On-line and off-line enrichment techniques combined with capillary electromigration separation methods in the analysis of highly hydrophilic analytes in biological and environmental samples

In der vorliegenden Arbeit werden kapillarelektromigrative Trennmethoden als alternative Strategien zu existierenden Verfahren zur Bestimmung extrem hydrophiler Analyte untersucht. Es wird gezeigt, dass die entwickelte Methodik (unter Einschluss unterschiedlicher on-line und off-line Anreicherungstechniken) sich sowohl zur Analyse von biologischen Flüssigkeiten als auch zur Analyse von Umweltproben eignet. Für diese Studie wurden daher als polare Analyte im Urin enthaltene Nukleoside und als mögliche Umweltkontaminanten diskutierte α-Aminocephalosporine als Modell-Analyte gewählt. Nukleoside verfügen über eine cis-Diol-Struktureinheit, die es ihnen bei alkalischem pH ermöglicht, negativ geladene Komplexe mit Tetrahydroxyborat-Ionen zu bilden. Über die effektive elektrophoretische Mobilität wird gezeigt, dass der Komplexierungsgrad sogar bei sehr geringer Tetraborat-Konzentration (2,5 mmol L-1) nahe 1 liegt. Weil die für die untersuchten Nukleoside mit Kapillarzonenelektrophorese (CZE) unter Verwendung eines Borat-Borsäure-Puffers erreichbare Auflösung nicht ausreichend ist, sind zur vollständigen Trennung der untersuchten Nukleoside alternative Herangehensweisen erforderlich: (i) ein anderer Trennmodus oder (ii) Pufferadditive, die eine veränderte Trennselektivität ermöglichen. In einem ersten Schritt wird daher zunächst ein Verfahren entwickelt, welches mizellare elektrokinetische Chromatographie (MEKC) mit dem kationischen Tensid 1-Tetradecyl-3-methylimidazoliumbromid (C14MImBr) in Gegenwart von Tetrahydroxyborat zur Trennung von in Urin enthaltenen Nukleosiden (nach Konversion in eine ionische Spezies) nutzt. Eine vollständige Trennung der hydrophilen Metabolite ist bereits bei geringer Konzentration des kationischen Tensids C14MImBr (20 mmol L-1) möglich (Hintergrundelektrolyt 5 mmol L-1 Dinatriumtetraborat, pH 9,38). Es wird untersucht, welche Gleichgewichte bei Verwendung von C14MImBr als kationischem Tensid für die Trennung der im Urin enthaltenen Nukleoside und die Steuerung des Retentionsfaktors (entgegengesetzt geladene PSP) verantwortlich sind. Es wird gezeigt, dass die negativ geladenen komplexierten Nukleoside mit den C14MImBr Mizellen hauptsächlich elektrostatische Wechselwirkungen eingehen, während hydrophobe Wechselwirkungen als vernachlässigbar betrachtet werden können. Diesem Ergebnis entspricht, dass erhöhte Retentionsfaktoren sowohl mit reduzierter Borat-Konzentration als auch mit heraufgesetztem pH-Wert erhalten werden. Unter Bedingungen maximaler Nukleosid-Mizell-Wechselwirkung (quantifiziert durch Ermittlung der Gleichgewichtskonstanten) wird unter Rückgriff auf kombinierte on-line Anreicherungstechniken ein optimiertes und vollständig validiertes MEKC Verfahren zur Identifizierung und Quantifizierung von Nukleosiden in Urin-Proben entwickelt. Es wird gezeigt, dass “pseudostationary ion-exchanger” sweeping hauptsächlich für den beobachteten Anreicherungsprozess verantwortlich ist. C14MImBr kann jedoch aufgrund der niedrigen Retentionsfaktoren für Adenosin (Ado) and Cytidin (Cyd) nicht effizient für das Sweeping dieser Analyte eingesetzt werden. Außerdem komigrieren diese Nukleoside mit Bestandteilen der Urin-Matrix. Daher wurde zusätzlich das anionische Tensid SDS für ein Alternativverfahren zur Bestimmung von Ado und Cyd nach Überführung in kationische Spezies (unter sauren pH-Bedingungen) unter Verwendung von “pseudostationary ion-exchanger” sweeping als on-line Anreicherungsprinzip herangezogen. Dieses Alternativverfahren wurde erfolgreich angewendet, um Ado und Cyd in Urinproben zu bestimmen. Ergänzend wurde nachgewiesen dass mit einem Hintergrundelektrolyten, der ein Alkyl- oder Arylboronat zusätzlich zu C14MImBr enthält, die Retentionsfaktoren aller untersuchten Nukleoside signifikant erhöht sind. Diese Verschiebung der Retentionsfaktoren zu höheren Werten wird der Wechselwirkung mit der zusätzlich über die Alkyl/Aryl-Gruppe des Boronats eingeführten hydrophoben Struktureinheit zugeschrieben (Komplexbildung des Boronats mit der cis-Diol-Gruppe des Nukleosids). Es wird gezeigt, dass die entwickelten Optimierungsstrategien in validierten Verfahren genutzt werden können, die eine Identifizierung und Quantifizierung der untersuchten Nukleoside in Urin-Proben ermöglichen (Nachweisgrenzen im Bereich von 0,1-0,2 mg L-1). In einem zweiten Schritt wird 2-Hydroxypropyl-β-cyclodextrin (2-HP-β-CD) als Puffer-Additiv in einem Borat-Borsäure-Puffer verwendet. Dieses Additiv ermöglicht eine Modifizierung der Trennselektivität und eine vollständige Trennung der untersuchten Nukleoside via CZE. Unter Nutzung der sehr hohen Komplexbildungskonstante zwischen Nukleosid und Tetrahydroxyborat unter gleichzeitiger Verwendung des selektivitätsmodifizierenden Additivs 2-HP-β-CD wird ein sehr empfindliches CZE-Verfahren entwickelt, dessen Nachweisgrenzen 10-40 μg L−1 erreichen. Dieses Verfahren stützt sich auf eine mehrstufige on-line Anreicherung unter Einbeziehung von dynamic pH junction, borate sweeping und large volume sample stacking (LVSS). Eine Validierung wird gemäß den Leitlinien der ICH durchgeführt. Das validierte Verfahren wird erfolgreich auf die Identifizierung und Quantifizierung von Nukleosiden in gespiktem und ungespiktem Urin angewandt. Die Ergebnisse dieses Teils der Untersuchungen werden nachfolgend auf die Analyse ausgewählter α-Aminocephalosporine in Oberflächenwasser übertragen. Unter Hinzuziehung einer hochempfindlichen Detektionsmethode (Laser-induzierte Fluoreszenz (LIF) Detektion) wird CZE mit LVSS-sweeping zur Analyse von Cefalexin und Cefadroxil in gespiktem Lahnwasser genutzt (Nachweisgrenzen 5-8 ng L-1)

Analysis of N-nitrosames in water using micellar electrokinetic chromatography (MEKC) and development of online and offline methods of extraction and enrichment of neutral polar analytes

Evaluation of the quality of the environment is essential for human wellness as pollutants in trace amounts can cause serious health problem. Nitrosamines are a group of compounds that are considered potential carcinogens and can be found in drinking water (as disinfection byproducts), foods, beverages and cosmetics. To monitor the level of these compounds to minimize daily intakes, fast and reliable analytical techniques are required. As these compounds are relatively highly polar, extraction and enrichment from environmental samples (aqueous) are challenging. Also, the trend of analytical techniques toward the reduction of sample size and minimization of organic solvent use demands new methods of analysis. In light of fulfilling these requirements, a new method of online preconcentration tailored to an electrokinetic chromatography is introduced. In this method, electroosmotic flow (EOF) was suppressed to increase the interaction time between analyte and micellar phase, therefore the only force to mobilize the neutral analytes is the interaction of analyte with moving micelles. In absence of EOF, polarity of applied potential was switched (negative or positive) to force (anionic or cationic) micelles to move toward the detector. To avoid the excessive band broadening due to longer analysis time caused by slow moving micelles, auxiliary pressure was introduced to boost the micelle movement toward the detector using an in house designed and built apparatus. Applying the external auxiliary pressure significantly reduced the analysis times without compromising separation efficiency. Parameters, such as type of surfactants, composition of background electrolyte (BGE), type of capillary, matrix effect, organic modifiers, etc., were evaluated in optimization of the method. The enrichment factors for targeted analytes were impressive, particularly; cationic surfactants were shown to be suitable for analysis of nitrosamines due to their ability to act as hydrogen bond donors. Ammonium perfluorooctanoate (APFO) also showed remarkable results in term of peak shapes and number of theoretical plates. It was shown that the separation results were best when a high conductivity sample was paired with a BGE of lower conductivity. Using higher surfactant concentrations (up to 200 mM SDS) than usual (50 mM SDS) for micellar electrokinetic chromatography (MEKC) improved the sweeping. A new method for micro-extraction and enrichment of highly polar neutral analytes (N-Nitrosamines in particular) based on three-phase drop micro-extraction was introduced and its performance studied. In this method, a new device using some easy-to-find components was fabricated and its operation and application demonstrated. Compared to conventional extraction methods (liquid-liquid extraction), consumption of organic solvents and operation times were significantly lower

Applications of chiral selectors and replaceable supports for capillary electrophoretic separations

The popularity of capillary electrophoresis (CE) as a separation technique has been established over the years. CE offers the advantages of high resolution, high separation efficiency, fast approaches of method development, a range of operational modes and low consumption of reagents. The strategy employed here for the development of chromatographic separations involved the utilization of experimental designs, multi-linear regression and response surface methodology to build empirical models that related the chromatographic quality to the factors influencing the separation. Separation of Nitrofuran antibiotics (NFAs) and their metabolites (NFMs) by using micellar electrokinetic capillary chromatography was successfully completed. The best conditions found to give optimum resolution from the optimization study was pH 9.0, 80 mM SDC concentration, 16 kV with running buffer consisting of 20 mM borate and 20 mM phosphate concentration using a 73 cm x 75 µm column, resulting in completely resolved NFAs and NFMs within 16 min. It is interesting that all the compounds can be reliably separated with the one mixture, and single CE condition. Whilst all antibiotics have shorter migration time than their respective derivatised metabolites, as a group apart from nitrofurantoin the antibiotics elute before the metabolites. The analytical figures of merit for CE analysis exhibited excellent reproducibility of absolute and relative migration times, and acceptable reproducibility of relative response areas. Successful separation of metabolite derivatives was achieved when the developed method was applied to a spiked prawn sample. The chiral separation of Triadimenol was successfully completed using micellar electrokinetic capillary chromatography. The best conditions found to give optimum resolution from the optimisation study were pH 6.0, 20% methanol, 50 mM SDS concentration, 18 kV with running buffer consisting of 20 mM borate and 20 mM phosphate concentration using a 64.5 cm x 50 µm column, resulted in baseline resolution of all Triadimenol isomers within 18 min. The optimised separation conditions were applied to a blank grape sample and to a spiked grape sample. No peaks were observed in the blank grape sample whereas the spiked grape sample had two diastereoismer peaks with poor detection sensitivity. Increase in detection sensitivity is necessary to determine the possibility of resolution of all the isomers of Triadimenol, in the spiked grape sample and the blank. Online preconcentration techniques were attempted to for Triadimenol isomer separation. When using online preconcentration technique of sweeping, a 30-fold increase in detection sensitivity of Triadimenol was observed compared to MEKC mode. However enantiomer separation was not possible with sulfated-β-CD chiral selector. The best conditions were found to be pH 2.5, 50 mM SDS concentration, -20 kV with running buffer consisting of 20 mM phosphate concentration, using a 64.5 cm x 50 µm column, resulting in diastereoisomer separation within 8 min. Final stage of the project was to create stationary phase beds in capillaries and micro-channels that could be removed and re-created, thus providing a fresh stationary phase. The replaceable stationary phase (RSP) can be used as an operating mode of CE/CEC. Preparation of reversible stationary phase (RSP) inside the capillary column was successfully performed using low methoxy pectin (LMP). LMP renders a capability of reversible thermogelation. Electroosmotic flow (EOF) and sufficient hydrophobicity of LMP gel allow separation of analyates. The porosity of LMP RSP was adequate to support EOF. Successful separation with good reproducibility of areas and migration times was obtained for Caffeine, Aspartame, Benzoic acid, Saccharine (CABS) mixture and NFAs. After performing continuous analyses, the aging of RSP was observed. Temperature was the 'switch' which applied to remove aged RSP. RSP was recreated for further analysis of analytes. RSP was UV transparent, capable of handling various analytes and diff erent buffer electrolytes including aqueous-organic solvents

Predicting Human Intestinal Absorption Using Chromatography and Spectroscopy

New drug entities (NDE) are constantly being developed with most of them intended for oral administration. For this reason, there is a need to estimate their absorption in order to save time and money that would be lost if the drug enters the clinical stage and is then found to exhibit poor absorption. For many years, the use of animals was the most abundant method for studying pharmacokinetics to predict parameters such as intestinal absorption. However, these methods are time consuming, and expensive as well as being ethically unfavourable. As a result, developing other methods to evaluate a drug’s pharmacokinetics is crucial. The aim of this work was to develop in vitro methods for estimation of human intestinal absorption (%HIA) to replace the use of the aforementioned, less favourable methods involving the use of animals. Among the developed methods in this thesis is a unique type of chromatography known as micellar liquid chromatography (MLC) using biosurfactants such as bile salts as a mobile phase. Furthermore, studies investigated the effect of a change in the stationary phase in addition to investigating the effect of the change in temperature on the elution of the analysed compounds. It was found that R2PRED for the developed MLC methods was in the range of 43.3 % - 91.12 %. Another developed method was a spectrophotometric method based on the use of the solubilising effects of bile salts, as well as their binding to compounds. Therefore, two spectrophotometric methods were developed, a solubilisation method and a double reciprocal method, and used in the prediction of %HIA. It was found that the solubilisation method had a better predictability for %HIA than that of the double reciprocal method where R2PRED was found to be 82.32 % and 61.90 % respectively. Finally, a permeation method was developed using the ability of NaDC to form a hydrogel under specific conditions and applying the investigated drugs in an infinite dose to the prepared hydrogels. This facilitated the determination of permeability coefficients (Kp) that were then used in the prediction of %HIA using the obtained model. The two developed permeation methods were found to have close values of R2PRED for % HIA where R2PRED of the permeation method using flow through cells was found to be 79.8 % while that of the permeation method using Franz cells was found to be 79.67 %. In summary, this work reports several unique models for the in vitro prediction of human intestinal absorption, potentially removing the need for animal testing to predict %HIA

Synthesis, characterization, and applications of novel pseudostationary phases in micellar capillary electrophoresis for separation of chiral and archiral compounds h[electronic resource]

The research presented in this dissertation involves the synthesis, characterization, and the use of novel surfactants, including both micelles and vesicles, as pseudostationary phases in micellar capillary electrophoresis (MCE) for the separation of achiral and chiral compounds. Separation of environmental pollutants such as 2 to 6-ring polycyclic aromatic hydrocarbons (PAHs) was achieved using poly(sodium undecylenic sulfate). A baseline separation of all 16 PAHs was possible for the first time in MCE by a single-surfactant system. In addition, a surfactant with a phosphated head group, i.e., di(2-ethylhexyl)phosphate (DEHP), was also introduced as a novel pseudostationary phase for separation of 21 weakly and strongly hydrophobic neutral compounds. Acetonitrile at a concentration of 30% (v/v) in combination with 100 mM DEHP gave optimum separation for a mixture of 21 benzene derivatives and PAHs in under 16 minutes. An application of cyclodextrin modified MCE was used for separation of twelve mono-methylbenz[a]anthracene positional isomers using a combination of poly-SUS and b-CD, g-CD or b-CD derivatives. Tartaric acid based vesicular surfactants were synthesized and utilized as novel pseudostationary phases in MCE. Linear solvation energy relationship model was applied to understand the fundamental nature of the solute-surfactant interactions and to investigate the effect of the type and the composition of pseudostationary phases on the retention mechanism and selectivity in MCE. The solute size has the largest influence on the solute retention in MCE. The hydrogen bond accepting ability of the solute is the second most important factor on retention and is the largest contributor towards the selectivity differences between pseudostationary phases used. Another study conducted was the synthesis of sodium N-undecanoyl L-leucinate and co-polymerization of SUL with SUS to make a variety of co-polymerized molecular micelles having both chiral (leucinate) and achiral (sulfate) head groups. These surfactants were applied as novel pseudostationary phases in MCE for separation of chiral and achiral compounds. Aggregation numbers and partial specific volumes of these surfactant systems were determined using fluorescence spectroscopy and densitometry, respectively. Thermodynamic parameters such as enthalpy, entropy, and Gibbs free energy changes upon transfer of analyte(s) from aqueous phase to the pseudostationary phase were also determined

Preconcentration strategies in capillary electrophoresis for the determination of pharmaceutical and personal care products

L'objectiu principal d'aquestaTesi Doctoral és el desenvolupament de diferents estratègies per disminuir els límits de detecció de l’electroforesicapil•lar per a la determinació de compostos farmacèutics i els productes de cura personal. Aquestes estratègies es basen en les tècniques de preconcentració electroforètiques i cromatogràfiques, i l'ús de l’espectrometria de masses com a sistema de detecció. Com a tècniques de preconcentració electroforètiques s'han estudiat les tècniques de samplestacking i sweeping, i com a tècnica de preconcentració cromatogràficas’ha avaluat l'acoblament en línia entre l'extracció en fase sòlida i l'electroforesicapil•lar (In-line SPE-CE). Entre elsPPCPs, aquesta tesi doctoral es centra específicament en els antiinflamatoris no esteroïdals(AINE), els parabens i els filtres ultraviolats. Un altre dels objectius d'aquestaTesi Doctoral és estudiarl’aplicabilitat de les metodologies desenvolupades per a l'anàlisi de mostres ambientals per determinar PPCP.The main objective of this Doctoral Thesis is the development of different strategies to decrease the detection limits of capillary electrophoresis for the determination of pharmaceutical and personal care products. These strategies are based on electrophoretic and chromatographic preconcentration techniques, and the use of mass spectrometry as a detection system. The electrophoretic preconcentration techniques studied included sample stacking techniques and sweeping while the chromatographic preconcentration technique evaluated was in-line coupling between solid phase extraction and capillary electrophoresis. With respect to PPCPs, this Doctoral Thesis focuses specifically on non-steroidal anti-inflammatory drugs (NSAIDs), parabens and UV-filters. Another objective of this Doctoral Thesis is to study the suitability of the developed methodologies for the determination of PPCPs in environmental samples

Synthesis of ionic liquids and their applications in capillary electrophoresis

Ph.DDOCTOR OF PHILOSOPH

Analysis of plant analytes using capillary electrophoresis and high performance liquid chromatography

Plants contain an enormous array of organic and inorganic components, the analysis for which may involve a wide range of methods. The focus of this study was to develop high performance liquid chromatography and capillary electrophoresis methods for the analysis of three classes of analytes: osmoregulants, minerals and amino acids. Firstly, this study explored the potential of capillary electrophoresis for the analysis of three very common osmoregulants (proline, glycine betaine and mannitol). A diverse array of methods has been reported for determining each of these analytes, however, the literature on osmoregulants and their analysis is quite disjointed and traverses both biological and chemistry fields. Therefore, a comprehensive review of this literature has been completed (Chapter 2). Considerably fewer methods are available for the simultaneous determination of these osmoregulants, compared to individual analysis. In chapter 3, a method is described for the simultaneous analysis of proline and betaine by capillary electrophoresis at low pH and specifically various cationic probes for the indirect detection of proline and betaine were explored. Sulfanilamide was identified as a suitable probe and was employed to quantify proline and betaine in spinach and beetroot. However, this method could not detect mannitol as it is not charged at low pH. In Chapter 4, a high performance liquid chromatography method for the simultaneous determination of all three osmoregulants is described. For separation, a NH2 column with formic acid and acetonitrile as the mobile phase were used. The high performance liquid chromatography evaporative light scattering detection method was applied to determine osmoregulants in Stylosanthes guianensis, Atriplex cinerea and Rhagodia baccata plant extracts. A complementary method, using a C18 column with heptafluorobutyric acid added to acetonitrile was used for verification of the analytes. Secondly, the potential for using capillary electrophoresis was investigated to simplify and shorten the complex sample preparation procedure. Chapter 5 describes a capillary electrophoresis method that allows direct injection from plant tissues. The experiments highlighted that uncontrolled hydrodynamic injection of sample on piercing of food sample resulted in non-reproducibility. The addition of hydroxypropylmethlycellulose to the background reduced the uncontrolled hydrodynamic injection up to 95% for all of the analytes. The sample was injected electrokinetically and an imidazole buffer consisting of hydroxypropylmethlycellulose was used for separation. The issue of reducing the reliance on prior separation is also relevant to minerals, thus the developed capillary zone electrophoresis-UV method was applied for the direct injection of inorganic cations from apple, mushroom, zucchini, green bean and strawberries. The applicability of the method across fruit varieties was determined by analysing four apple varieties including red delicious, fuji, pink lady and royal gala. Thirdly, the potential of the direct injection method was explored for the analysis of amino acids in zucchini. As amino acids are present at low concentrations and lack a chromophore, a more sensitive detector, capacitively coupled contactless conductivity, and pre-concentration of amino acids using isotachophoresis (leading electrolyte = HCl, terminating electrolyte = hydroxyproline) was performed. The separation of amino acids was carried using acetic acid. For minimising uncontrolled hydrodynamic injection poly(ethylene oxide) was used. Using this method sensitive detection of amino acids was possible (Chapter 6). In short, the developed methods allow for quick, inexpensive, sensitive and efficient analysis of plant components