Comparison of capillary electrophoresis and reversed-phase liquid chromatography for determination of the enantiomeric purity of an M3 antagonist.

The chiral separation of an M3 antagonist was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins and by reversed-phase liquid chromatography with derivatized cellulose, derivatized amylose, and two protein stationary phases. Operational parameters for each technique, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH and type, organic modifiers, injection mode and temperature were varied in order to achieve a desired elution order and to meet a 0.1% limit of quantitation (LOQ) criteria. Based on the advantages and disadvantages of each technique, a practical CE method using sulfated gamma-cyclodextrin was selected. The method was validated in terms of linearity, LOQ, accuracy, ruggedness and precision.

Mechanistic aspects of chiral discrimination on an amylose tris(3,5-dimethylphenyl)carbamate.

The separation of [2R-[2alpha(R*),3alpha]]-5-[[2-[1-[3,5-bis-(trifluoromethyl)phenyl]ethoxy]-3(S)-4-fluorophenyl)4-morpholinyl]-methyl]-N,N-dimethyl-1H-1,2,3-triazole-4-methanamine hydrochloride from its enantiomer was achieved on an amylose tris-3,5-dimethylphenyl carbamate stationary phase. The retention of the enantiomers is dominated by weak hydrogen bonds while the enantioselectivity is governed by other kinds of interactions, e.g., inclusion in the amylose carbamate chains. Van't Hoffplots of 1nalpha vs. reciprocal temperature were non-linear and could be divided into two linear regions. One region at low temperature (5 degrees C- approximately 20 degrees C) and another one between 25 degrees C-70 degrees C with the change in slope occurring between 16 degrees C and 20 degrees C. DSC experiments suggested that the behavior can be attributed to breakage of H-bonds triggering a conformational change. Molecular simulation indicated a correlation between the interaction energies and the elution order obtained experimentally. The most retained enantiomer (R,R,S-enantiomer) interacts with the stationary phase through a hydrogen bond between the triazole proton and the C=O groups of the stationary phase, as well as through an inclusion in the cleft of the stationary phase. The other enantiomer exhibits a bifurcated H-bond between the triazolic proton and the C=O groups of the stationary phase leading to a less stable complex.

Chiral capillary electrophoretic analysis of the enantiomeric purity of a pharmaceutical compound using sulfated beta-cyclodextrin.

A practical chiral capillary electrophoresis method using randomly sulfated beta-cyclodextrin was developed for the quantitative determination of the chiral purity of a pharmaceutical compound. A systematic method development approach was conducted by modifying selected parameters such as the concentration of the chiral selectors, buffer pH, organic modifiers, buffer concentrations and type, temperature and applied voltage. The results of the investigation permitted an improved understanding of the separation mechanism. Two facile strategies for the reversal of the enantiomer elution order are also described. The optimized method was validated in terms of variability of the chiral selector, linearity, sensitivity, accuracy, recovery, ruggedness, and precision.

Enantiomeric separation of enzymatic hydrolysis products of dihydropyrimidinone methyl ester with cationic cyclodextrin by capillary electrophoresis.

The achiral separation of dihydropyrimidinone (DHP) methyl ester and its corresponding carboxylic acid and the chiral separation of their respective enantiomers were achieved in a single analysis using capillary electrophoresis (CE) with quaternary ammonium-beta-cyclodextrin (QA-beta-CD) as a chiral buffer additive. Separation of the DHP methyl ester from the corresponding carboxylic acid was achieved because the acid was negatively charged at pH 8.3 of the running buffer and the ester is neutral. Upon the addition of QA-beta-CD, the enantiomers of the acid and ester were well resolved before and after the electroosmotic flow, respectively. In addition, the minor DHP methyl ester enantiomer (R isomer) was well separated from several impurities. This CE system was used to monitor the progress of a bioresolution reaction that utilizes an enzyme to convert the R isomer of the ester to its corresponding acid. The quantities of all four enantiomers can be determined using a single set of CE conditions. In addition, it is demonstrated that samples can be directly injected into the capillary without sample pretreatment due to the fact that the coating of the cationic CD on the capillary surface prevents adsorption of the positively charged enzyme. The effects of other experimental parameters such as type of CDs, concentration of CDs, pH, temperature, and the preconditioning of capillary were also studied.

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein.

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.

Chromatographic separation of 3,4-difluorophenylacetic acid and its positional isomers using five different techniques.

The separation of five positional isomers from 3,4-difluorophenylacetic acid was investigated using normal- and reversed-phase high-performance liquid chromatography, capillary zone electrophoresis, gas chromatography and supercritical fluid chromatography. Operating parameters of each technique, such as temperature, type of stationary phase, mobile phase pH, ionic strength, organic modifiers and additives were varied in order to elucidate the separation mechanisms. Based on the advantages and disadvantages of each methodology, a simple and practical RPLC method was selected. The method was validated in terms of linearity, limit of detection, accuracy, recovery, ruggedness and precision.