Antonio Salgado

Separation of propranolol enantiomers by CE using sulfated beta-CD derivatives in aqueous and non-aqueous electrolytes: Comparative CE and NMR study.

Separations using CE employing non‐aqueous BGE are already as well established as separations in aqueous buffers. The separation mechanisms in achiral CE with non‐aqueous BGEs are most likely similar to those in aqueous buffers. However, for the separation of enantiomers involving their interaction with chiral buffer additives, the interaction mechanisms might be very different in aqueous and non‐aqueous BGEs. While the hypothesis regarding distinct mechanisms of enantiomer separations in aqueous and non‐aqueous BGEs has been mentioned in several papers, no direct proof of this hypothesis has been reported to date. In the present study, the enantiomers of propranolol were resolved using CE in aqueous and non‐aqueous methanolic BGEs with two single isomer sulfated derivatives of β‐CD, namely heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD and heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD. The enantiomer migration order of propranolol was inverted when an aqueous BGE was replaced with non‐aqueous BGE in the case of heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD but remained the same in the case of heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD. The possible molecular mechanisms leading to this reversal of enantiomer migration order were studied by using nuclear overhauser effect spectroscopy in both aqueous and non‐aqueous BGEs.

About the role of enantioselective selector-selectand interactions and the mobilities of diastereomeric associates in enantiomer separations using CE

It is generally accepted that the selective binding of enantiomers of the chiral analyte to a chiral selector is necessary for enantioseparations in CE, whereas the role of mobility differences between the temporary diastereomeric associates formed between the enantiomers and the chiral selector has been commonly neglected. One of the authors of this study suggested in 1997 that the mobility difference between the diastereomeric associates of two enantiomers with the chiral selector may be solely responsible for a separation of enantiomers in CE and enantioselective selector–selectand binding may be not necessarily required. Several indirect confirmations of this hypothesis have been described in the literature within the last few years but a dedicated study proving this concept has not been published yet. The present data obtained for the two chiral antimycotic drugs ketoconazole and terconazole by CE and NMR spectroscopy unequivocally support this concept.

Capillary electrophoretic and nuclear magnetic resonance studies on the opposite affinity pattern of propranolol enantiomers towards various cyclodextrins

In the present study the migration order of the propranolol enantiomers with various native CDs and neutral and charged CD derivatives was examined in capillary electrophoresis (CE). The reversal of the enantiomer migration order was observed due to sulfation of beta-CD on its primary hydroxy groups. The structures of intermolecular selector-select and temporary diastereomeric associates in solution were elucidated based on 1D rotating frame nuclear Overhauser effect spectroscopy (1D ROESY) experiments. Major structural differences were observed between the propranolol complexes with native beta-CD and heptakis(6-O-sulfo)-beta-CD.

Separation of enantiomers of norephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: Comparative CE and NMR studies

In this study, the enantiomer migration order (EMO) of norephedrine (NEP) in the presence of various CDs was investigated by CE. NMR and CE techniques were used to analyze the mechanism of the chiral recognition between NEP enantiomers and four CDs, i.e., native α‐CD, β‐CD, heptakis(2,3‐di‐O‐acetyl‐6‐O‐sulfo)‐β‐CD (HDAS‐β‐CD), and heptakis(2,3‐di‐O‐methyl‐6‐O‐sulfo)‐β‐CD (HDMS‐β‐CD). EMO was reversed in the presence of α‐CD and β‐CD, although only minor differences in the structures of the complexes formed between NEP and these CDs could be derived from rotating frame nuclear Overhauser experiments (ROESY). The complexes between the enantiomers of NEP and the sulfated CDs, HDMS‐β‐CD, and HDAS‐β‐CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS‐β‐CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.

Comparative NMR and MS studies on the mechanism of enantioseparation of propranolol with heptakis(2,3‐diacetyl‐6‐sulfo)‐β‐cyclodextrin in capillary electrophoresis with aqueous and non‐aqueous electrolytes

In our recent studies, the reversal of the enantiomer migration order (EMO) was observed with heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD (HDMS‐β‐CD) when aqueous electrolyte was changed with nonaqueous electrolyte in CE. One‐dimensional rotating frame nuclear Overhauser effect spectroscopy experiments prevailed that an inclusion complex was formed between the analyte and the chiral selector in the aqueous buffer, whereas an external complex resulted when a methanolic electrolyte was employed. In the case of the similarly substituted heptakis (2,3‐diacetyl‐6‐sulfo)‐β‐CD (HDAS‐β‐CD), the external complex was observed in the aqueous buffer but an inclusion complex was formed in methanolic electrolyte. In contrast to heptakis (2,3‐dimethyl‐6‐sulfo)‐β‐CD, no reversal of the enantiomer migration order was observed with HDAS‐β‐CD. In the present study, further mechanisms of enantioselective recognition and separation of propranolol enantiomers with HDAS‐β‐CD were investigated by using different techniques of nuclear magnetic resonance spectroscopy and high‐resolution mass spectrometry. To the best of our knowledge, enantioselective nuclear Overhauser effect was observed for the first time in this study.

Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies.

The enantiomer migration order (EMO) of ephedrine was investigated in the presence of various CDs in CE. The molecular mechanisms of chiral recognition were followed for the ephedrine complexes with native α‐ and β‐CD and heptakis(2,3‐di‐O‐acetyl‐6‐O‐sulfo)‐β‐CD (HDAS‐β‐CD) by CE, NMR spectroscopy and high‐resolution MS. Minor structural differences were observed between the complexes of ephedrine with α‐ and β‐CD although the migration order of enantiomers was opposite when these two CDs were applied as chiral selectors in CE. The EMO was also opposite between β‐CD and HDAS‐β‐CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS‐β‐CD. The latter CD was advantageous as chiral CE selector not only due to its opposite electrophoretic mobility compared with that of the cationic chiral analyte, but also primarily due to its enhanced chiral recognition ability towards the enantiomers of ephedrine.

Comparative enantioseparation of talinolol in aqueous and non-aqueous capillary electrophoresis and study of related selector–selectand interactions by nuclear magnetic resonance spectroscopy

The enantiomers of the chiral β-blocker drug talinolol were separated with two single component sulfated β-cyclodextrin (CD) derivatives, namely heptakis (2,3-di-O-methyl-6-sulfo)-β-CD) (HDMS-β-CD) and heptakis (2,3-di-O-acetyl-6-sulfo)-β-CD) (HDAS-β-CD), in aqueous and non-aqueous capillary electrophoresis (CE). The enantiomer affinity pattern of talinolol toward these two CDs was opposite in both aqueous and non-aqueous CE. However, the enantiomer affinity pattern for a given CD derivative did not change when aqueous buffer was replaced with non-aqueous background electrolyte. The structures of the analyte-selector complexes in both, aqueous and non-aqueous electrolytes were studied using rotating frame nuclear Overhauser effect (ROESY) NMR spectroscopy. Inclusion complex formation between the enantiomers of talinolol and HDAS-β-CD was confirmed in aqueous buffer, while the complex between the enantiomers of talinolol and HDMS-β-CD was of the external type. The complex of the talinolol enantiomers with HDAS-β-CD in non-aqueous electrolyte was also of the external type. In spite of external complex formation excellent separation of the enantiomers was observed in non-aqueous CE.

Comparative Enantioseparation of Ketoprofen with Trimethylated α-, β-, and γ-Cyclodextrins in Capillary Electrophoresis and Study of Related Selector–Selectand Interactions Using Nuclear Magnetic Resonance Spectroscopy

The enantiomers of ketoprofen were separated by capillary electrophoresis using the (2,3,6-tri-O-methyl)-derivatives of α-, β-, and γ-cyclodextrin (CyD) as chiral selectors. The affinity pattern of the ketoprofen enantiomers toward these CyDs changed depending on their cavity size. Thus, with hexakis (2,3,6-tri-O-methyl)-α-CyD and heptakis (2,3,6-tri-O-methyl)-β-CyD, the R enantiomer of the drug migrated first, whereas the enantiomer migration order was reversed in the presence of octakis(2,3,6-tri-O-methyl)-γ-CyD. The change in the migration order was rationalized on the basis of changes in the structure of the complexes between the ketoprofen enantiomers and the chiral selectors as derived from nuclear magnetic resonance spectroscopy experiments.

Applications of nuclear magnetic resonance spectroscopy for the understanding of enantiomer separation mechanisms in capillary electrophoresis

This review deals with the applications of nuclear magnetic resonance (NMR) spectroscopy to understand the mechanisms of chiral separation in capillary electrophoresis (CE). It is accepted that changes observed in the separation process, including the reversal of enantiomer migration order (EMO), can be caused by subtle modifications in the molecular recognition mechanisms between enantiomer and chiral selector. These modifications may imply minor structural differences in those selector-selectand complexes that arise from the above mentioned interactions. Therefore, it is mandatory to understand the fine intermolecular interactions between analytes and chiral selectors. In other words, it is necessary to know in detail the structures of the complexes formed by the enantiomer (selectand) and the selector. Any differences in the structures of these complexes arising from either enantiomer should be detected, so that enantiomeric bias in the separation process could be explained. As to the nature of these interactions, those have been extensively reviewed, and it is not intended to be discussed here. These interactions contemplate ionic, ion-dipole and dipole-dipole interactions, hydrogen bonding, van der Waals forces, π-π stacking, steric and hydrophobic interactions. The main subject of this review is to describe how NMR spectroscopy helps to gain insight into the non-covalent intermolecular interactions between selector and selectand that lead to enantiomer separation by CE. Examples in which diastereomeric species are created by covalent (irreversible) derivatization will not be considered here. This review is structured upon the different structural classes of chiral selectors employed in CE, in which NMR spectroscopy has made substantial contributions to rationalize the observed enantioseparations. Cases in which other techniques complement NMR spectroscopic data are also mentioned.

Investigation on the enantioseparation of duloxetine by capillary electrophoresis, NMR, and mass spectrometry

The enantiomeric separation of the antidepressant drug duloxetine was investigated by CE using 15 neutral CDs as chiral selectors. Among them, (2‐hydroxypropyl)‐β‐CD and methyl‐γ‐CD gave rise to the highest enantioresolution. The enantiomer migration order for duloxetine was found to be reversed depending on the CD employed: R‐duloxetine was the first‐migrating enantiomer for (2‐hydroxypropyl)‐β‐CD while it was the second‐migrating enantiomer for methyl‐γ‐CD. NMR and MS experiments were performed in order to justify this behavior. Although the elucidation of the structure of the enantiomer–CD complexes was not possible, their averaged stoichiometry was studied and their apparent and averaged equilibrium constants were calculated. The results obtained showed that the chiral separation of duloxetine by CE depends not only on the thermodynamic stability of the enantiomer–chiral selector complexes but also on their electrophoretic mobility.