Rozprawy doktorskie na temat „Liquid chromatography research”

The three factors of the resolution (Rs)equation(see Equation 1.1)were explored in this thesis. During the course of the research project, an important aim was to explore separation processes that would lead to an increase in productivity without sacrificing Rs. To that end, an increase in the retention factor (k)to enhance Rs was deemed detrimental to the cycle time, hence the production rate, particularly when preparative separations are involved. Consequently the primary objectives were to (i)prepare more efficient columns and (ii)investigate new strategies in manipulating selectivity. The significance of the work contained in this thesis is highlighted in 27th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2003)held in Nice, France between 15-19 June, 2003. Many of the papers presented significantly compared to chapters contained in this research
Doctor of Philosophy (PhD)

The three factors of the resolution (Rs)equation(see Equation 1.1)were explored in this thesis. During the course of the research project, an important aim was to explore separation processes that would lead to an increase in productivity without sacrificing Rs. To that end, an increase in the retention factor (k)to enhance Rs was deemed detrimental to the cycle time, hence the production rate, particularly when preparative separations are involved. Consequently the primary objectives were to (i)prepare more efficient columns and (ii)investigate new strategies in manipulating selectivity. The significance of the work contained in this thesis is highlighted in 27th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2003)held in Nice, France between 15-19 June, 2003. Many of the papers presented significantly compared to chapters contained in this research

The alternative fuel biodiesel is produced from the transesterification of vegetable oils or animal fat to fatty acid methyl esters. Pomona has a reactor on campus that can be used to run this reaction and produce biodiesel. The use of biodiesel has been found to lower air pollutant and greenhouse gas emissions, but can be potentially harmful to the engines if it contains impurities. This paper proposes a method using high-performance liquid chromatography to test the quality of biodiesel. This method utilizes instrumentation and materials that are available in Pomona College's Chemistry Department, requires very little sample preparation, and is relatively safe, as long as general lab safety practices are followed. This method can also be used to optimize the procedure used to make the biodiesel. An optimized production procedure and a test method to assess the final product will ensure high quality fuel that can be used with confidence in diesel engines. This will likely add strength to proposals to increase the use of the on-campus reactor and produce biodiesel for campus grounds equipment from waste vegetable oil.

Cis-diol-containing metabolites have attracted increasing attention in recent years. These metabolites widely exist in the body fluids and tissues. They play important roles in the structure, function and metabolic activity of cells. Some of them are related to cell proliferation and metabolic processes. And they have been used to denote a state of disease as potential biomarkers. Several methods have been developed for the analysis of cis-diol-containing metabolites. However, these methods faced a challenge to separate and detect isomers of these compounds, particularly for compounds with low abundance and high polarity. Therefore, novel methods were necessary to improve the separation and detection sensitivity of this kind of metabolites. With this aim, chemical derivatization methods were developed for cis-diol-containing metabolite detection by using liquid chromatography-mass spectrometry in this project. These methods were optimized and validated to achieve the optimal reaction conditions. And they were applied to study real-world biological systems, including the changes of modified nucleosides in hepatocellular carcinoma (HCC) nude mice and toxic effects of bisphenol A (BPA) exposure. Firstly, the derivatization reaction of cis-diol compounds with acetone were optimized. Factors that affected reaction efficiency were investigated by reacting guanosine (G) with acetone. The optimal reaction conditions were validated by detecting four acetonides of urinary nucleosides by using LC-MS/MS. The results showed that the approach had good linearity, accuracy and precision. The recoveries were ranged from 92.9% to 103.5%. It indicated that the assay was reproducible. The robust method should be potentially useful for the analysis of modified nucleosides and other cis-diol-containing metabolites in biological samples. The validated derivatization method was applied to determine urinary nucleosides by LC-MS. This method not only improved the retention of nucleosides on reversed-phase column, but also reduced the matrix effect from urine samples and enhanced detection sensitivity of mass spectrometry. Isotope labeling method with acetone-d6 and multivariate statistical analysis enabled the positive identification of 56 nucleosides, including 52 modified nucleosides. The obtained results indicated that the derivatization method was practical, fast and effective for the identification of urinary nucleosides. It was successfully applied to study the changes of urinary nucleosides in nude mice bearing HCC. Some significantly changed nucleosides were identified as potential biomarkers. Subsequently, this approach was modified by employing parallel reaction monitoring (PRM) method which was based on high resolution MS to detect urinary nucleosides in rats exposed to BPA. Comparing to the data acquired by triple quadrupole MS with neutral loss scanning, higher specificity and sensitivity were achieved by using PRM scanning mode. Therefore, more nucleosides were identified by using the method in urine samples (from 56 up to 66). The changes of the detected nucleosides were studied in the rats exposed to BPA. Various trends of modified nucleosides were observed with different dose BPA exposure. Specifically, the high-dose exposure group was the most strongly affected. The biomarker of RNA oxidation, 8-hydroxyguanosine (8-oxoG), showed significant change in this group. It proved that BPA exposure could induce RNA damage when the dose of BPA was beyond a certain amount. Except for nucleosides, other cis-diol-containing metabolites, such as carbohydrates, were also studied by using the derivatization method. Acetone and acetone-d6 were applied to label the cis-diol metabolites. Based on the chemical isotope labeling, cis-diol metabolites were easily recognized from urine samples. Influence of BPA exposure on these metabolites was investigated by comparing different doses of BPA administration on rats. Analytes showed noticeable difference were highlighted. Pathway analysis indicated that galactose metabolism, nucleoside and its analogues metabolism were disturbed. The derivatization method was extended to quantify nucleotides in plasma samples. According to the specific physical-chemical properties of nucleotides, the method was improved to fit the requirement of analysis by using 1,1-Dimethoxycyclohexane (DMCH) as derivatization agent and formic acid (FA) as catalyst. Tip micro-columns packed with TiO2 were used for selective adsorption of nucleotides in the plasma. Then in-situ derivatization were carried out to change the polarity of targeted compounds. LC-MS analysis of the derivatization products were employed without using ion-pairing reagents. This method exhibited a high selectivity for the extraction of nucleotides. After derivatization, retention of nucleotides on reversed-phase C18 column was improved. Complete separation of nucleotides with the same base was achieved. The peak shape was symmetrical and the tailing was eliminated by using high pH mobile phase. The method settled the problems of nucleotide detection, which were poor retention, trailing, in-source fragmentation and contamination of ion-pairing reagents. The quantitative method was successfully applied to determine the content of nucleotides in plasma samples of rats exposed to BPA. It was simple and fast, as well as good selectivity and stability. It could be extended to detection of other phosphorylated metabolites with similar structure. To our best knowledge, it was the first time to employ derivatization methods to detect cis-diol-containing metabolites. The methods decreased the matrix effects of complex biological samples, and also decreased the polarity of cis-diol-containing metabolites. The changes of properties not only improved the chromatographic separation, but also enhanced the MS intensities. The methods overcame the problems of cis-diol-containing metabolite detection on reversed-phase column. They were successfully applied to study the changes of cis-diol-containing metabolites of HCC and toxic effects of BPA exposure. The method might be extended to determine other cis-diol-containing metabolites in urine samples as well as in cells, tissues and plasma samples. It might be valuable for the understanding of the roles of cis-diol-containing metabolites in in cell metabolism.

In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

The advent of liquid chromatography tandem mass spectrometry (LC-MS/MS) coupled with soft ionization method has expanded the scope for measurement of steroids from biological matrices with higher accuracy, specificity, sensitivity and requires less sample preparation. The overall aim of this study is to develop and validate LC-MS/MS methods to measure steroids from biological samples for clinical research studies. A sensitive and specific LC-MS/MS assay was developed and validated to measure steroids from dried blood spots (DBS) samples to assess the feasibility and pharmacology of subcutaneous (sc) injection of androgen ester in healthy men using DBS for frequent sampling. The study demonstrated a sustained release of this androgen ester which suggests that sc injections of testosterone esters may prove to be pharmacologically effective. A LC-MS/MS assay was also developed to measure urinary androgens and estrogen in adolescents and subsequently to relate the changes in the urinary sex hormones over 12 months to the standard anthropometric markers of puberty. We found that the urine hormone measurements correlated cross-sectionally and longitudinally with age, anthropometry and Tanner stage. We also investigated whether first morning void urine hormones in growing adolescents require adjustments for urine dilution/concentration and, if so, whether urinary creatinine or specific gravity (SG) are better adjustments. The study demonstrates that urine steroid and LH concentrations in first morning void samples of adolescents are not significantly influenced by hydration status and may not require adjustments; however, if desired, both creatinine and SG adjustments are equally suitable. We also assessed whether commercially available luteinizing hormone (LH) immunoassays (immunochemiluminometric, ICL and immunofluorometric, IF) previously validated for human blood samples is suitable for urine samples kept at prolonged frozen (4 years). We found that both immunoassays are suitable for urinary LH measurements with ICL assay being more robust for quantitative urinary LH measurement such as for anti-doping purpose whereas the IF could be applicable for research studies where urine LH levels are compared within-study but not in absolute terms.

Note:
Clinical chemistry is a medical discipline whose aim is to diagnose and assess disease by analysis of biological specimens. Modem laboratories can perform several hundred different tests using many different methods developed over the last century. The classical, more traditional assays are typically labour-intensive, not multiplexed (only measure one analyte or disorder per assay), expensive, require a long turnaround time, and may not provide adequate sensitivity and specificity. Developments in mass spectrometry (MS) and related technologies over the last two decades have provided solutions for many if not all of these shortcomings. While MS based applications have not yet been widely implemented in clinical chemistry laboratories, current developments will encourage the replacement of traditional methods as well as the expansion of clinically diagnostic endpoints. Indeed, modem MS can be used to simultaneously analyze and quantitate multiple biomarkers in a single analysis. Currently, no other technique exists that can provide a comparable multiplexed analysis. In this thesis, current MS and related technologies were developed and applied to several important but distinct clinical chemistry applications. [...]
La chimie clinique est une discipline medicale qui a pour but de diagnostiquer la presence et la progression d'une maladie par l'analyse d'echantillons biologiques. Les laboratoires modemes peuvent executer des centaines d'analyses en utilisant plusieurs methodes developpees au courrant des cent demieres annees. Les essaisc1assiques, et plus traditionnels, sont souvent laborieux, non multiplexe (mesurent seulement un analyte par essai), cher, exige un long temps de rotation et risque de ne pas fournir une specificite adequate. Pendant les deux dernieres decennies, les developpements dans Ie domaine de la spectrometrie de masse (MS) et les technologies rattachees ont foumi des solutions a plusieurs, pour ne pas dire tous, manques retrouves dans les methodes d'analyse traditionnelles.

Thesis (Ph.D.)--University of Florida, 2004.
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There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2016.
The presence of estrogenic hormones in the environment has been a subject of concern in recent years; they have been classified as “emerging pollutants” and may pose a potential risk for human consumption. Hormones have been detected in ground and surface water at low concentrations. These compounds contaminate the surface and ground water via waste water treatment plants (WWTP) and may elicit endocrine disruption to organisms. Because these compounds are available at low concentration, robust analytical methods are required to quantify these compounds in water and environmental samples. The common method for the analysis of hormones in water samples is Gas Chromatography (GC) coupled to Mass Spectrometer (MS). The challenge with GC-MS is the required lengthy derivatisation step that involves toxic chemicals. The first part of this case study was to develop a method to determine trace concentrations of the Estrone (E1), 17α-Estradiol (E2α), 17 β-Estradiol (E2β) and 17α-Ethinylestradiol (EE2) hormones using Ultra-Fast Liquid Chromatography Mass Spectrometry (UFLC-MS-MS). Using the developed method, the second part of the case study was to determine the concentrations of the hormones in raw and potable water samples from the Vaal River catchment area in the South of Johannesburg, South Africa. Analytes were extracted by solid phase extraction (SPE C18 Sorbent, 200 mg/6mℓ cartridges) and subjected to Ultra-Fast Liquid Chromatography coupled to Mass Spectrometer (UFLC-MS-MS) for identification and quantification. Optimum SPE parameters were 1000 mℓ of sample percolated, at flow rate of 10 mℓ/min, sample pH of above 7, 7.5 mℓ of methanol as elution solvent followed by solvent reduction to 250 μℓ. The limits of quantification were in a range of 0.24 to 0.32 ng/ℓ for all analytes. Accuracy was 95.6, 93.8, 97.6 and 100.9% for 17α-Ethinylestradiol, 17α-Estradiol, 17β-Estradiol and estrone, respectively. In raw water samples taken during the rainy wet season, estrone was detected at concentrations of 0.90 and 4.43 ng/ℓ. However, drinking water samples no presence of hormones with the exception of M-B12 sample point where the estrone amount of 2.88 ng/ℓ was detected. This is potentially due to fact that conventional water treatment plants are able to remove the compounds during water purification process depending on the concentration levels.
LG2017

Bioethanol fermentation is an important process that is reducing the global demand on fossil fuels and remains a field of research for the foreseeable future. Carbohydrates are sourced from crops and hydrolyzed into simpler sugars then fermented into ethanol. Fermentation of sugars sourced from food crops presents a sustainability issue with a growing population. Fermentation of lignocellulosic material is more sustainable since it is sourced from non-food crops or wastes. It is comprised of a variety of both pentose and hexose sugars. The composition and ratio of these varies depending on the source of the material. Accurate analysis of the material is essential for both the monitoring of the hydrolysis and its fermentation to valuable end-products such as ethanol. Although there have been major advances in novel fermentation processes and the discovery and construction of novel microorganisms, development of methods for analysis of these complex substrates and their fermentation to ethanol has not advanced as rapidly. High Performance Liquid Chromatography (HPLC) is one of the most common techniques for the analysis of these complex substrates. The resolution of popular HPLC modes was compared and no mode was found to have complete separation of common fiber sugars. Free solution Capillary Electrophoresis / capillary zone electrophoresis (CE) is used and recognized in both research and industry as a viable technique for the separation of carbohydrates. Recent studies on the use of direct UV detection for determination of underivatized carbohydrates have shown great promise and, in this work, the technique was applied to lignocellulosic plant fiber analysis as well as monitoring its fermentation to ethanol and sugar alcohols. All resolution values with CE were higher than 0.5, in contrast to any HPLC mode investigated. The running cost of HPLC, for this application, is also much higher than CE. Determination of carbohydrates from lignocellulosic fiber by both HPLC on a cation exchange resin and by CE resulted in values 17-22 % higher with CE than HPLC. The influence of the counter-ion in the BackGround Electrolyte (BGE) was found to affect the resolution and time of the separation. 130 mM KOH was shown to be effective for a fast separation of simple mixtures and a mixture of 65 mM NaOH and 65 mM LiOH achieved a better resolution with more complex carbohydrate mixtures than the other BGE’s studied. In a quantitative study on fermentation samples, CE, HPLC and High Performance Anion Exchange Chromatography (HPAEC) closely agreed within experimental error (less than 7 % difference from the average total detected amount).