<p>Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. </p><p>The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for <i>1S,2R</i>-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. </p><p>By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity <i>R</i>-timolol compared with injection without transient isotachophoresis.</p><p>The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone. </p>

Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for 1S,2R-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity R-timolol compared with injection without transient isotachophoresis. The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone.

In this study, resolution of racemic mandelic acid by enyzme enhanced ultrafiltration (EEUF) was studied. In order to develop a methodology, bovine serum albumin (BSA) was used as a model protein for polymer enhanced ultrafiltration (PEUF) experiments and the enzyme S-mandelate dehydrogenase was used for EEUF experiments. To be used for enzyme enhanced ultrafiltration experiments, the gene which is responsible from the production of S-mandelate dehydrogenase was isolated from Pseudomonas putida, expressed in Escherichia coli and the recombinant enzyme was produced. For PEUF experiments, effects of pH and ligand ratio were investigated. Total retention of mandelic acid increased with decrease in pH and total retention of mandelic acid reached to a maximum value of 74.4% at pH 4.3. For EEUF experiments, pH and ligand ratio effect on total retention, enantiomeric excess, enantioselectivity were investigated. Although apoenzyme was tried to be obtained by diafiltration and conversion was tried to be prevented, conversion occured especially at high pH values. To create the apoenzyme effect, three methods were studied. Enzyme conversion was prevented by sodium sulfite inhibition but enzyme did not retain mandelic acid. By oxygen saturation of enzyme, conversion was prevented, binding was achieved but enzyme showed no enantioselectivity. When the enzyme was diafiltrated at pH 10.0, total mandelic acid retention, enantiomeric excess and enantioselectivity reached to 77.2%, 38.9%, 2.27, respectively and the enzyme selectivity was reversed as R-selective.

A duloxetina é um potente inibidor duplo da recaptação de serotonina e norepinefrina, disponível como enantiômero puro, sob a forma S-duloxetina e comercializado como pellets em cápsulas. O R enantiômero da duloxetina é também um inibidor da recaptação, no entanto, este é menos potente que seu isômero, sendo considerado como impureza enantiomérica. Este trabalho teve como objetivos o desenvolvimento e a validação de métodos analíticos para o controle de qualidade da duloxetina e de sua respectiva impureza enantiomérica por cromatografia líquida de alta eficiência (CLAE), e por eletroforese capilar (EC). A resolução dos enantiômeros da duloxetina foi realizada a partir da utilização de fase estacionária quiral, baseada celulose, por CLAE. A separação por EC foi desenvolvida a partir da utilização de hidroxipropil-β-ciclodextrina (HPβCD) como seletor quiral. A validação dos métodos foi efetuada de acordo com os guias de validação disponíveis na literatura e os métodos propostos foram considerados específicos, lineares, precisos, exatos e robustos. A análise comparativa entre os métodos desenvolvidos demonstrou não haver diferença estatisticamente significativa na quantificação do enantiômero S-duloxetina. / Duloxetine is a double potent inhibitor of serotonin and norepinephrine reuptake, available as a pure enantiomer, in the S-duloxetine form and marketed as pellets into capsules. The R enantiomer of duloxetine is also an inhibitor of reuptake, however, less potent and being considered enantiomeric impurity. This work aimed the development and validation of analytical methods for quality control of duloxetine and its respective enantiomeric impurity by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The resolution of the enantiomers of duloxetine was performed with the use of chiral stationary phase, based on cellulose, by HPLC. The separation by CE was developed with the use of hydroxypropyl-β-cyclodextrin (HPβCD) as chiral selector. The method validation was performed according to the guides available in the literature and the proposed methods were considered specific, linear, precise, accurate and robust. The comparative analysis between the methods developed showed no statistically significant difference in the quantification of S-duloxetine enantiomer.

A duloxetina é um potente inibidor duplo da recaptação de serotonina e norepinefrina, disponível como enantiômero puro, sob a forma S-duloxetina e comercializado como pellets em cápsulas. O R enantiômero da duloxetina é também um inibidor da recaptação, no entanto, este é menos potente que seu isômero, sendo considerado como impureza enantiomérica. Este trabalho teve como objetivos o desenvolvimento e a validação de métodos analíticos para o controle de qualidade da duloxetina e de sua respectiva impureza enantiomérica por cromatografia líquida de alta eficiência (CLAE), e por eletroforese capilar (EC). A resolução dos enantiômeros da duloxetina foi realizada a partir da utilização de fase estacionária quiral, baseada celulose, por CLAE. A separação por EC foi desenvolvida a partir da utilização de hidroxipropil-β-ciclodextrina (HPβCD) como seletor quiral. A validação dos métodos foi efetuada de acordo com os guias de validação disponíveis na literatura e os métodos propostos foram considerados específicos, lineares, precisos, exatos e robustos. A análise comparativa entre os métodos desenvolvidos demonstrou não haver diferença estatisticamente significativa na quantificação do enantiômero S-duloxetina. / Duloxetine is a double potent inhibitor of serotonin and norepinephrine reuptake, available as a pure enantiomer, in the S-duloxetine form and marketed as pellets into capsules. The R enantiomer of duloxetine is also an inhibitor of reuptake, however, less potent and being considered enantiomeric impurity. This work aimed the development and validation of analytical methods for quality control of duloxetine and its respective enantiomeric impurity by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The resolution of the enantiomers of duloxetine was performed with the use of chiral stationary phase, based on cellulose, by HPLC. The separation by CE was developed with the use of hydroxypropyl-β-cyclodextrin (HPβCD) as chiral selector. The method validation was performed according to the guides available in the literature and the proposed methods were considered specific, linear, precise, accurate and robust. The comparative analysis between the methods developed showed no statistically significant difference in the quantification of S-duloxetine enantiomer.

This diploma thesis deals with the application of two permanently positively charged monosubstituted β-cyclodextrin derivatives (PEMEDA-β-CD, PEMPDA-β-CD) as chiral selectors in capillary electrophoresis. Use of PEMPDA-β-cyclodextrin in capillary electrophoresis has not been reported in literature. Properties of PEMEDA-β- cyclodextrin are already known, but its application for separation of amino acid enantiomers has not been published yet. Cyclodextrin derivatives were tested as additives in different buffers of different pH and with eventual addition of organic modifier. As suitable background electrolyte 15 mmol·l-1 borate buffer, pH = 9.5 without organic modifier was chosen. Furthermore the influence of chiral selector on separation and eventual enantioseparation of chosen analytes was evaluated. Addition of cyclodextrin derivatives in concentration range 0.0 - 5.0 was tested. Fourteen anionogenic analytes, including native amino acids, N- blocked amino acids and profens, were detected with UV-VIS detector at optimal wavelength 214, 254 or 280 nm. Both chiral selectors were suitable for enantioseparation of N-boc-D,L-tryptophan, which was baseline separated at concentration of selector as low as 0.5 mmol·l-1 . Tested amino acids blocked with terc-butoxycarbonyl and D,L-ketoprofen were partially...

Capillary zone electrophoresis was used for chiral separation of eleven diquat derivatives. These N-heteroaromatic dications containing structural motif of 2,2'-bipyridine have recently been studied for their interesting electrochemical properties as well as for the axial chirality of their molecules. The combination of these properties could potencially lead to interesting applications in the future. For enantioseparation of diquats (DQ) commercially available randomly sulfated α-, β-, and γ-cyclodextrins with high degree of substitution were used. A succesfull chiral separation was achieved using all of the three sulfated cyclodextrins as chiral selectors (CS). Baseline enantioseparation was achieved for 82 %, 91 % respectively 100 % of the analyzed DQ in the presence of HS-α-CD, HS-β-CD, HS-γ-CD respectively. The highest separation efficiency and resolution were obtained in the backround electrolyte containing 22 mmol/L NaOH, 35 mmol/L H3PO4 (pH2,5) and 6 mmol/L HS-β-CD. Using three available nonracemic DQ an identification of the particular M- and P-enantiomers was done for the three corresponding DQ structures. Apparent stabillity constants of complexes of the DQ derivatives with above mentioned cyclodextrins as CS were determined by means of capillary affinity electrophoresis. The stability...

In this work, an enantioselective HPLC method with UV and fluorimetric detection was developed and subsequently optimized for chiral separation of four aminoacids (D/L-alanine, D/L-valine, D/L-leucine, D/L-isoleucine) in native and particularly in derivatized form with an emphasis on enantioseparation of D-analogues. Retention and enantioseparation behavior of studied analytes was investigated on three chiral stationary phases based on teicoplanin (Chirobiotic T, Chirobiotic T2) and teicoplanin aglycone (Chirobiotic TAG). At the Chirobiotic T column, enantioseparations of underivatized aminoacids were performed with UV detection at 205 nm in the mobile phases methanol/water with different volume ratios. Baseline separation of L- and D-forms was achieved, however, the sensitivity of detection was very low. In order to increase detection sensitivity, derivatization of aminoacids was performed using 9-fluorenylmethyl chloroformate (FMOC-Cl) and the derivatization procedure was monitored on Chirobiotic T column with fluorimetric detection (λEx = 254 nm, λEm = 314 nm) in a buffered mobile phase methanol/0.5% TEAA buffer, pH 6.0 40/60 (v/v). In terms of derivatization, volume ratio D/L-aminoacid/derivatization agent 1/1 with ten times higher concentration of derivatization agent was found to be the most suitable....

<p>Statistical experimental designs have been used for method development and optimisation of separation. Two reversed phase HPLC methods were optimised. Parameters such as the pH, the amount of tetrabutylammonium (TBA; co-ion) and the gradient slope (acetonitrile) were investigated and optimised for separation of erythromycin A and eight related compounds. In the second method, a statistical experimental design was used, where the amounts of acetonitrile and octane sulphonate (OSA; counter ion) and the buffer concentration were studied, and generation of an α-plot with chromatogram simulations optimised the separation of six analytes.</p><p>The partial filling technique was used in capillary electrophoresis to introduce the chiral selector Cel7A. The effect of the pH, the ionic strength and the amount of acetonitrile on the separation and the peak shape of R- and S-propranolol were investigated.</p><p>Microemulsion electrokinetic chromatography (MEEKC) is a technique similar to micellar electrokinetic chromatography (MEKC), except that the microemulsion has a core of tiny droplets of oil inside the micelles. A large number of factors can be varied when using this technique. A screening design using the amounts of sodium dodecyl sulphate (SDS), Brij 35, 1-butanol and 2-propanol, the buffer concentration and the temperature as factors revealed that the amounts of SDS and 2-propanol were the most important factors for migration time and selectivity manipulation of eight different compounds varying in charge and hydrophobicity. SDS and 2-propanol in the MEEKC method were further investigated in a three-level full factorial design analysing 29 different compounds sorted into five different groups. Different optimisation strategies were evaluated such as generating response surface plots of the selectivity/resolution of the most critical pair of peaks, employing chromatographic functions, simplex optimisation in MODDE and 3D resolution maps in DryLab™.</p><p>Molecular descriptors were fitted in a PLS model to retention data from the three-level full factorial design of the MEEKC system. Two different test sets were used to study the predictive ability of the training set. It was concluded that 86 – 89% of the retention data could be predicted correctly for new molecules (80 – 120% of the experimental values) with different settings of SDS and 2-propanol.</p><p>Statistical experimental designs and chemometrics are valuable tools for the development and optimisation of analytical methods. The same chemometric strategies can be employed for all types of separation techniques.</p>

=RESUME= Trois approches différentes ont été comparées pour lestimation de lincertitude de mesure, à savoir celles basées sur des études inter-laboratoire, sur la robustesse et sur la validation. Pour ce faire, deux techniques analytiques séparatives, la chromatographie liquide haute performance (CLHP) et lélectrophorèse capillaire (EC), ont été utilisées pour la détermination de lénantiomère R-timolol dans des échantillons de maléate de S-timolol. Loptimisation de la méthode de CLHP a été effectuée sur une phase chirale à base de cellulose modifiée selon une approche multivariée. En ce qui concerne lEC, une séparation chirale convenable a été obtenue en milieu non aqueux en utilisant de lheptakis(2,3-di-O-méthyl-6-O-sulfo)-β-cyclodextrine comme sélecteur chiral en combinaison avec le camphosulfonate. Les deux méthodes ont été validées pour lusage prédéfini en appliquant la stratégie du profil dexactitude, ce qui a permis également destimer lincertitude de mesure. Par la suite, un test de robustesse a été effectué pour les deux méthodes. Linfluence des paramètres opératoires a été évaluée en considérant non seulement les réponses qualitatives mais surtout les réponses quantitatives. Les résultats de ces dernières (teneurs en R-timolol) ont servi à lestimation de lincertitude de mesure, selon le guide ISO 5725-2. Ce même guide a été utilisé pour évaluer la reproductibilité des résultats obtenus lors des études inter-laboratoire menées avec les deux méthodes. Lincertitude estimée à partir de la reproductibilité, a été trouvée dépendante de la concentration, comme observé également lors des études de validation et de robustesse. Il apparaît que lincertitude obtenue en robustesse prédit très bien celle obtenue en inter-laboratoire et constitue donc une alternative intéressante à cette dernière. Par contre, lincertitude associée à la validation est quant à elle différente de celle des autres approches. Cependant, elle reste parfaitement valable pour autant que le protocole de validation soit en accord avec la routine et que la méthode ne quitte pas le laboratoire qui la validée. Lors de la comparaison des deux méthodes, lincertitude obtenue en CLHP a été trouvée plus faible que celle obtenue en EC. =SUMMARY= Three different approaches (inter-laboratory, robustness and validation) have been applied to the estimation of uncertainty and compared. For that purpose, two analytical separation techniques, namely high performance chromatography liquid (HPLC) and capillary electrophoresis (CE), have been used for the determination of R-timolol enantiomer in S-timolol maleate samples. The optimisation of the HPLC method was carried out on a chiral stationary phase containing modified cellulose, by applying a multivariate approach. Concerning the CE method, a suitable chiral separation was obtained in a nonaqueous medium using heptakis(2,3-di-O-méthyl-6-O-sulfo)-β-cyclodextrin as chiral selector in combination with camphorsulfonate. The two methods were validated for the intended use by applying the strategy of the accuracy profile, which could be used additionally to estimate the uncertainty of measurement. Then, a robustness test was performed for the two methods. The influence of the operating parameters was assessed considering not only the qualitative responses but mainly the quantitative ones (the R-timolol content). The latter were used to estimate the uncertainty of measurement according to the ISO 5725-2 guide. The same guide was applied to evaluate the reproducibility of results obtained in inter-laboratory studies carried out with the two methods. The uncertainty was found to be concentration dependent, as also observed in validation and in robustness studies. The uncertainty obtained by robustness studies predicts well that obtained in inter-laboratory studies and can be proposed as an alternative to the latter. On the other hand, the estimation of uncertainty obtained with the validation studies leads to lower values than those obtained with the two other approaches but is still acceptable as long as the analytical method is used in a single laboratory. When comparing the two analytical methods, the uncertainty obtained in LC was found to be lower than that obtained in CE.