Separation of tetrahydrozoline enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of chiral recognition mechanisms.

The recognition power and affinity pattern of various cyclodextrins (CD) towards the enantiomers of tetrahydrozoline (THZ) were studied using capillary electrophoresis (CE). As expected, affinity of THZ enantiomers and selectivity of recognition towards CD derivatives was strongly dependent on the cavity size and substituent type and pattern on the CD rims. Not only were the affinity strength and selectivity of recognition affected by the size of the cavity and chemistry of the CDs but also the affinity pattern. Another interesting example of opposite affinity pattern of enantiomers towards α- and ß-CD was observed here. In addition, opposite affinity pattern of THZ enantiomers was seen towards ß-CD and its acetylated derivatives, while methylation of ß-CD did not affect the affinity pattern of THZ enantiomers. In order to get more information about structural mechanisms of the multivariate dependences mentioned above, rotating frame Overhauser enhancement spectroscopy (ROESY) and computation techniques were used. Significant differences between the structure of THZ complexes with different CDs with both methods were encountered. Good correlations between experimentally determined and computed structure of complexes, as well as between computed complex stabilities and enantiomer migration order (EMO) in CE were observed.

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, ß-CD, heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD), and heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD). EMO was reversed in the presence of α-CD and ß-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-ß-CD, and HDAS-ß-CD, were substantially different. However, EMO of NEP was identical in the presence of these CDs. HDAS-ß-CD proved to be the most suitable chiral selector for the CE enantioseparation of NEP.

Comparative high-performance liquid chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by coating totally porous and core-shell silica particles.

This article reports comparative high-performance liquid chromatographic separations of enantiomers with chiral stationary phases (CSPs) prepared by coating cellulose tris(4-chloro-3-methylphenylcarbamate) on totally porous and on core-shell type silica of comparable particle diameter. Several interesting observations were made: (1) the selectivity of separation was higher on core-shell type CSP compared to totally porous CSP at comparable content of chiral selector (polysaccharide derivative); (2) much flatter dependence of plate height on the mobile phase flow rate was observed for columns packet with CSP prepared with core-shell silica compared to the ones packed with CSPs prepared with totally porous particles; (3) at low mobile phase flow rates core-shell CSP provided lower resolving ability compared to a commercially available CSP having four times higher content of chiral selector along with higher retention of chiral analytes. However, at high flow rates core-shell type CSP performed similarly or better than the commercial column in regards of plate count (N) and peak resolution (R(s)) per column length and within a given total analysis time. The advantage of CSP prepared with core-shell silica is obvious from the viewpoint of plate numbers and resolution calculated per unit time (i.e. speed of analysis).

Enantiomeric separation of FMOC-amino acids by nano-LC and CEC using a new chiral stationary phase, cellulose tris(3-chloro-4-methylphenylcarbamate).

A novel polysaccharide-based chiral stationary phase (CSP), cellulose tris(3-chloro-4-methylphenylcarbamate), also known as Sepapak-2 or Lux Cellulose-2, has been evaluated for the enantiomeric separation of FMOC derivatives of amino acids. After mobile-phase optimization in nano liquid chromatography (nano-LC) the column enabled the enantiomeric separation of 19 out of 23 amino acids tested, indicating the high chiral recognition power of this new CSP. Subsequently, a comparison of the driving force employed (pressure or voltage) was carried out comparing nano-LC and CEC under the same conditions. Better peak efficiencies and resolution were observed by using CEC experiments, which enabled the chiral discrimination of 20 out of 23 amino acids tested. Finally, in order to show the potential of this new CSP, the determination of the content and the enantiomeric purity of the non-protein amino acid citrulline in food supplements was performed. For that purpose, the method was optimized, evaluated and applied to different commercial samples.

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 ß-CD and heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD) by CE, NMR spectroscopy and high-resolution MS. Minor structural differences were observed between the complexes of ephedrine with α- and ß-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 ß-CD and HDAS-ß-CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS-ß-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 NMR and MS studies on the mechanism of enantioseparation of propranolol with heptakis(2,3-diacetyl-6-sulfo)-ß-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)-ß-CD (HDMS-ß-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)-ß-CD (HDAS-ß-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)-ß-CD, no reversal of the enantiomer migration order was observed with HDAS-ß-CD. In the present study, further mechanisms of enantioselective recognition and separation of propranolol enantiomers with HDAS-ß-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.

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 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 beta-CD, namely heptakis (2,3-diacetyl-6-sulfo)-beta-CD and heptakis (2,3-dimethyl-6-sulfo)-beta-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)-beta-CD but remained the same in the case of heptakis (2,3-diacetyl-6-sulfo)-beta-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.

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography.

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous-organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.

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.

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography.

Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.

Separation of enantiomers of deprenyl with various CDs in CE and the effect of enantiomer migration order on enantiomeric impurity determination of selegiline in active ingredients and tablets.

Opposite affinity pattern of enantiomers of the antiparkinsonian chiral drug deprenyl (DEP) was observed towards various neutral and charged derivatives of -CD. The effect of the enantiomer migration order on the LOD of enantiomeric impurity of R-DEP (selegiline) was studied for the standard substances and in the tablets from three different suppliers. The influence of injection mode on the LOD of a minor enantiomeric impurity was also studied and the CE method was compared with the pharmacopoeial HPLC method using a commercially available chiral column Chiralcel OD-H. The optimized CE method was more suitable for low-level enantiomeric impurity determination in selegiline compared to the pharmacopoeial HPLC method.

Comparative enantioseparations with native beta-cyclodextrin, randomly acetylated beta-cyclodextrin and heptakis-(2,3-di-O-acetyl)-beta-cyclodextrin in capillary electrophoresis.

Comparative enantioseparations were performed with three neutral cyclodextrins (CDs) in capillary electrophoresis (CE). In particular, native beta-CD was compared with single component heptakis(2,3-di-O-acetyl)-beta-CD (HDA-beta-CD) and randomly acetylated beta-CD (Ac-beta-CD) with the emphasis on the enantiomer migration order. The opposite affinity of the enantiomers of several chiral analytes was observed towards native beta-CD and its acetylated derivatives. The enantiomer affinity pattern of some chiral analytes was also opposite towards the two acetylated derivatives of beta-CD. In the case of the chiral drug clenbuterol (CL) an attempt was made to evaluate the possible structural reasons of the affinity reversal using one- and two-dimensional as well as transverse rotating frame nuclear Overhauser effect spectroscopy (ROESY). Significant differences were observed between the structure of the CL complexes with beta-CD and HDA-beta-CD.