Tesis doctoral de María Castro Puyana
Nowadays, chiral analysis is one of the most interesting aspects of separation techniques due to the impact that chirality has in different fields such as the pharmaceutical, agrochemical, environmental or food analysis. In fact, the individual determination of the enantiomers of a chiral compound is very important due to the different biological activity that they may have. Capillary electrophoresis (ce) has shown in the last years to be a powerful technique for the resolution of enantiomers due to its high separation efficiency and versatility, short analysis time, simplicity, and low consumption of reagents and samples. Electrokinetic chromatography (ekc) is a mode of ce enabling enantiomeric separations through the addition of a chiral selector to the electrolytic solution. in this work, new contributions of ekc with uv and mass spectrometry detection in the field of chiral analysis are presented. With this aim, new analytical methodologies have been developed enabling the rapid and simple enantiomeric separation of chiral compounds with a high interest in different fields including drug or food analysis or astrobiology. electrokinetic chromatography with uv detection using cyclodextrins (cds) as chiral selectors in the separation buffer has been employed in order to achieve the enantiomeric separation of a group of antifungal compounds. The influence of some experimental conditions, such as temperature, chiral selector concentration, and ph, on the enantiomeric separation was studied. The developed methods enabled the first enantiomeric separation of terconazole, the first ekc separation of the four enantiomers of itraconazole, and the improvement of the enantiomeric separation of ketoconazole, miconazole, bifonazole, sulconazole and econazole in terms of analysis time or resolution with respect to previous methods developed by ce or hplc. In addition, the analytical methodologies developed for the enantiomeric analysis of ketoconazole and itraconazole have been successfully applied to the quantization of both compounds in pharmaceutical formulations. Moreover, the enantioselective recognition of six azole compounds (ketoconazole, terconazole, miconazole, econazole, bifonazole and sulconazole) with three neutral β-cds has been discussed on the basis of the determination of the apparent binding constants for each pair enantiomer-cd, and the inversion in the migration order for the enantiomers of two of the antifungals studied (ketoconazole and terconazole) with the cd concentration in the separation medium has been reported for the first time. the affinity pattern of the enantiomers of deprenyl (an antiparkinsonian drug) has been studied towards different cds using ekc with uv detection, which enabled to obtain new data on the effect of cavity size and nature of substituents in the cds. Among the three native cds, only β-cd has enabled the enantiomeric separation of deprenyl. Moreover, opposite migration order for the enantiomers of deprenyl was observed for different neutral and charged β-cds. While r-deprenyl was the first-migrating enantiomer when using β-cd and methylated β-cds, the use of acetylated β-cds originated that s-deprenyl migrated before r-deprenyl. The importance of the enantiomer migration order for the determination of enantiomeric impurities has been shown, since the developed method was applied to the determination of the minor enantiomeric impurity (s-deprenyl) in pharmaceutical formulations of selegiline (r(-)-deprenyl). Finally, the ekc method developed has been compared with the pharmacopoeial hplc method, appearing to be more suitable for low-level enantiomeric impurity determination in selegiline. the development of ekc-uv-esi-ms analytical methodologies has enabled, for the first time, the detection and the identification of trans-ketoconazole as the main impurity of cis-ketoconazole in standards as well as in pharmaceutical formulations. Ekc-uv has enabled the detection, enantiomeric separation and quantization of trans-ketoconazole while ekc-esi-ms has made possible the identification and quantization of the impurity in cis-ketoconazole after an optimization of esi-ms conditions. the first enantiomeric separation of a short-chain tricarboxylic acid has been achieved in this work. In fact, the enantiomers of 3-carboxy adipic acid have been separated for the first time by ekc with uv detection in spite of the low uv absorption of this compound. An analytical methodology has been developed using a macrocyclic antibiotic (vancomycin) as chiral selector in the separation buffer and the partial filling technique to avoid vancomycin reaching the detection window. To achieve the sensitivity needed to determine enantiomeric excesses of 3-carboxy adipic acid adsorbed on mineral samples, a strategy was proposed consisting of a field-amplified sample stacking together with the use of a bubble capillary and uv detection at low wavelengths. Results obtained showed that an enantioselective adsorption of the enantiomers of 3-carboxy adipic acid on minerals took place, which may point a possible mechanism for prebiotic origins of biological homoquirality. finally, an ekc-esi-ms method has been developed for the separation of carnitine enantiomers in order to combine the enormous performance of ce for chiral separations with the selectivity and structural information provided by ms2 experiments. Optimization of the separation conditions was performed by ekc-uv and included the study of the influence of the type and concentration of chiral selector, ph and buffer concentration. A pre-capillary derivatization with 9-fluorenylmethoxycarbonyl (fmoc) was achieved with the following purposes: (i) to introduce a chromophore group into the molecule to allow its uv detection, (ii) to facilitate the interaction with the cds needed to obtain a chiral discrimination, and (iii) to obtain a better sensitivity in ms analysis. Optimization of ekc-esi-ms analysis enabled the successful application of the method to the analysis of l- and d-carnitine in infant formulas. A sample treatment involving ultrafiltration prior to fmoc derivatization was required prior to injection of samples in the ekc-esi-ms system.
Datos académicos de la tesis doctoral «Nuevas contribuciones de la cromatografia electrocinetica con detecciones uv y de espectrometria de masas en el campo de las separaciones quirales. new contributions of electrokinetic chromatography with uv and mass spectrometry detection in the field of chiral separations«
- Título de la tesis: Nuevas contribuciones de la cromatografia electrocinetica con detecciones uv y de espectrometria de masas en el campo de las separaciones quirales. new contributions of electrokinetic chromatography with uv and mass spectrometry detection in the field of chiral separations
- Autor: María Castro Puyana
- Universidad: Alcalá
- Fecha de lectura de la tesis: 14/12/2007
Dirección y tribunal
- Director de la tesis
- María Luisa Marina Alegre
- Tribunal
- Presidente del tribunal: ángel Ríos castro
- elisa Blanco gonzález (vocal)
- bezhan Chankvetadze (vocal)
- alejandro Cifuentes gallego (vocal)