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<p>Gas chromatography (GC) has been utilized for the study of enantiomer resolution of the atropisomers of PCBs, o,p´-DDD and o,p´-DDT. Different substituents and concentrations of cyclodextrin, capillary dimensions and type of stationary phase films have been investigated to achieve the resolution of as many of the atropisomers on one column as possible. The results indicated that the butyl substitution of 6-hydroxyl and the methyl substitution of 2- and 3-hydroxyl were the most promising for the enantiomeric separation. Using Capillary Electrophoresis (CE), the trimers and monomers of PM-β-CDs were compared for enantiomeric resolution, as well as comparing the cationic PMMA-β-CD with the anionic HS-β-CD. In these studies the trimer did not show an improved resolution for mepivacaine, when compared to the equimolar concentration of the monomer. The cationic CD gave increased resolution values for ibuprofen when compared to the anionic CD. A scheme for reversing enantiomeric elution order of both the basic propranolol and acidic ibuprofen is also presented, with the aim of facilitating the detection of impurities in a high sample loading. The detection of 1% of each enantiomer of propranolol, and 1% of R(-)-ibuprofen, was demonstrated, with elution prior to the tailing peak of the corresponding enantiomer. Dimethylacrylamide-coated capillaries were used in this work, and the stability of this coating was demonstrated, giving a highly reduced electroosmotic flow for up to six months. Enantiomeric baseline separations of omeprazole and 5-hydroxyomeprazole have also been achieved with both CE and Open Tubular Capillary Electrochromatography (OT-CEC) methods. With CE-UV, both a non-aqueous method (using HDMS-β-CD) and an aqueous method (using HS-β-CD) were used for enantiomeric resolution of the two racemates. Resolution of omeprazole was also achieved using CE-Electrospray Ionization-Mass Spectrometry (ESI-MS). In OT-CEC, avidin was immobilized on the inside surface of a fused silica capillary and was employed as chiral selector for the enantiomeric baseline resolution of omeprazole and 5-hydroxyomeprazole.</p>

This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam. The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected. The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam. The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam. The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples. The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.

Stereochemistry plays an important role in nature because biologically important molecules such as amino acids, nucleotides and sugars, only exist in enantiomerically pure forms. Semiochemicals carry messages, between the same species (pheromones) and between different species (allelochemicals). Both pheromones and allelochemicals can be used as environmentally friendly pest management. Many semiochemicals, i.e. behaviour modifying chemicals, consist of pure or well-defined mixtures of stereoisomers, where some of the other stereoisomers can be repellent. It is therefore important to be able to separate them to produce a synthetic pheromone in a mixture that is attractive. Pine sawflies are a family of insects that in some cases can be severe defoliators of conifer trees. Diprion pini, Diprion similis and Neodiprion sertifer are severe pests for these trees and have got the most attention in pine sawfly pheromone studies. The pheromone precursors are stored in the female body as long-chain secondary alcohols, which, when released, are esterified to acetates or propionates. The alcohols are chiral, and normally one of the stereoisomer is the main pheromone component, sometimes possible together with other stereoisomers as essential minor components. Bicyclus is a genus of African butterflies, and especially Bicyclus anynana has become a popular model for the study of life history evolution, morphology, mating choice and genetics. The wing pattern of Bicyclus differs depending on the season, with large eyespots during the rain-season and small or absent spots during the dry season.  Euglossa is one of the genera among the orchid bees in the Neotropics that does not produce its own pheromone. Instead, the males collect fragrances from orchids and other sources and store them in a pocket in their hind legs. Both Bicyclus and Euglossa use semiochemicals similar to pine sawflies, and thus can be analysed by the same methods. Pheromones and other semiochemicals in insects are often present in low amounts in a complex matrix, and purification of the sample before chemical analysis is often required. A common method is gradient elution on a solid phase silica column. Separation of stereoisomers can be achieved either by using a column with a chiral stationary phase (CSP) or with pre-column derivatisation using a column with an achiral stationary phase (ASP) or a combination of both, with mass detection as the dominant detection method. The purpose of this work has been to improve the purification method, find suitable methods to separate the stereoisomers of secondary alcohols, and to apply this on extracts of insects. By selecting the right fractions to collect during gradient elution the purification method was optimised. To reduce plasticizer contamination from ordinary columns, solid phase columns of Teflon or glass were used. For pre-column derivatisation of different chiral alcohols various acid chlorides were tested. For the pine sawfly pheromone precursors enantiopure (2S)-2-acetoxypropionyl chloride was the best choice. To separate some of the stereoisomers achiral 2-naphthoyl chloride was used. For derivatisation of 6,10,14-trimethylpentadecan-2-ol (R)-trans-chrysanthemoyl chloride was the best choice. The derivatised alcohols were separated on different columns, both chiral and non-chiral. Varian FactorFour VF-23ms was chosen as a general-purpose column, the Agilent HP-88 column was the best column with an ASP of those tested, and the Chiraldex B-PA column (CSP) was the only one that could separate all eight stereoisomers of derivatised 3,7-dimethylundecan-2-ol, 3,7-dimethyldodecan-2-ol, and 3,7-dimethyltridecan-2-ol. To determine the stereoisomeric purity of standard solutions used in field experiments and extracts of different species of insects the optimised methods were applied. For extracts from B. anynana, Euglossa and Neodiprion lecontei this work describe the first determination of the stereochemistry of some of their semiochemicals. For the determination of the stereochemistry of chiral semiochemicals the methods for purification and separation presented herein have shown to be of great value. The results will hopefully contribute to a better understanding of the communication among insects, and ultimately to a more environmentally friendly pest control. / Många naturligt förekommande kemiska ämnen finns som två spegelbilder av varandra, ungefär som höger och vänster hand. Dessa kan ha helt olika egenskaper och det är därför viktigt att kunna separera dem. Insekter och andra djur använder olika doftämnen för att kommunicera med varandra, om det är inom samma art kallas de för feromoner. De kan bestå av ett ämne eller en blandning av flera. Dessa doftämnen kan man även använda för att på ett miljövänligt sätt bekämpa skadeinsekter. En fälla med syntetiskt feromon för en viss insekt lockar endast till sig den arten, medan alla andra är opåverkade. Eftersom dessa ämnen ofta finns som spegelbilder där kanske bara den ena är aktiv och den andra rent av frånstötande, måste man kunna separera dem för att framställa ett syntetiskt feromon som är attraktivt. Målet med detta arbete har varit att bestämma feromonet hos olika arter av tallsteklar som kan vara svåra skadedjur på tallskog. De metoder som tagits fram har även tillämpats på några arter av afrikanska fjärilar samt orkidébin från Centralamerika eftersom de använder snarlika doftämnen. Att få fram feromonet från en insekt är lite som att leta efter in nål i en höstack eftersom de ofta bara innehåller några miljarddels gram per individ. Provet behöver först renas, och en del av arbetet i det här projektet har gått ut på att ta fram en lämplig reningsmetod. Huvudfokus har dock varit på att ta fram metoder som kan separera och identifiera det eller de ämnen, och spegelbilder av dessa, som doftämnena består av. När lämpliga metoder tagits fram har extrakt av olika insektsarter analyserats. I några fall är det första gången som deras feromon bestämts i detalj. Resultaten kan förhoppningsvis bidra till en ökad kunskap om insekters sätt att kommunicera, och i slutändan till miljövänligare bekämpning av skadeinsekter.

Micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC) are two of the major capillary electrophoresis (CE) modes that have been interfaced to mass spectrometry (MS) for sensitive and selective analysis of chiral compounds. This research combines these two modes and expands their applications in chiral CE analysis. Chapter 1 is a review of amino acid based molecular micelles used in MEKC-MS for enantioselective analysis over the past five years. In this chapter, a typical MEKC-MS experiment setup as well as detailed standard operating procedure in synthesis of molecular micelles and running a typical MEKC-MS experiment using the molecular micelles is discussed. Chapter 2 described a multivariate MEKC-MS optimization for the simultaneous analysis of two negatively charged model chiral compounds in negative ion mode with molecular micelles. In this chapter, a central composite design (CCD) is used to first construct a series of experiments to optimize all the important MEKC-MS parameters. Next, response surface methodology (RSM) was used to analyze the interactions between the factors, picking up the best separation and detection conditions, predicting the result of the chiral separation/MS detection, and finally running the actual experiment and comparing the chromatographic results with the predicted parameters. Chapter 3 demonstrates a similar multivariate MEKC-MS optimization for analysis of a positively charged model chiral compound in a positive ion mode. The same CCD and RSM methods were used to optimize the separations and MS sensitivity. Chapter 4 describes a chiral analysis of four neutral benzoin derivatives (hydrobenzoin, benzoin, benzoin methyl ether, and benzoin ethyl ether) using MEKC coupled to atmospheric pressure photo-ionization mass spectrometry (APPI-MS). The same multivariate experimental design strategy was used to optimize the MEKC as well as APPI-MS parameters. Simultaneous chiral separation of all four benzoin derivatives was achieved with high detection sensitivity compared to UV-detection. Chapter 5 introduces a novel one-pot synthesis scheme for an acryloyl-terminated, carbamate-linked surfactant-bound monolith with leucine head group and different chain lengths. The method promises to open up the discovery of new amino acid based polymeric monoliths for chiral separations and enhanced chemoselectivity for simultaneous chiral separations and enhanced detection in CEC and CEC-MS. In Chapter 6, five amide-linked surfactant-bound monoliths with different chain lengths and head groups (leucine, valine, and phenylalanine) were synthesized and characterized. Enantioseparation of several test compounds was achieved by CEC using the monolithic columns. One of the chiral surfactant, sodium 11-acrylamidoundecanoyl-L-leucinate (SAAUL), was polymerized in aqueous solution under 60Co radiation to form molecular micelle poly-SAAUL. MEKC experiments were carried out with the poly-SAAUL molecular micelle to separate ten cationic chiral compounds. The result was compared with the CEC separation using the AAUL monolithic column. This study is the first comparison of chiral CEC and MEKC with the same surfactant monomer, which has the capability of forming both chiral stationary phase for CEC and chiral pseudophase for MEKC.

The major innovation of this work is an establishment of a novel chiral separation process using preferential crystallization coupled with a membrane barrier. This hybrid process was proved to be promising from a significant increase in product yield and purity compared to existing chiral separation processes. This work sets up a process design platform to extend the use of this hybrid process to a separation of other mixtures. This novel process especially is a promising alternative for chiral separation of pharmaceutical compounds which include more than fifty percent of approved drugs world-wide. A better performance chiral separation technique contributes to cut the operating cost and to reduce the price of chiral drugs.

Gas chromatography (GC) has been utilized for the study of enantiomer resolution of the atropisomers of PCBs, o,p´-DDD and o,p´-DDT. Different substituents and concentrations of cyclodextrin, capillary dimensions and type of stationary phase films have been investigated to achieve the resolution of as many of the atropisomers on one column as possible. The results indicated that the butyl substitution of 6-hydroxyl and the methyl substitution of 2- and 3-hydroxyl were the most promising for the enantiomeric separation. Using Capillary Electrophoresis (CE), the trimers and monomers of PM-β-CDs were compared for enantiomeric resolution, as well as comparing the cationic PMMA-β-CD with the anionic HS-β-CD. In these studies the trimer did not show an improved resolution for mepivacaine, when compared to the equimolar concentration of the monomer. The cationic CD gave increased resolution values for ibuprofen when compared to the anionic CD. A scheme for reversing enantiomeric elution order of both the basic propranolol and acidic ibuprofen is also presented, with the aim of facilitating the detection of impurities in a high sample loading. The detection of 1% of each enantiomer of propranolol, and 1% of R(-)-ibuprofen, was demonstrated, with elution prior to the tailing peak of the corresponding enantiomer. Dimethylacrylamide-coated capillaries were used in this work, and the stability of this coating was demonstrated, giving a highly reduced electroosmotic flow for up to six months. Enantiomeric baseline separations of omeprazole and 5-hydroxyomeprazole have also been achieved with both CE and Open Tubular Capillary Electrochromatography (OT-CEC) methods. With CE-UV, both a non-aqueous method (using HDMS-β-CD) and an aqueous method (using HS-β-CD) were used for enantiomeric resolution of the two racemates. Resolution of omeprazole was also achieved using CE-Electrospray Ionization-Mass Spectrometry (ESI-MS). In OT-CEC, avidin was immobilized on the inside surface of a fused silica capillary and was employed as chiral selector for the enantiomeric baseline resolution of omeprazole and 5-hydroxyomeprazole.

A malária é a doença que mais mortes causa no mundo. O uso de fármacos antimaláricos representa a solução mais eficaz para o combate e controle da doença e o interesse pelo desenvolvimento de novos fármacos é grande devido a problemas de resistência. Existe uma grande variedade de fármacos antimaláricos, sendo que muitos deles são quirais, vendidos e administrados como mistura racêmica. Nas últimas décadas, houve um aumento no interesse quanto aos aspectos farmacodinâmicos e farmacocinéticos de fármacos quirais, devido ao conhecimento de que um dos isômeros pode ser mais ativo ou mais tóxico que o outro. Sendo assim, tem-se a necessidade do desenvolvimento de métodos analíticos enantiosseletivos, e a eletroforese capilar tem emergido como uma técnica de separação quiral com alto poder de resolução. Além disso, a inexistência de métodos oficiais para fármacos antimaláricos e os poucos estudos que relatam aplicações na análise de formulações farmacêuticas, demandam o desenvolvimento e validação de novos métodos para tal finalidade. Este trabalho tem como objetivo o desenvolvimento de dois métodos analíticos, não quiral e quiral, para a determinação de fármacos antimaláricos em formulações farmacêuticas por eletroforese capilar. O método não quiral utilizou eletroforese capilar de zona, sendo otimizado para a separação de 7 fármacos antimaláricos (cloroquina, hidroxicloroquina, primaquina, quinidina, quinina, quinacrina e mefloquina) com um eletrólito composto por 45 mmol L-1 de tampão citrato, pH 4,50, e apresentou um tempo de análise de 10 minutos, permitindo a separação dos diastereoisômeros quinina e quinidina sem a adição de aditivos. O método quiral utilizou eletroforese capilar de zona modificada por ciclodextrina e foi otimizado para separação enantiosseletiva de cloroquina, mefloquina, hidroxicloroquina, primaquina, quinina e quinidina com um eletrólito composto por 50 mmol L-1 de tampão citrato, e 2% de S-&#946;-CD, pH 2,7. A separação dos fármacos antimaláricos e seus enantiômeros foi alcançada com tempo de análise de 12 minutos. Os métodos desenvolvidos foram validados de acordo com os protocolos oficiais, apresentando características adequadas e foram aplicados na determinação de cloroquina, hidroxicloroquina e mefloquina em formulações farmacêuticas. Como figuras de mérito para o método não quiral, tem-se: linearidade (R2 > 0,99), LD (7,43 - 24,4 &#181;mol L-1), LQ (22,5 - 73,8 &#181;mol L-1), precisão intermediária (0,76 - 1,7% RSD), recuperação (97,8 - 102,2%). Para o método quiral, tem-se: linearidade (R2 > 0,99), LD (7,43 - 9,58 &#181;mol L-1), LQ (22,8 - 29,0 &#181;mol L-1), precisão intermediária (0,50 - 1,8% RSD), recuperação (97,7 - 102,5%). Um ensaio de robustez para ambos os métodos foi realizado para comparar os resultados obtidos aplicando-se pequenas variações de tensão e temperatura. Observou-se que não existe uma diferença significativa entre os resultados, a um nível de confiança de P = 95 %. / Malaria is a disease that causes a large number of deaths worldwide. The use of antimalarial drugs is the most effective solution to combat and control the disease and interest in the development of new antimalarial drugs is still of great importance due to resistance issues. There is a wide variety of antimalarial drugs, many of which are chiral, sold and administered as racemic mixtures. In recent decades there has been an increased interest regarding the pharmacodynamic and pharmacokinetic aspects of chiral drugs, due to the knowledge that one of the isomers can be more active or more toxic than the other. Thus, there is a need for the development of enantioselective analytical methods, and capillary electrophoresis has emerged as a chiral technique separation with high resolving power. Moreover, the lack of official methods for antimalarial drugs and the few studies reporting applications in the analysis of pharmaceutical formulations, demands the development and validation of new methods for this purpose. This work aims at the development of two analytical methods, non-chiral and chiral, for the determination of antimalarial drugs in pharmaceutical formulations by capillary electrophoresis. The non-chiral method used capillary zone electrophoresis, being optimized for the separation of 7 antimalarial drugs (chloroquine, hydroxychloroquine, primaquine, quinidine, quinine, quinacrine and mefloquine) with an electrolyte consisting of 45 mmol L-1 of citrate buffer, pH 4.50, and had an analysis time of 10 minutes, allowing separation of isolated diasteroisomers quinine and quinidine without any additives. The chiral method used capillary zone electrophoresis modified by cyclodextrin and was optimized for enantioselective separation of chloroquine, mefloquine, hydroxychloroquine, primaquine, quinine and quinidine with an electrolyte consisting of 50 mmol L-1 citrate buffer and 2% S-&#946;-CD, pH 2.7. The separation of the antimalarial drugs and their enantiomers was achieved in less than 12 minutes. The proposed methods were validated following official protocols, with adequate results and were used for determination of chloroquine, hydroxychloroquine, and mefloquine in pharmaceutical formulations. Figures of merit for the non-chiral method include: linearity (R2> 0.99), LD (7.43 to 24.4 &#181;mol L-1), LQ (22.5 to 73.8 &#181;mol L-1), intermediary precision (0.76 to 1.7% RSD), and recovery (from 97.8 to 102.2%). For the chiral method, we have: linearity (R2> 0.99), LD (7.43 to 9.58 &#181;mol L-1), LQ (22.8 to 29.0 &#181;mol L-1), intermediary precision (0.50 to 1.8% RSD), and recovery (from 97.7 to 102.5%). For both methods a robustness test was performed to compare the results obtained by applying slight variations in voltage and temperature. It was observed that there is no significant difference between the results, a confidence level P = 95%.

The aim of this work was to develop and to optimize HPLC method for enantioseparation of newly synthesized derivatives of amino acids. The set of these analytes contained four N- blocked derivatives of D,L-Phenylalanine, three N-unblocked derivatives of D,L- Phenylalanine, , one methylated derivative of D,L-Tyrosine and D,L-Tyrosine. Two separation modes i.e. reversed phase and polar-organic modes and two columns i.e. Chirobiotic® T and Chirobiotic® T2 were used. Chiral stationary phases of these columns were composed of macrocyclic antibiotic teicoplanin coated on silica gel support. As mobile phases in revesed phase mode, methanol and acetate buffer were used with Chirobiotic® T column. The most suitable concentration were 20 mM and the most suitable pH value were 4.00. Under the above mentioned conditions eight analytes of ten were separated and the optimal conditions were found. Polar-organic mode and Chirobiotic® T column were suitable only for enantioseparation of three N-blocked derivatives of D,L- Phenylalanine. Mobile phases were composed of methanol with small additions of triethylamine and acetic acid. Chirobiotic® T2 column in reversed phase mode was not suitable for tested set of analytes. Only partial separation of D,L-Tyrosine derivative and one N-unblocked D,L-Phenylalanine...

This diploma thesis is focused on the comparison of enantioselective potential of chiral stationary phases based on derivatized cellulose by high performance liquid chromatography (HPLC). Polysaccharide - based chiral stationary phases are suitable choice for enantioseparation of various chiral compounds. In this work Chiralpak IB column containing cellulose tris(3,5-dimethylphenylcarbamate) selector immobilized onto silica gel and Chiralpak IC column containing cellulose tris(3,5- dichlorophenylcarbamate) selector immobilized onto silica gel were used. Their ability of enantioseparation was tested on 28 structurally different chiral analytes. Reversed- phase separation mode was used for enantioseparation. Mobile phases were composed of organic modifiers acetonitrile or methanol and the aqueous part was selected according to the nature of analytes. Acidic analytes were separated in mobile phases containing aqueous solution of formic acid (pH 2.1). Analytes of bifunctional nature were separated in the presence of aqueous solution of formic acid (pH 2.1), 10 mM ammonium acetate buffer (pH 8.8) or 100 mM solution of KPF6. Mobile phases containing 10 mM ammonium acetate buffer (pH 8.8) or 100 mM solution of KPF6 were used for enantioseparation of alkaline chiral analytes. Twelve chiral analytes were...