The diploma thesis is focused on the development of in-line micro-electromembrane extraction (EME) coupled to capillary electrophoresis (CE) for the analysis of selected drugs in body fluids. Up to now, direct coupling of EME to CE has been demonstrated on diluted river samples only [1]. Although the published set-up has been implemented within a commercial CE it suffers from several drawbacks that can have a negative impact on the analysis of samples with higher complexity. The instrumental arrangement presented in this thesis eliminates these deficiencies. The experimental part is based on the optimization of fundamental extraction and separation conditions for the analysis of model basic drugs (nortriptyline, haloperidol, loperamide) with the use of transient isotachophoresis (tITP) principle. The extraction conditions were optimized for electro-driven transport of basic analytes from complex matrices (urine) through free liquid membrane followed by injection step utilized by electrokinetic supercharging (EKS), which focused target analytes into the CE capillary. Optimized conditions have been applied on blood in the form of dry blood spots, which are highly attractive samples in the current clinical analysis. The repeatability of the measurements was

This thesis describes studies on the analysis of arsenic (As) in human biological samples, mainly urine but also hair and fingernails using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry (GF-AAS). The relationship between ethnicity and arsenic metabolism was investigated for the first time for a population in the United Kingdom. This investigation has been carried out through comparative analysis of arsenic in human urine, hair and fingernails in volunteers from three different ethnic groups (Whites, Asians and Somali Black-Africans) who are only exposed to background levels of arsenic. Results obtained with 63 volunteers showed ethnic differences in urinary arsenic excretion as well as differences in arsenic levels in fingernail samples. The averages of total arsenic levels for the Somali Black-Africans (urine 7.2 µg/g creatinine; fingernails 723 µg/kg) are significantly (P< 0.05) different from both the Asians (urine 20.6 µg/g creatinine; fingernails 153.9 µg/kg) and Whites (urine 24.5 µg/g creatinine; fingernails 177.0 µg/kg). The Somali group also shows a higher percentage (50%) of dimethylarsinate (DMA) and a lower percentage (48%) of arsenobetaine (AB), compared to Asians (16% DMA and 83% AB) and Whites (22% DMA and 77% AB). The effect of fasting on urinary arsenic species distribution was also investigated by monitoring urine samples from 29 Ramadan fasting volunteers, with each volunteer providing a sample at the beginning (RF1) and at the end (RF2) of an approximately 12 hours fast. The results obtained showed the frequency of MA detection for RF2 was 12 and 2-fold higher than for the non-fasting and RF1 groups, respectively. This suggests fasting may alter the pattern of arsenic metabolism and excretion. However, there was no significant difference (P> 0.05) in the average of total level of arsenic for RF1 (18.3 µg/g creatinine) and RF2 (17.7 µg/g creatinine). A relationship between excretion of arsenic and selenium in individuals exposed to background levels of arsenic and selenium was investigated through analysis of urine samples from 93 volunteers from Leicester, UK. A positive correlation between arsenic and selenium was found and the As:Se ratio was 0.7 ± 0.4. The intra-individual variation of As:Se ratio does not alter significantly over time, as determined by monitoring urine samples from a volunteer over a period of one year. Furthermore, within a single day, with urine samples collected at the beginning and after a 12-hour fast, the As:Se ratio was found to be similar (0.7 ± 0.5). These findings suggest a close relationship between these two metalloids, the biological significance of which needs to be explored in the future.

Salmonella (S.) enterica est reconnue comme le principal agent causal de la salmonellose chez l’homme et les animaux. La distribution épidémiologique de cette infection implique souvent des régions géographiques éloignées; il est ainsi nécessaire de posséder des méthodes fiables afin de pouvoir discriminer des souches responsables d’une épidémie. En raison des limites de la méthode de typage sérologique, de nombreuses méthodes de génotypage moléculaire ont été développées. En particulier, la méthode par PCR couplée à l’électrophorèse en champ pulsé, qui est utilisée pour la séparation et la caractérisation des molécules d’ADN, et le génotypage par analyse de répétition en tandem polymorphe ou MLVA (Multiple-Locus VNTR Analysis), sont des méthodes modernes qui permettent d’étudier le polymorphisme et la diversité génétique des souches de S. enterica liées à une épidémie. Dans notre étude, onze marqueurs contenant des régions de répétitions en tandem polymorphique (VNTR : Variable Number Tandem Repeats) sélectionnés à partir du génome de S. enterica Typhimurium LT2 ont été utilisés pour évaluer la diversité génétique de 206 souches de S. enterica sélectionnées entre 2001 et 2007. Ces salmonelles sont représentées par 31 sérotypes, ont été isolées à partir de trois sources: hommes, aliments cuits et crus. Chaque souche a été isolée à partir d’échantillon unique et n’était liée à aucun épisode d’intoxication alimentaire ou à une épidémie de salmonellose connue. La technique MLVA a permis de sous typer 107 génotypes regroupés dans un dendrogramme en deux branches distintes dont la première et constituée par Salmonella Typhi et la deuxième par les 30 autres sérotypes liés entre eux par un ancêtre commun. Parmi les sérotypes, quatre ont été répartis dans deux à cinq branches phylogénétiques. La représentation de la variation allélique des sérotypes de S. enterica a utilisé l’arbre minimum couvrant sans racine. Des variations alléliques pour des sérotypes de S. enterica précédemment ou nouvellement décrits ont été identifiées et des variants génétiques ont été répartis en types ou en variants MLVA à loci uniques, en variants différents par un locus (SLVs), en variants différant par deux loci (DLVs) et des variants différant par plus de deux loci. Quatre marqueurs (STTR3, STTR5, STTR8 et Sal20) ont présenté un indice de diversité élevé (DI> 0,80). En résumé, la technique MLVA peut être appliquée pour étudier le profil génétique de S. enterica avec une grande diversité de sérotypes. / Epidemiological distribution of this infection often involves large areas of geographically distant, and reliable methods to discriminate strains responsible for an epidemic are necessary. Due to limitations of serological typing method, many molecular genotyping methods have been developed. Some molecular methods and their applications are: PCR coupled to PFGE, which is used for the separation and characterization of molecular profiles, and MLVA (Multiple-Locus VNTR Analysis) genotyping, or so called analysis of polymorphic tandem repeats are modern methods that allow study of the polymorphic genetic diversity and discrimination of Salmonella strains related or unrelated to epidemics. In our study, 11 markers containing polymorphic tandem repeats (VNTR: Variable Number Tandem Repeats) selected from the genome of S. enterica Typhimurium LT2 were used to assess the genetic diversity of 206 strains of S. enterica selected in 2001-2007 period. These are represented by 31 Salmonella serotypes selected from three sources: human, food and animals. Each strain was isolated from a single sample and was not related to an episode of epidemic of salmonellosis. The technique MLVA has allowed subtyping of 107 genotypes grouped in a dendrogram into two distinct dispersion trees, the first for serotype Typhi and the second for the other 30 serotypes devided within two subgroups derived from a common ancestor. Four serotypes were dispersed in two to five phylogenetic branches. The representation of the allelic variation of serotypes of S. enterica used a minimum spanning tree. Allelic variations in the serotypes of S. enterica, previously or newly described, were identified and genetic variants were distributed in MLVA types in unique locus variants, in single locus variants or in variants different by a locus (SLVs), in variants different by two loci (DLVs) and in different variants by more than two loci. Four markers (STTR3, STTR5, STTR8, and Sal20) have shown a high Diversity Index (DI> 0.80). In summary, MLVA can be applied to study the genetic profile of S. enterica with a wide variety of serotypes.

Métodos espectrofotométricos em condições estacionária e em fluxo para determinação de Zn(II) foram desenvolvidos com base na formação do complexo de Zn(II) com di-2-piridil cetona saliciloilhidrazona (DPKSH), em meio de etanol-água 50% (V/V) e pH 4,5. Os complexos de Zn(II) com DPKSH foram caracterizados utilizando diferentes técnicas e as respectivas constantes de formação (&#946;l 1,07x105 e &#946;2 1,05x1010) e absortividades molares, em 376 nm, (&#949;l 1,1x104 e &#949;2 4,5x104) determinadas. Os parâmetros para o método em condição estacionária foram otimizados e, a seguir, construiu-se a respectiva curva analítica. Obteve-se uma faixa de linearidade (lei de Beer) de (0,0293 a 2,01)x10-5mol. L-1, absortividade molar média (em 376 nm, em meio de etanol-água 50% (V/V) e pH 4,5) de 4,83x104 mol-1.L.cm-1 e limite de detecção igual a 1,24x10-7 mol.L-1 (8,11 &#181;g.L-1). Avaliou-se a interferência de 43 íons. Alguns destes que apresentaram interferência positiva são comuns em preparações farmacêuticas e amostras biológicas, porém tal interferência pode ser facilmente eliminada. Aplicou-se o método desenvolvido à preparações farmacêuticas e amostras biológicas. Comparando-se os resultados com a técnica de absorção atômica com chama, obteve-se grande concordância. O método espectrofotométrico em fluxo foi desenvolvido a partir de alguns parâmetros previamente estudados e estabelecidos. Construiu-se a curva analítica e obteve-se uma faixa de linearidade de (0,332 a 7,04)x 10-5 mol.L-1 e limite de detecção 7,46x10-7 mol.L-1 (48,8 &#181;g.L-1). Aplicou-se o método em preparações farmacêuticas e comparando-se os resultados com a técnica de absorção atômica com chama observou-se grande concordância. Com a finalidade de aplicar o método em fluxo a amostras biológicas, foi acoplada ao sistema uma coluna com resina de troca iônica no lugar da alça de amostragem. Após avaliar alguns parâmetros, construiu-se a curva analítica que apresentou uma faixa de linearidade de (0,126 a 3,15)x10-5 mol.L-1 e limite de detecção 2,12x10-7 mol.L-1 (13,9 &#181;g.L-1). / Spectrophotometric methods in stationary and flow conditions were developed for Zn(II) determination based on the complex formation of Zn(II) with di-2-pyridyl ketone salicyloylhydrazona (DPKSH), in ethanol-water 50% (V/V) medium and pH 4.5. The complexes Zn(II)/DPKSH were characterized by several methods and the respective formation constants (&#946;1 1.07x105 and &#946;2 1.05x1010) and molar absorptivities, in 376 nm, (&#949;1 1.1x104 and &#949;2 4.5x104) were determinated. Parameters for the stationary method were optimized and, the analytical curve was obtained. A linear behavior (Beer\'s law) was verified in the range (0.0293 and 2.01)x10-5mol.L-1, the medium molar absorptivity (in 376 nm, ethanol-water 50% (V/V) medium and pH 4.5) and the detection limit are 4.83x104 mol-1.L.cm-1 and 1.24x10-7 mol.L-1 (8.11 ppb), respectively. The interference of 43 ions was evaluated. Some of them that showed present a positive interference are commom in pharmaceutical formulations and biological samples, but such interference could be easily eliminated. The developed method was applied to pharmaceutical formulations and biological samples. The results complied well with atomic absorption (flame atomization) technique. A spectrophotometric method in flow was developed upon some previously established parameters. The analytical curve was determined and a linear range was verified between (0.332 to 7.04)x10-5 mol.L-1, and the detection limit 7.46x10-7 mol.L-1 ( 48.8 &#181;g.L-1). This method was applied to pharmaceutical formulations and the results showed a good agreement with atomic absorption (flame atomization) technique. Aiming at employing the flow method to biological samples an ion-exchange resin column was adapted substituting the sampling handle. After evaluating some parameters the analytical curve was determined and it displayed a linear range of (0.126 to 3.15)x10-5 mol.L-1, and detection limit 2.12x10-7 mol.L-1 (13.9 &#181;g.L-1).

<p>This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.</p><p>Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. </p><p>Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).</p><p>Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.</p><p>Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.</p>

<p>Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems.</p><p>Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions.</p><p>An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD < 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis.</p><p>Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (> 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers.</p><p>Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).</p>

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper. Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil). Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE. Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.

Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems. Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions. An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD &lt; 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis. Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (&gt; 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers. Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).

The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices. Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 734

Phospholipids are an important group of polar lipids constituting the main component of cell membranes. Their proportion may vary depending on many factors of the surrounding environment in which the cell is located. Determination of membrane phospholipids is essential in many scientific, industrial and economic sectors. The aim of this work has been to develop a separation method for determination of membrane phospholipids by which it was possible to analyze phosphorylated parts of phospholipids and fatty acids from one sample. Comprehensive gas chromatography with mass detection (GC×GC-MS) was chosen for the assay. This method allows the separation of the entire sample on two serially connected different columns, among which is the interface called modulator. The preparation of the sample includes a cleavage of the phospholipid molecule by the enzyme phospholipase C, which released the phosphorylated polar headgroups. These polar parts had to be derivatized before analysis. The principle of the chosen derivatization consisted in the use of two different silylation agents (hexamethyldisilazane and N,O-Bis(trimethylsilyl) trifluoracetamide) in two steps. Conditions were selected for efficient separation of the silylderivatives of phosphorylated headgroups using GC×GC-MS using a cryogenic...