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An experimental design approach is described to evaluate the main electrophoretic parameters involved in the enantioseparation of pharmaceuticals by capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (ESI-MS). For all experiments, the partial-filling technique was applied to avoid the chiral selector entering in the mass spectrometer ion source with a negative effect on the electrospray performance. To carry out enantioseparation, a volatile buffer constituted of 20 mM ammonium acetate at pH 4.0, and a polyvinyl alcohol-coated capillary were used. Methadone was employed as the model compound and three different cyclodextrins (CDs), namely sulfobutyl ether-beta-CD, carboxymethylated-beta-CD and hydroxypropyl-beta-CD, were selected in order to study the countercurrent process. Two different experimental designs were chosen: (i) a full-factorial design to examine the effects and significance of the investigated factors, and (ii) a central composite face-centered design to establish the mathematical model of the selected responses in function of experimental factors. The chiral selector concentration, percentage of the capillary filled with the chiral selector, and drying gas nebulization pressure were three relevant factors taken into consideration. For each CD, the methadone enantiomeric resolution, apparent selectivity, and migration time of the second enantiomer were established as responses. The latter were systematically related to experimental parameters with the help of multiple linear regression. It is noteworthy that the behaviour was different in function of the chiral selector charge. Results revealed that the nebulization pressure involved in the electrospray process and the CD concentration had a significant effect on the enantiomeric resolution, while the effect of the separation zone length was less pronounced. Finally, response surfaces were drawn from the mathematical model and experimental conditions were selected to allow a robust determination of methadone enantiomers by CE-MS.

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.

A capillary zone electrophoresis method was developed for the enantioseparation of celiprolol enantiomers, using a sulfated beta-cyclodextrin (beta-CD) as a chiral selector. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A central composite design was applied to optimize the method and four parameters were selected for this study: the buffer pH, the buffer concentration, the sulfated beta-CD concentration and the temperature. Resolution between celiprolol enantiomers as well as analysis time and generated current were established as responses. For each response, a model was obtained by a second-degree mathematical expression. From the models, the most favorable conditions were determined by optimizing the resolution between celiprolol enantiomers and by setting the two other responses at threshold values. Response surfaces were also used to assess the robustness of the analytical method around the optimal region. Successful results were obtained with a 52 mM acetate buffer at pH 4.0 in the presence of 3.0 mM sulfated beta-CD at a temperature of 19.5 degrees C. Under these optimized conditions, baseline separation of the celiprolol enantiomers was achieved in less than 10 min. The method showed good validation data in terms of precision, accuracy and linearity, and was found to be suitable in determining celiprolol enantiomers in pharmaceutical preparations and in biological fluids.

The simultaneous determination of atropine and scopolamine derivatives, which have similar structures, was investigated by using capillary zone electrophoresis. The effects of buffer pH, buffer concentration and hydroxypropyl-beta-cyclodextrin concentration on migration time and resolution of the investigated compounds were systematically studied. The selected electrophoretic buffer consisted of a 80 mM sodium citrate pH 2.5, containing 2.5 mM hydroxypropyl-beta-cyclodextrin as the complexing agent. Quantitative analysis was validated by testing the reproducibility of the method, giving a relative standard deviation less than 1 and 2% for the intermediate precision of migration times and peak area ratios, respectively. The linearity of the method was assessed between 50 and 150% of the theoretical content (coefficient of correlation greater than 0.99). The proposed method was found to be suitable and accurate for the determination of these basic drugs in pharmaceutical preparations.