Nonaqueous capillary electrophoretic separation of basic enantiomers using octakis(2,3-O-dimethyl-6-O-sulfo)-gamma-cyclodextrin, a new, single-isomer chiral resolving agent

The enantiomers of 34 pharmaceutical weak-base analytes were separated by nonaqueous capillary electrophoresis in acidic methanol background electrolytes using the sodium salt of the new, single-isomer chiral resolving agent, octakis(2,3-O-dimethyl-6-O-sulfo)-gamma-cyclodextrin (ODMS). The effective mobilities, separation selectivities and peak resolution values of the weak-base analytes were determined as a function of the ODMS concentration in the 0-40 mM range and were found to follow the theoretical predictions of the charged resolving agent migration model (CHARM model) modified for ionic strength effects. Fast, efficient separations were achieved for both comparatively small and large enantiomers.     

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.     

Enantiomer separations by nonaqueous capillary electrophoresis using octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The newest member of the single-isomer isomer sulfated cyclodextrin family, octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma-CD) was used for the first time as a resolving agent for the nonaqueous capillary electrophoretic separation of the enantiomers of 26 weak base pharmaceuticals in an acidic methanol background electrolyte. The solubility limit of ODAS-gamma-CD at room temperature proved to be 55 mM in this background electrolyte, which afforded good, fast enantiomer separations for most of the basic drugs tested. For all the bases studied, the effective mobilities and separation selectivities were found to follow the predictions of the charged resolving agent migration model of electrophoretic enantiomer separations. The effective mobilities of the weakly binding weak bases remained cationic throughout the entire 0 to 45 mM ODAS-gamma-CD concentration range; separation selectivities increased as the ODAS-gamma-CD concentration was increased. The effective mobilities of the moderately binding weak bases became anionic in the 2.5 to 45 mM ODAS-gamma-CD concentration range; separation selectivities first increased as the effective mobilities approached zero, then decreased again as the ODAS-gamma-CD concentration was increased further. The effective mobilities of the strongly binding weak bases became anionic in the 0 to 2.5 mM ODAS-gamma-CD concentration range; separation selectivities decreased as the ODAS-gamma-CD concentration was increased above 2.5 mM.     

Experimental verification of a predicted, hitherto unseen separation selectivity pattern in the nonaqueous capillary electrophoretic separation of weak base enantiomers by octakis (2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of cationic enantiomers with single-isomer multivalent anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (mu0(RCD)/mu0(SCD)), and parameter a, the complexation-induced alteration of the analyte's mobility (mu0(RCD)/mu0). In addition to the previously observed discontinuity in separation selectivity that occurs as mu(eff) of the less mobile enantiomer changes from cationic to anionic, a new feature, a separation selectivity maximum was predicted to occur in the resolving agent concentration range where both enantiomers migrate cationically provided that (i) K(RCD)/K(SCD) <1 and mu0(RCD)/mu0(SCD) >1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) > 1, or (ii) K(RCD)/K(SCD) >1 and mu0(RCD)/mu0(SCD) <1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) <1. This hitherto unseen separation selectivity pattern was experimentally verified during the nonaqueous capillary electrophoretic separation of the enantiomers of four weak base analytes in acidic methanol background electrolytes with octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gammaCD) as resolving agent.     

Capillary electrophoretic separation of enantiomers in a high-pH background electrolyte by means of the single-isomer chiral resolving agent octa(6-O-sulfo)-gamma-cyclodextrin

The new, alkali-stable, single-isomer, sulfated gamma-cyclodextrin, the sodium salt of octa(6-O-sulfo)-gamma-cyclodextrin (OS) was used for the first time to separate the enantiomers of non-ionic, acidic, basic and ampholytic analytes by capillary electrophoresis in high-pH aqueous background electrolytes. The effective mobilities and separation selectivities were found to follow trends similar to those observed earlier in acidic aqueous background electrolytes. OS proved to be a broadly applicable chiral resolving agent and afforded adequate peak resolution values with short separation times for a number of non-ionic, weak acid, weak base and ampholytic analytes.     

Electrophoretic enantiomer separations at high pH using the new,single-isomer octakis(2,3-dimethyl-6-O-sulfo)-gamma-cyclodextrin as chiral resolving agent

The latest, single-isomer, sulfated γ-cyclodextrin, the sodium salt of octakis(2,3-dimethyl-6-O-sulfo)-γ-cyclodextrin that is stable in basic media was used to separate the enantiomers of neutral, weak acid and weak base analytes by capillary electrophoresis in high pH aqueous background electrolytes. The effective mobilities and separation selectivities were found to follow trends similar to those observed earlier in acidic aqueous background electrolytes. Octakis(2,3-dimethyl-6-O-sulfo)-γ-cyclodextrin proved to interact with all three analyte types less strongly than other single-isomer sulfated cyclodextrins do under comparable conditions.     

Synthesis,analytical characterization and use of octakis(2,3-di-O-methyl-6-O-sulfo)-gamma-cyclodextrin,a novel,single-isomer,chiral resolving agent in low-pH background electrolytes

The third member of the family of single-isomer, sulfated gamma-cyclodextrins, the sodium salt of octakis(2,3-di-O-methyl-6-O-sulfo)-gamma-cyclodextrin has been synthesized, analytically characterized and used for the capillary electrophoretic separation of the enantiomers of nonionic, weak acid and weak base analytes in low-pH aqueous background electrolytes. Though octakis(2,3-di-O-methyl-6-O-sulfo)-gamma-cyclodextrin complexes less strongly with many of the analytes tested than the other members of the single-isomer, 6-O-sulfo gamma-cyclodextrin family, such as octa(6-O-sulfo)-gamma-cyclodextrin and octakis(2,3-di-O-acetyl-6-O-sulfo)-gamma-cyclodextrin, it offers excellent separation selectivities, often complementary to those of both the single-isomer, 6-O-sulfo beta-cyclodextrins and 6-O-sulfo gamma-cyclodextrins. Rapid, efficient enantiomer separations were observed for a large number of structurally diverse analytes in acidic aqueous background electrolytes.     

Nonaqueous capillary electrophoretic separation of basic enantiomers using heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin.

The enantiomers of 40 basic analytes, mostly pharmaceuticals, were separated by nonaqueous capillary electrophoresis in acidic methanol background electrolytes using the sodium salt of heptakis(2,3-dimethyl-6-sulfato)-beta-cyclodextrin (HDMS-beta-CD). The effective mobilities, separation selectivities, and peak resolution values were determined as a function of the HDMS-beta-CD concentration in the 0-40 mM range and were found to follow the theoretical predictions of the charged resolving agent migration model (CHARM model). Fast, efficient enantiomer separations were achieved for a large number of both very hydrophobic and hydrophilic weak bases.     

Mechanistic study on the opposite migration order of clenbuterol enantiomers in capillary electrophoresis with beta-cyclodextrin and single-isomer heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin

Opposite migration order was observed for the enantiomers of the chiral beta2-adrenergic drug clenbuterol (CL) in capillary electrophoresis (CE) when resolved with native beta-cyclodextrin (beta-CD) and heptakis (2,3-diacetyl-6-sulfo)-beta-CD (HDAS-beta-CD). The possible mechanisms of the affinity reversal of the CL enantiomers depending on the structure of the CD were studied using 1H-nuclear magnetic resonance (1H-NMR) spectrometry and one-dimensional rotating frame nuclear Overhauser and exchange spectrometry (1-D ROESY). Significant differences were observed between the structure of the (+/-)-CL complexes with beta-CD and HDAS-beta-CD.     

NMR spectra and dynamics of the sodium salt of per(6-thiobenzoic acid)-gamma-cyclodextrin

The solution-state NMR spectra of a per-6-substituted gamma-cyclodextrin show some interesting dynamic properties. At high temperature (353 K), the (1)H NMR spectrum shows dynamic averaging of the different conformations. This averaging is no longer observed on cooling of the cyclodextrin solution to 278 K, resulting in NMR spectra with a large (1)H and (13)C chemical shift dispersion. The complete assignment of the eight unique glucosyl residues was achieved using COSY, HSQC and exchange spectroscopy. A ROESY spectrum, with a short mixing time to reduce the effects of exchange, gives correlations that lead to the determination of the connectivity of all eight glucosyl residues. On the NMR time-scale, the cyclodextrin is highly dynamic; the lower temperature minimum energy conformation has one of the aromatic rings self-complexed and a distorted cyclodextrin torus.     

Thermodynamic study on the gas chromatographic separation of the enantiomers of aromatic alcohols using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin as a stationary phase

Gas chromatographic separation of the enantiomers of nineteen structurally related aromatic alcohols was investigated as a function of temperature using a heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin-coated capillary column. Thermodynamic parameters were determined and compared with those obtained with the nonchiral, reference stationary phase, OV-1701. While the -deltaH and -deltaS values for the more retained enantiomers of all nineteen alcohols are comparable on the chiral stationary phase used, the -delta(deltaH) and -delta(deltaS) values are considerably different. Of all the solutes tested, enantiodiscrimination was the greatest for the 2,6-difluoro-alpha-methylbenzyl alcohol.     

Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier

Summary Guaran, a neutral polysaccharide, has been used as a buffer modifier to improve the separation of basic proteins and drugs. Migration reproductibility, peak shape and efficiency were improved when 0.1% guaran was added to the buffer. The concentration of guaran, ionic strength, and pH of buffer solution were optimized to obtain the optimum separation of proteins. Possible separation efficiencies of 700,000 plates per meter were obtained for test proteins. The relative standard deviation (% RSD) of the migration time of all test proteins was less than 0.5%. Improved separation of β-blockers was also observed when guaran was added to the buffer.     

Capillary zone electrophoretic chiral discrimination using 6-O-(2-hydroxy-3-trimethylammoniopropyl)-beta-cyclodextrin as a chiral selector

A charged highly water-soluble CD derivative, 6-O-(2-hydroxy-3-trimethylammoniopropyl)-beta-CD (herein noted as 6-HPTMA-beta-CD) was synthesized and successfully used as a chiral selector for enantiomeric separation of some acidic compounds by CZE in an uncoated capillary. Substitution with 2-hydroxy-3-trimethylammoniopropyl groups at the primary hydroxyl group of the CD was aimed at influencing the magnitude and selectivity of analyte-CD interactions. The behavior of 6-HPTMA-beta-CD was compared with that of the commercially available quaternary ammonium-beta-CD (QA-beta-CD) under the same separating conditions. The experiments were carried out using a BGE consisting of 50 mM phosphate in the pH range of 4-6 by adding a relatively low concentration of chiral selector (less than 10 mM). The effects of the concentration of CD and the pH of the electrolyte on the resolution of these compounds were studied.     

Capillary electrophoretic separation of tricyclic antidepressants using a polymer-coated capillary and beta-cyclodextrin as an electrolyte additive

A new method for the CE separation of nine tricyclic antidepressants (TCAs), viz. amitriptyline, clomipramine, desipramine, doxepin, fluphenazine, imipramine, nortriptyline, promazine, and thioridazine, is described. The capillary was statically coated with a layer of poly(N,N-dimethylacrylamide) (PDMA) to suppress the EOF, and beta-CD was used as an additive in the BGE solution. The optimal resolution of nine TCAs was obtained by using a 1% v/v PDMA-coated capillary and a BGE solution of 50 mM sodium phosphate buffer (pH 3.0) containing 0.5 mM beta-CD. Efficiencies were typically >10(5 )plates/m. Complete separation of nine TCAs could be achieved in about 28 min; the two diastereomers of doxepin and the two enantiomers of thioridazine could also be separated. The RSD values of migration time and peak area of the TCAs were in the ranges 0.5-0.8 and 3.3-4.9% (n = 10), respectively. In combination with a suitable sample clean-up technique, such as hollow fiber-based liquid phase microextraction (HF-LPME), the polymer-coated capillary can be employed for the CE-UV analysis of TCAs in human plasma.     

Determination of enantiomeric impurity of linezolid by capillary electrophoresis using heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin

A method for the enantioseparation of linezolid, the first compound of a truly new class of antibiotics-the oxazolidinones, was developed. The elaborated method of linezolid enantiomers separation was successfully performed using an anionic single-isomer cyclodextrin-heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) as a resolving agent with the help of the charged resolving agent migration model (CHARM model). The best results were obtained with 27.5mM HDAS-beta-CD dissolved in 50mM borate buffer, pH 9.0, 15 degrees C, normal polarity. The facile strategies for the reversal of the enantiomers elution order are also described. Afterwards, the optimized method was validated in terms of sensitivity, linearity, accuracy and precision.     

Synthesis,analytical characterization and initial capillary electrophoretic use in acidic background electrolytes of a new,single-isomer chiral resolving agent: hexakis(2,3-di-O-acetyl-6-O-sulfo)-alpha-cyclodextrin

The sodium salt of hexakis(2,3-di-O-acetyl-6-O-sulfo)-alpha-CD (HxDAS), the first member of the family of single-isomer, fully sulfated alpha-CDs, has been synthesized and used for the initial capillary electrophoretic separation of the enantiomers of nonionic, weak acid, weak base, and ampholytic analytes. HxDAS complexes less strongly with many of the analytes tested than the analogous beta- and gamma-cyclodextrin derivatives, namely, heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS) and octakis(2,3-di-O-acetyl-6-O-sulfo)-gamma-cyclodextrin (ODAS). Nevertheless, it facilitated the separation of the enantiomers of a large number of weak electrolyte and nonelectrolyte analytes in acidic aqueous background electrolytes. For all analytes, the effective mobilities and separation selectivities as a function of the background electrolyte concentration of HxDAS followed the trends that were found for HDAS and ODAS.     

Use of the new, single-isomer, hexakis(2,3-diacetyl-6-O-sulfo)-alpha-cyclodextrin in acidic methanol background electrolytes for nonaqueous capillary electrophoretic enantiomer separations

The new, single-isomer, sulfated alpha-cyclodextrin, the sodium salt of hexakis(2,3-diacetyl-6-O-sulfo)-alpha-cyclodextrin (HxDAS), was used for the first time in acidic methanol background electrolytes (BGEs) to separate the enantiomers of weak base analytes by nonaqueous capillary electrophoresis (NACE). The concentration dependence of the effective mobilities and separation selectivities followed trends similar to those observed earlier in acidic methanol background electrolytes with heptakis(2,3-diacetyl-6-O-sulfo)-beta-cyclodextrin (HDAS) and octakis(2,3-diacetyl-6-O-sulfo)-gamma-cyclodextrin (ODAS). In general, interactions between the weak base analytes and HxDAS were weaker than with HDAS and ODAS. For some of the weak base analytes, separation selectivities observed in acidic aqueous and acidic methanol background electrolytes were complementary to each other, permitting the eventual separation of enantiomers that could not be achieved otherwise.     

Non-aqueous capillary electrophoretic enantiomer separations using the tetrabutylammonium salt of heptakis(2,3-O-diacetyl-6-O-sulfo)-cyclomaltoheptaose, a single-isomer sulfated beta-cyclodextrin highly-soluble in organic solvents

The tetrabutylammonium salt of heptakis(2,3-di-O-acetyl-6-O-sulfo)-cyclomaltoheptaose, a single-isomer sulfated beta-cyclodextrin that is adequately soluble in a number of protic and aprotic polar solvents was synthesized on the large scale and used for the capillary electrophoretic separation of the enantiomers of weak bases in acidic acetonitrile background electrolytes. The effective mobilities and separation selectivities observed for these analytes followed trends similar to those found with other single-isomer sulfated cyclodextrins in acidic methanol background electrolytes. Enantiomer separations obtained with the tetrabutylammonium and sodium salts of heptakis(2,3-di-O-acetyl-6-O-sulfo)-cyclomaltoheptaose were different indicating, for the first time, that selection of the counter ion of the single-isomer sulfated cyclodextrin is also of importance for the separation of enantiomers.     

Heptakis(6-amino-6-deoxy)-beta-cyclodextrin as a chiral selector for the separation of anionic analyte enantiomers by capillary electrophoresis

A hepta-substituted beta-cyclodextrin bearing seven amino groups, heptakis(6-amino-6-deoxy)-beta-cyclodextrin (per-6-NH2-beta-CD) was successfully used as a chiral selector for the enantioseparation of different anionic analytes. The running buffer pH and chiral selector concentration were the studied parameters crucial in achieving the maximum possible enantioresolution. Enantiomeric separation of a mixture of seven carboxybenzyl-amino acids was achieved in 24 min. Excellent resolution was obtained for carboxybenzyl-tryptophan (Rs = 11.2).