the Dns-AAs were determined from the effective mobilities (μeff) measured at
125
several pHs. The effective mobilities were plotted against the applied pHs and the intercept of 5
Page 5 of 25
126
the fitted curve on the abscissa gave the pI of the given analyte (Fig. S1, Table S1) [44,45]. Predicted logP values were calculated by ChemAxon’s Marvin Calculator Plugin
128
software (version 5.9.2; URL: http://www.chemaxon.com/marvin) called as “Marvin” in the
129
followings.
ip t
127
130 3. Results and discussion
cr
131 132
Chiral separations of 11 Dns-AA enantiomer pairs (Table 1) were studied by the
134
cationic PMMABCD. Separations were carried out in the pH 3-9 range and by applying the
135
selector in the 2.5 mM to 20 mM concentration range. Results are summarized in (Table 2,
136
Table S2). Using these conditions eight enantiomer pairs can be resolved at least at baseline
137
or better and additional two pairs can be separated partially. Separations can be carried out in
138
reasonable running times. Changes in resolution of Dns-AAs as a function of pH and
139
PMMABCD concentration is discussed in details as follows.
142 143 144 145
an
M
d
te
141
Ac ce p
140
us
133
[Table 1] [Table 2]
3.1. pH dependence of resolution of Dns-AA enantiomers
Changes in the chiral separations of the enantiomers of Dns-AAs were investigated by
146
varying the pH between pH 3 and pH 9. In order to evaluate charge relationships between the
147
host and guest molecules in this pH range the pI values of the analytes were measured by CE.
148
The pI values of the Dns-AAs are between 3.46-3.79 (Table 1) being in good agreement with
149
literature data (Table S1) [46]. Consequently, the Dns-AAs are positively charged below and
150
negatively charged above these values while the PMMABCD is positively charged
6
Page 6 of 25
151
permanently below pH 9. The effects of the pH on chiral separations of Dns-AAs were
152
studied by investigating several combinations of different charge states of the selector and
153
selectands (at pH 3, 4, 6 and 9). Migrations of the Dns-AAs were accelerated (μcompl>μfree) by PMMABCD at pHs
155
above their pI values (Table S2). The negatively charged Dns-AAs if complexed with
156
PMMABCD migrate faster than the free analytes alone due to the decrease in their negative
157
charges and increase in their mass (i.e. hydrodynamic radius). Using this cationic selector
158
below the pIs of the Dns-AAs the complexes gain positive charges not only from the analyte
159
but also from the selector, in addition their sizes are increased. Therefore their mobility
160
changes are not substantial and could not be predicted well due to those counter-effects.
an
us
cr
ip t
154
Resolutions show maxima at pH 4 or at pH 6 (Table 2, Fig. 1) where PMMABCD is
162
cationic and the Dns-AAs are at least partially negatively charged. The highest selectivities
163
can be observed for Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp and Dns-Thr
164
at pH 4 and for Dns-Met, Dns-Val and Dns-Asp at pH 6. Resolutions are decreased at pH 9
165
where PMMABCD is partially in uncharged form and the Dns-AAs are negatively charged.
166
Resolutions are weaker too at pH 3 where the selector is fully positively charged and the
167
analytes are partially cationic.
d
te
Ac ce p
168
M
161
In general, the amount of the D-amino acids is substantially lower than that of their L
169
pairs in nature. In most of our cases the migration order of the enantiomers (EMO) is D,L in
170
the entire pH range investigated. So that EMO is advantageous because the evaluation of
171
peaks is more accurate when the smaller component migrates before the major one.
172 173
[Fig. 1]
174 175
3.2. CD concentration dependence of resolution of Dns-AA enantiomers
7
Page 7 of 25
176 Selectivities increase with increasing selector concentrations in most cases of the Dns-
178
AAs investigated. Our finding that the maximal selectivities are in the high concentration
179
range of the selector corresponds [2,47,48] to the weak interactions between PMMABCD and
180
Dns-AAs (Kassoc ≤ 10 M-1) estimated by the well known non-linear regression of the
181
electrophoretic mobility shifts [49-53].
cr
ip t
177
Whereas, increasing the selector concentration results in the improvement of
183
separations usage of PMMABCD in concentrations substantially higher than 20 mM is not
184
recommended because of longer analysis times and peak distortions. In addition, due to the
185
elevated current other counterproductive effects of Joule heating can also occur. Furthermore,
186
it is noteworthy that the pH and selector concentration dependencies for Dns-Nle and Dns-
187
Phe deviate from the others (Fig. 1) because in these two cases the migration time of the
188
peaks of the enantiomers overlaps with the time of the electroosmotic flow at high (15-20
189
mM) concentrations of PMMABCD at pH 4.
te
d
M
an
us
182
Baseline or better separation can be achieved for some of the enantiomer pairs of these
191
Dns-AAs even with low concentrations of PMMABCD. In the presence of only 2.5 mM-10
192
mM concentrations of the selector Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp
193
and Dns-Met exhibited good resolution with further improvements with elevated CD
194
concentrations. In the group of Dns-AAs with nonpolar aliphatic sidechains the higher is the
195
lipophilicity the better is the resolution. 15 mM - 20 mM concentrations of PMMABCD are
196
needed for the resolution of the more polar (cf. the logP values in Table 1) Dns-AAs (namely:
197
Dns-Val, Dns-Thr and Dns-Asp). Unfortunately, enantiomers of the polar Dns-Glu can not be
198
separated at all with PMMABCD.
Ac ce p
190
199
Limit of detection (LOD at signal to noise ratio S/N = 3) and the limit of quantitation
200
(LOQ at S/N = 10) were determined for several dansylated D- and L-amino acids (Table 3).
8
Page 8 of 25
The LOD and LOQ values were between 0.2 - 3 µg/ml and 6 - 10 µg/ml, respectively. Most
202
biological samples contain D- and L-amino acid enantiomers approximately in a ratio of
203
1:100 [54]. Accordingly, the sensibility of our system for the detection of the minor (D-Dns-
204
AA) component followed by the major ‘L’ enantiomer is demonstrated here with some
205
examples (Dns-Phe, Dns-Trp, Dns-Leu, Dns-Val, Dns-Nva and Dns-Met) where the
206
enantiomer ratios is also 1 to 100. In these setups (20 mM PMMABCD at pH 6.0) the
207
concentrations of D-Dns-AAs is around at their LOQ values (10 µg/ml) and those of their ‘L’
208
pairs were 1 mg/ml (Fig. 2).
us
cr
ip t
201
an
209 210
[Table 3] [Fig. 2]
212
M
211
3.4. Resolution of an artificial mixture of Dns-AAs
d
213
The resolution power of PMMABCD is further studied by mixture of Dns-AAs. From
215
our enantiomer pairs we chose those eight Dns-AAs which were separated at baseline at least.
216
Migration times of the enantiomers were taken into account too in selecting optimal running
217
parameters (pH and CD concentration) in order to obtain separated peaks for each constituent
218
of the mixture. Despite that in some cases pH 4 can provide more improved resolution for the
219
individual Dns-AAs than pH 6 the number of the enantiomer pairs resolved is greater at pH 6
220
than at pH 4. Consequently, we have chosen 20 mM PMMABCD at pH 6.0 and 7 enantiomer
221
pairs separated well and two further peaks partially of that mixture containing 8 pairs (Fig. 3).
222
[Fig. 3]
Ac ce p
te
214
223 224 225
9
Page 9 of 25
226
4. Conclusions
227 The amino and methyl substituted βCD derivative PMMABCD is introduced as an
229
excellent cationic selector for Dns-AAs having nonpolar aliphatic and aromatic side chains
230
and a usable selector for some Dns-AAs with polar uncharged or charged groups. The
231
advantage of using PMMABCD is that it consumes a small amount of CD derivative, the
232
separations can be carried out in reasonable running times, and it provides the preferable ‘DL’
233
migration order for the resolution of Dns-AAs at pHs above their pI values. Furthermore, the
234
chiral recognition ability of PMMABCD is proved by the separation of a mixture of Dns-
235
AAs.
an
us
cr
ip t
228
238
M
236
239
We gratefully acknowledge Dr. Róbert Iványi, Katalin Tuza (Cyclolab R&D Ltd.) as well as
240
Prof. Miklós Simonyi, Dr. Gábor Tárkányi, Mrs. Ilona Kawka (RCNS, HAS) and Dr.
241
Zsuzsanna Lakatos for their helpful contributions. The authors thank for the financial support
242
from the following grants: NKFP_A3-2008-0211 NATURSEP, TECH-09-AI-2009-0117
243
NanoSEN9, the Hungarian Scientific Research Fund (OTKA, grant numbers K-100134 and
244
NN-110214) and the “Lendület” Program of the Hungarian Academy of Sciences (LP2013-
245
55/2013).
te
d
Acknowledgements
246
Ac ce p
237
247
Appendix A. Supplementary data
248
Supplementary data associated with this article can be found, in the online version, at doi:
249 250
The authors have declared no conflict of interest.
10
Page 10 of 25
251 5. References
253
[1] G. Gübitz, M.G. Schmid, Chiral separation principles in capillary electrophoresis, J.
254
Chromatogr. A 792 (1997) 179-225.
255
[2] S. Fanali, Enantioselective determination by capillary electrophoresis with cyclodextrins
256
as chiral selectors, J. Chromatogr. A 875 (2000) 89-122.
257
[3] G. Blaschke, B. Chankvetadze, Enantiomer separation of drugs by capillary
258
electromigration techniques , J. Chromatogr. A 875 (2000) 3-25.
259
[4] B. Chankvetadze, Enantioseparations by using capillary electrophoretic techniques. The
260
story of 20 and a few more years, J. Chromatogr. A 1168 (2007) 45-70.
261
[5] P. Jac, G.K.E. Scriba, Recent advances in electrodriven enantioseparations, J. Sep Sci. 36
262
(2013) 52-74.
263
[6] L. Qi, Y. Chen, M. Xie, Z. Guo, X. Wang, Separation of dansylated amino acid
264
enantiomers by chiral ligand-exchange CE with a zinc(II) L-arginine complex as the selecting
265
system Electrophoresis 29 (2008) 4277-4283.
266
[7] D.A. Tsioupi R-I. Stefan-van-Staden, C.P. Kapnissi-Christodoulou, Chiral selectors in CE:
267
Recent developments and applications, Electrophoresis 34 (2013) 178-204.
268
[8] B. Chankvetadze, G. Endresz, G. Blaschke, Charged cyclodextrin derivatives as chiral
269
selectors in capillary electrophoresis, Chem. Soc. Review, 25 (1996) 141-148.
270
[9] V. Cucinotta, A. Contino, A. Giuffrida, G. Maccarrone, M. Messina, Application of
271
charged single isomer derivatives of cyclodextrins in capillary electrophoresis for chiral
272
analysis, J. Chromatogr. A 1217 (2010) 953-967.
273
[10] Z. Juvancz, R. Bodáné-Kendrovics, R. Iványi, L. Szente, The role of cyclodextrins in
274
chiral capillary electrophoresis, Electrophoresis 29 (2008) 1701-1712.
Ac ce p
te
d
M
an
us
cr
ip t
252
11
Page 11 of 25
[11] Y. Tanaka, S. Terabe, Enantiomer separation of acidic racemates by capillary
276
electrophoresis using cationic and amphoteric beta-cyclodextrins as chiral selectors, J.
277
Chromatogr. A 781 (1997) 151-160.
278
[12] H. Yamamura, Y. Yamada, R. Miyagi, K. Kano, S. Araki, M. Kawai, Preparation of β-
279
Cyclodextrin Derivatives Possessing Two Trimethylammonio Groups on Their Primary
280
Hydroxy Sides as Chiral Guest Selectors, J. Incl. Phenom. Macrocycl. Chem. 45 (2003) 211–
281
216.
282
[13] W. Tang, I. W. Muderawan, S.-C. Ng, H. S. O. Chan, Enantiomeric separation of 8
283
hydroxy, 10 carboxylic and 6 dansyl amino acids by mono(6-amino-6-deoxy)-beta-
284
cyclodextrin in capillary electrophoresis, Anal. Chim. Acta 554 (2005) 156-162.
285
[14] W. Tang, I. W. Muderawan, T.-T. Ong, S.-C. Ng, Enantio separation of dansyl amino
286
acids by a novel permanently positively charged single-isomer cyclodextrin: Mono-6-N-
287
allylammonium-6-deoxy-beta-cyclodextrin chloride by capillary electrophoresis, Anal. Chim.
288
Acta 546 (2005) 119-125.
289
[15] Sz. Béni, T. Sohajda, G. Neumajer, R. Iványi, L. Szente, B. Noszál, Separation and
290
characterization of modified pregabalins in terms of cyclodextrin complexation, using
291
capillary electrophoresis and nuclear magnetic resonance, J. Pharm. Biomed.Anal. 51
292
(2010) 842-852.
293
[16] A. Giuffrida, A. Contino, G. Maccarrone, M. Messina, V. Cucinotta The 3-amino-
294
derivative of gamma-cyclodextrin as chiral selector of Dns-amino acids in electrokinetic
295
chromatography, J. Chromatogr. A 1216 (2009) 3678-3686.
296
[17] P. Liu, W. He, X-Y.,Qin, X-L. Sun, H. Chen, S-Y. Zhang, Synthesis and application of a
297
novel
298
tetramethylenediamine)-beta-cyclodextrin as chiral selector in capillary electrophoresis
299
Chirality 22 (2010) 914-921.
Ac ce p
te
d
M
an
us
cr
ip t
275
single-isomer
mono-6-deoxy-6-((2S,3S)-(+)-2,3-O-isopropylidene-1,4-
12
Page 12 of 25
[18] A. Giuffrida, R. Caruso, M. Messina, G. Maccarrone, A. Contino, A. Cifuentes, V.
301
Cucinotta, Chiral separation of amino acids derivatised with fluorescein isothiocyanate by
302
single isomer derivatives 3-monodeoxy-3-monoamino-beta- and gamma-cyclodextrins: the
303
effect of the cavity size J. Chromatogr. A 1269 (2012) 360-365.
304
[19] Y. Dai, S. Wang, J. Zhou, Y. Liu, D. Sun, J. Tang, W. Tang, Cationic cyclodextrin as
305
versatile chiral selector for enantiomeric separation in capillary electrophoresis, J.
306
Chromatogr. A, 1246 (2012) 98-102.
307
[20] S. Wang, Y. Dai, J. Wu, J. Zhou, J. Tang, W. Tang, Methoxyethylammonium
308
monosubstituted β-cyclodextrin as the chiral selector for enantioseparation in capillary
309
electrophoresis, J. Chromatogr. A 1277 (2013) 84-92.
310
[21] J. Zhou, F. Ai, B. Zhou, J. Tang, S.-C. Ng, W. Tang, Hydroxyethylammonium
311
monosubstituted cyclodextrin as chiral selector for capillary electrophoresis, Anal. Chim.
312
Acta 800 (2013) 95-102.
313
[22] H. Wan, L.G. Blomberg, Chiral separation of amino acids and peptides by capillary
314
electrophoresis, J. Chromatogr. A 875 (2000) 43-88.
315
[23] V. Poinsot, M.-A. Carpene, J. Bouajila, P. Gavard, B. Feurer, F. Couderc, Recent
316
advances in amino acid analysis by capillary electrophoresis, Electrophoresis 33 (2012) 14-
317
35.
318
[24] V. Poinsot, C. Bayle, F. Couderc, Recent advances in amino acid analysis by capillary
319
electrophoresis, Electrophoresis 24 (2003) 4047-4067.
320
[25] M. Chiari, M. Cretich, G. Crini, L. Janus, M. Morcellet, Recent advances in amino acid
321
analysis by capillary electrophoresis, J. Chromatogr. A 894 (2000) 95-103.
322
[26] R. Iványi, L. Jicsinszky, Z. Juvancz, Permethyl monoamino beta-cyclodextrin a new
323
chiral selective agent for capillary electrophoresis, Chromatographia 53 (2001) 166-172.
Ac ce p
te
d
M
an
us
cr
ip t
300
13
Page 13 of 25
[27] L. Jicsinszky, R. Iványi, Catalytic transfer hydrogenation of sugar derivatives,
325
Carbohydr. Polym. 45 (2001) 139-145.
326
[28] R. Iványi, L. Jicsinszky, Z. Juvancz, Chiral separation of pyrethroic acids with single
327
isomer permethyl monoamino beta-cyclodextrin selector, Electrophoresis 22 (2001) 3232-
328
3236.
329
[29] A.M. Abushoffa, M. Fillet, A.C. Servais, P. Hubert, J. Crommen, Enhancement of
330
selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of
331
oppositely charged cyclodextrins in capillary electrophoresis, Electrophoresis 24 (2003) 343-
332
350.
333
[30] B. Tőkés, L. Ferencz, P. Buchwald, G. Donázh-Nagy, Sz. Vancea, N. Sántha, E. L. Kis,
334
Structural studies on the chiral selector capacity of cyclodextrin derivatives, J. Biochem.
335
Biophys. Methods 70 (2008) 1276–1282.
336
[31] A.G. Cârje; Z. Juvancz, R. Iványi, V. Schurig, B. Tőkés, Comparative study on chiral
337
separation of pyrethroic acids with amino and neutral cyclodextrine derivatives, Acta
338
Medica Marisiensis 57 (2011) 52-54.
339
[32] G. Tárkányi, K. Németh, R. Mizsei, O. Tőke, J. Visy, M. Simonyi, L. Jicsinszky, J.
340
Szemán, L. Szente, Structure and stability of warfarin-sodium inclusion complexes formed
341
with permethylated monoamino-beta-cyclodextrin, J. Pharm. Biomed.Anal. 72 (2013) 292-
342
298.
343
[33] R.M. Callejón, A.M. Troncoso, M.L. Morales, Determination of amino acids in grape-
344
derived products: A review, Talanta 81 (2010) 1143-1152.
345
[34] M. Wcisło, D. Compagnone, M. Trojanowicz, Enantio selective screen-printed
346
amperometric biosensor for the determination of D-amino acids, Bioelectrochemistry 71
347
(2007) 91-98.
Ac ce p
te
d
M
an
us
cr
ip t
324
14
Page 14 of 25
[35] X. He, X. Cui, M. Li, L. Lin, X. Liu, X. Feng, Highly enantioselective fluorescent sensor
349
for chiral recognition of amino acid derivatives, Tetrahedron Lett. 50 (2009) 5853-5856.
350
[36] H. Zahradníčková, P. Hušek, P. Šimek, P. Hartvich, B. Maršálek, I. Holoubek,
351
Determination of D- and L-amino acids produced by cyanobacteria using gas chromatography
352
on Chirasil-Val after derivatization with pentafluoropropyl chloroformate, Anal. Bioanal.
353
Chem. 388 (2007) 1815-1822.
354
[37] Y. Miyoshi, R. Koga, T. Oyama, H. Han, K. Ueno, K. Masuyama, Y. Itoh, K. Hamase,
355
HPLC analysis of naturally occurring free D-amino acids in mammals, J. Pharm. Biomed.
356
Anal. 69 (2012) 42-49.
357
[38] M. Herrero, C., Simo, V. Garcia-Canas, S. Fanali, A. Cifuentes, Electrophoresis 31
358
(2010) 2106-2114.
359
[39] K. Hamase, Sensitive two-dimensional determination of small amounts of D-amino acids
360
in mammals and the study on their functions, Chem. Pharm. Bull. 55 (2007) 503-510.
361
[40] M. Friedman, Origin, Microbiology, Nutrition, and Pharmacology of D-Amino Acids,
362
Chem. Biodiversity 7 (2010) 1491-1530.
363
[41] K. Hamase, Analysis and biological relevance of D-amino acids and relating compounds,
364
J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3077-3077.
365
[42] H. Ohide, Y. Miyoshi, R. Maruyama, K. Hamase, K. R. Konno, D-Amino acid
366
metabolism in mammals: Biosynthesis, degradation and analytical aspects of the metabolic
367
study, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3162-3168.
368
[43] S. Fanali, Controlling enantioselectivity in chiral capillary electrophoresis with inclusion-
369
complexation, J. Chromatogr. A 792 (1997) 227-267.
370
[44] D. Koval, V. Kasicka, J. Jiracek, M. Collinsová, Determination of pK(a) values of
371
diastereomers of phosphinic pseudopeptides by CZE, Electrophoresis 27 (2006) 4648-4657.
Ac ce p
te
d
M
an
us
cr
ip t
348
15
Page 15 of 25
[45] S.K. Poole, S. Patel, K. Dehring, H. Workman, C.F. Poole, Determination of acid
373
dissociation constants by capillary electrophoresis, J. Chromatogr. A 1037 (2004) 445-454.
374
[46] A. Bianchi-Bosisio, P.G. Righetti, N.B. Egen, M. Bier, Isoelectric focusing of
375
dansylated amino-acids in immobilized pH gradients, Electrophoresis 7 (1986) 128-133.
376
[47] A. Rizzi, Fundamental aspects of chiral separations by capillary electrophoresis,
377
Electrophoresis 22 (2001) 3079-3106.
378
[48] S. Wren, The separation of enantiomers by capillary electrophoresis, Chromatographia
379
54 (2001) S1-S95.
380
[49] S. G. Penn, D. M. Goodall, J. S. Loran, Differential Binding of tioconazole enantiomers
381
to hydroxypropyl-beta cyclodextrin studied by capillary, J. Chromatogr. 636 (1993) 149-152.
382
[50] K. L. Rundlett, D. W. Armstrong, Examination of the origin, variation, and proper use of
383
expressions for the estimation of association constants by capillary electrophoresis, J.
384
Chromatogr. A 721 (1996) 173-186.
385
[51] S. A.C. Wren, Mobility measurements on dansylated amino acids, J. Chromatogr. A 768
386
(1997) 153-159.
387
[52] A. Salvador, E. Varesio, M. Dreux, J-L. Veuthey, Binding constant dependency of
388
amphetamines with various commercial methylated beta-cyclodextrins, Electrophoresis 20
389
(1999) 2670-2679.
390
[53] Y. Tanaka, S. Terabe, Estimation of binding constants by capillary electrophoresis, J.
391
Chromatogr. A 768 (2002) 81-92.
392
[54] Zs. Wagner, T. Tábi, T. Jakó, G. Zachar, A. Csillag, É. Szökő, Chiral separation and
393
determination of excitatory amino acids in brain samples by CE-LIF using dual
394
cyclodextrin system, Anal. Bioanal. Chem. 404 (2012) 2363-2368.
Ac ce p
te
d
M
an
us
cr
ip t
372
395
16
Page 16 of 25
395
Figure captions
396 Fig. 1. Changes in selectivities (αt,migr) of several Dns-AAs as a function of pH and selector
398
concentration
399
Fig. 2. Separations of D- and L-Dns-AA enantiomers in a concentration ratio of 1:100 (10
400
µg/ml and 1 mg/ml, respectively) in the presence of 20 mM PMMABCD at pH 6
401
Fig. 3. Separation of Dns-AA mixture by 20 mM PMMABCD at pH 6
cr
ip t
397
us
402
an
403
Ac ce p
te
d
M
404
17
Page 17 of 25
404 405
Table 1: Structure, isoelectric point (pI) and lipophilicity (logP) of dansyl-amino acids H3C N O S
R group
COOH NH
pI
R
O
Nonpolar, aliphatic R group, linear sidechain: Dns-DL-α-aminobutyric acid (Dns-Abu)
-CH2CH3
Dns-DL-norvalin (Dns-Nva)
-(CH2)2CH3
Dns-DL-norleucine (Dns-Nle)
-(CH2)3CH3
-2.54
3.67
-2.11
3.67
-1.52
-CH(CH3)2
3.70
-2.26
-CH2CH(CH3)2
3.63
-1.52
-CH2C6H5
3.67
-1.38
-CH2C8NH6
3.79
-1.05
-(CH2)2SCH3
3.55
-1.87
-CHCH3OH
3.66
-2.94
-CH2COOH
3.46
-3.89
-(CH2)2COOH
3.50
-3.69
cr
3.68
Nonpolar, aliphatic R group, branched sidechain:
us
Dns-DL-valine (Dns-Val) Dns-DL-leucine (Dns-Leu) Nonpolar, aromatic R group:
an
Dns-DL-phenylalanine (Dns-Phe) Dns-DL-tryptophan (Dns-Trp) Nonpolar, uncharged R group:
M
Dns-DL-methionine (Dns-Met) Polar, uncharged R group: Dns-DL-threonine (Dns-Thr) Dns-DL-aspartic acid (Dns-Asp)
Ac ce p
te
Dns-DL-glutamic acid (Dns-Glu)
d
Polar, negatively charged R group:
406 407 408
logP
*
ip t
H3C
18
Page 18 of 25
Table 2: Migration times (t1), selectivities (α) and resolutions (Rs) of Dns-AAs at pH 9-3 in the presence of 2.5-20 mM PMMABCD as chiral selector pH 2.5
DnsLeu
DnsPhe
DnsTrp
DnsThr DnsAsp
411 412 413 414 415 416 417 418 419 420
20
2.5
5
10
20
2.5
5
10
15
20
n.a.
4.63
4.79
5.23
n.a.
1.004
1.010
n.a.
n.a.
nr
0.5
1.5
5.90
6.54
6.83
7.33
7.79
1.000
1.012
1.025
1.042
1.073
nr
1.8
3.9
6.5
11.1
4
12.19
14.19
17.21
19.23
20.30
1.000
1.009
1.039
1.085
1.131
nr
3
n.a.
n.a.
14.67
15.49
19.16
n.a.
n.a. 1.000
1.008
1.000
n.a.
9
4.22
4.30
4.47
4.64
5.07
1.000
1.000
1.000
1.000
1.012
nr
6
5.59
5.94
6.32
6.51
7.58
1.000
1.013
1.029
1.042
1.092
nr
4
12.21
14.02
16.14
17.22
17.59
1.000
1.016
1.068
1.133
1.179
3
12.56
12.65
20.52
22.62
26.37
1.000
1.000
1.000
1.000
1.016
1.0
4.0
8.1
11.8
n.a.
nr
nr
nr
nr
nr
nr
1.7
2.1
4.6
8.0
12.9
nr
1.6
6.8
13.2
17.3
nr
nr
nr
nr
0.8
n.a.
n.a.
0.6
2.5
4.9
0.9
5.2
10.1
14.0
19.5
9
n.a.
n.a.
4.45
4.55
4.89
n.a.
1.018
1.032
6
5.49
5.68
5.92
6.05
6.40
1.006
1.032
1.068
1.088
1.160
4
12.14
13.20
13.42
14.84
17.52
1.015
1.065
1.197
1.149
n.a.
1.9
5.5
20.4
20.3
n.a.
3
n.a.
n.a.
15.91
17.24
21.66
n.a.
n.a. 1.022
1.067
1.060
n.a.
n.a.
1.8
2.8
3.6
9
n.a.
n.a.
4.56
4.73
5.19
n.a.
6
5.62
6.07
6.62
7.01
8.45
1.000
4
11.53
13.43
16.16
18.85
20.59
3
n.a.
n.a.
15.64
16.60
n.a.
9
4.17
4.24
4.41
4.58
5.08
1.003
1.005
1.007
1.006
6
5.85
6.66
6.91
7.27
7.91
1.004
1.000
1.000
1.013
4
11.99
13.96
16.89
18.46
19.17
1.000
1.010
1.059
1.137
3
12.91
13.19
24.46
27.14
34.52
1.000
1.000
1.012
1.023
9
4.15
4.21
4.23
4.25
4.58
1.142
1.000
1.019
6
5.63
5.95
5.66
5.77
6.07
1.016
1.071
1.115
4
11.43
12.26
12.70
16.23
17.24
1.021
1.085
1.216
n.a.
3
13.05
13.16
22.41
24.90
31.77
1.000
1.000
1.015
1.017
9
4.16
4.26
4.48
4.64
5.11
1.000
1.000
1.000
1.000
6
5.47
5.89
6.37
6.64
7.33
1.000
1.000
1.014
1.019
4
n.a. 12.21
12.49
12.69
n.a. 1.000
us
n.a. 1.005
ip t
n.a.
6
cr
n.a. 1.000
15
9
3
DnsMet
15
Rs
an
DnsVal
10
1.000
1.000
n.a.
n.a.
nr
nr
nr
1.000
1.000
1.016
nr
nr
nr
nr
2.3
1.000
n.a. 1.000
1.000
1.000
nr
n.a.
nr
nr
nr
n.a.
n.a. 1.000
1.000
n.a.
n.a.
n.a.
nr
nr
n.a.
1.004
0.5
0.9
1.2
1.5
0.6
1.035
0.6
nr
nr
2.0
4.4
1.220
nr
1.2
6.0
12.8
19.5
1.036
nr
nr
1.0
1.7
6.9
1.039
1.059
0.9
nr
3.4
5.9
6.7
1.148
1.168
2.5
10.5
16.3
21.2
21.2
n.a.
nr
7.4
27.1
n.a.
n.a.
1.000
nr
nr
1.0
1.4
nr
1.000
nr
nr
nr
nr
nr
1.045
nr
nr
2.1
1.4
7.4
1.000
M
DnsNle
5
d
DnsNva
d
Ac ce p
DnsAbu
αe
t1 (min)
te
408 409 410
14.33
n.a.
17.53
17.33
20.05
23.72
n.a. 1.006 1.000
9
n.a.
n.a.
4.59
4.70
5.14
n.a.
6
5.96
6.78
6.92
7.20
7.94
1.000
1.000
1.026
n.a.
1.092
n.a.
0.8
2.8
n.a.
8.7
1.000
1.000
1.006
nr
nr
nr
nr
nr
1.011
1.021
n.a.
n.a.
nr
1.3
3.2
1.021
n.a. 1.000 1.044
1.071
1.134
nr
3.3
6.8
10.9
17.0
n.a. 1.000
1.000
1.000
1.000
n.a.
nr
nr
nr
nr
n.a. 1.000
1.000
1.010
n.a.
n.a.
nr
nr
0.5
4
n.a.
n.a.
n.a.
n.a.
n.a.
3
n.a.
n.a.
18.25
20.60
24.35
n.a.
4
12.74
14.96
19.15
24.44
29.21
1.000
1.000
1.000
1.000
1.015
nr
nr
nr
nr
1.0
6
9.88
11.43
13.31
16.10
19.79
1.000
1.000
1.000
1.011
1.016
nr
nr
nr
1.0
1.4
a) n.a. not available b) nr not resolved; Rs < 0.5 c) t1 migration time of the faster enantiomer d) Selector concentrations applied is given in mM in the second row of the header e) Selectivity is given as follows: α = t migr,2/tmigr,1 f) The enantiomer migration order is D,L in the majority of the cases. The selectivity values are underlined where the enantiomer migration order is reversed (L,D).
19
Page 19 of 25
Table 3: Limit of detection (LOD) and quantitation (LOQ) of D- and L-Dns AAs 421 D-Dns-AA LOD (µg/ml)
L-Dns-AA LOQ (µg/ml)
LOD (µg/ml)
LOQ (µg/ml)
0.4
5.8
0.4
6.4
Dns-Nva
0.2
5.7
0.2
6.3
Dns-Nle
0.1
5.2
0.1
6.5
Dns-Val
1.7
7.9
1.7
7.7
Dns-Leu
1.5
7.4
1.5
Dns-Phe
1.3
6.2
1.3
Dns-Trp
1. 8
6.1
1.8
6.1
Dns-Met
3.0
10.0
2.9
10.2
ip t
Dns-Abu
cr
420
7.5
us
7.5
Ac ce p
te
d
M
an
422 423
20
Page 20 of 25
6.5 7.0 7.5 8.0
cr
8.5
Migration time (min) 9.0
Dns-L-Met
Dns-L-Nva Dns-L-Leu Dns-L-Trp Dns-D-Val Dns-Abu Dns-L-Val
i
(OCH3)6 and (NH2)1
Dns-Abu Dns-D-Trp Dns-D-Leu
Dns-Nle
us
C(2)
Dns-D-Met
M an
Dns-L-Phe
(OCH3)7
Dns-D-Nva
ed
ce pt
C(3)
Dns-Nle
Dns-D-Phe
(CH3O)7
Ac
EOF
Absorbance (220 nm)
*Graphical Abstract C(6)
9.5 Page 21 of 25
20
20
cr
20
i
Figure(s)
1.100 10
1.125 1.150 1.175
Dns-Abu
5
1.050 1.075 1.100
10
1.125
5
1.200
Dns-Nva
1.225 3
4
5
6 pH
7
8
9
3
1.250
20
1.050 1.075 1.125 1.150 1.175
Dns-Leu
5
1.200
6 pH
7
8
9
5
6 pH
7
8
9
1.250
Ac
4
20
3
4
1.100 10
1.125 1.150 1.175
Dns-Nle
1.200 1.225
3
4
5
6 pH
7
8
9
1.100 1.150 1.175 1.200
1.000 1.025 15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Trp
1.200 1.225
1.225 5
6 pH
7
8
9
1.250
20
1.075
Dns-Phe
1.050 1.075
1.250
1.125
5
1.025
15
1.225
1.050
10
1.000
5
1.200
1.025
1.225
3
1.175
1.000
15
ce pt
1.100
10
CD concentration (mM)
1.025 15
5
ed
20 1.000
CD concentration (mM)
4
1.150
CD concentration (mM)
1.075
15
CD concentration (mM)
1.050
1.025
us
15
1.000
M an
1.025
CD concentration (mM)
CD concentration (mM)
1.000
1.250
3
4
5
6 pH
7
8
9
1.250
CD concentration (mM)
1.000 1.025
15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Met
1.200 1.225
3
4
5
6 pH
7
8
9
1.250
Page 22 of 25
Fig. 1
20
20
cr
20
i
Figure(s)
1.100 10
1.125 1.150 1.175
5
Dns-Abu
1.050 1.075 1.100
10
1.125
5
1.200
Dns-Nva
1.225 3
4
5
6 pH
7
8
9
3
1.250 20
15
1.050 1.075 1.100
10
1.125 1.150
5
1.175
Dns-Leu
CD concentration (mM)
1.025
1.200
5
6 pH
7
8
9
1.250
Ac
4
20
7
8
9
4
1.050 1.075 1.100
10
1.125 1.150 1.175
Dns-Nle
1.200
1.225
1.225 3
1.250
4
5
6 pH
7
8
9
1.025 1.050 1.075 1.100
1.175
Dns-Phe 6 pH
7
8
1.200
1.000 1.025 15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Trp
1.225 9
1.250
20
1.150
5
1.025
15
5
1.200
1.125
5
3
1.175
1.000
1.000
10
1.225
3
6 pH
15
ce pt
CD concentration (mM)
1.000
5
ed
20
4
1.150
CD concentration (mM)
1.075
15
CD concentration (mM)
1.050
1.025
us
15
1.000
M an
1.025
CD concentration (mM)
CD concentration (mM)
1.000
1.250
3
4
5
6 pH
7
8
1.200 1.225 9
1.250
CD concentration (mM)
1.000 1.025
15
1.050 1.075 1.100
10
1.125 1.150 1.175
5
Dns-Met
1.200 1.225
3
4
5
6 pH
7
8
9
1.250
23 of 25 Fig. 1 Page in grey
7.5 8.0 8.5
8.5
Migration time (min) 9.0 8.5
8.5
9.0
7.5 8.0
Migration time (min)
9.0
Dns-L-Nva
Dns-D-Nva
Absorbance (220 nm)
8.0
Dns-L-Met
8.0
8.0
us
cr
Absorbance (220 nm)
9.0
8.5
9.5
Migration time (min)
Dns-L-Val
Dns-D-Val
M an
Dns-L-Phe
Dns-D-Phe
Absorbance (220 nm)
i
Fig. 2
Dns-D-Met
7.5 7.5
Dns-L-Trp
7.0
Absorbance (220 nm)
ed Dns-D-Trp
Migration time (min)
Dns-L-Leu
7.0
ce pt
6.5
Dns-D-Leu
Absorbance (220 nm)
6.0
Ac
Absorbance (220 nm)
Figure(s)
Migration time (min)
9.5 10.0
Migration time (min)
9.0 9.5
10.0
Page 24 of 25
6.5 7.0 7.5 8.0
cr
8.5
Migration time (min) 9.0
Dns-L-Met
Dns-L-Nva Dns-L-Leu Dns-L-Trp Dns-D-Val Dns-Abu Dns-L-Val
i
(OCH3)6 and (NH2)1
Dns-Abu Dns-D-Trp Dns-D-Leu
Dns-Nle
C(2)
us
(OCH3)7
Dns-D-Met
M an
Dns-L-Phe
C(6)
Dns-D-Nva
ed
ce pt
C(3)
Dns-Nle
Dns-D-Phe
(CH3O)7
Ac
EOF
Absorbance (220 nm)
Figure(s)
Fig. 3
9.5 Page 25 of 25
