Tesis sobre el tema "Analytical chemistry"

Diss. (sammanfattning) Umeå : Umeå universitet, 1996, Härtill 4 uppsatser

The analysis of pesticide residues in fruit, vegetables, rape seed and water has been improved using developments in sample handling and analytical techniques. The method development is associated with analytical difficulties, since pesticides currently used in agriculture represent a variety of chemical classes having very different physico chemical properties. The method development also encounters difficulties when many various commodity classes with different characteristics are studied. The main task in pesticide residue analysis has been to provide multi residue methods, and traditionally GC has been the main analytical technique.

In order to regulate the use of hazardous pesticides, the EU commission introduces strict maximum residue levels (MRL). The need for improved sample handling and detection techniques are, however, high due to handling of lower detection limits, complex matrices and the need of more efficient sample throughput. Of the new techniques introduced as alternative techniques to the traditional extraction techniques, pressurised fluid extraction (PFE) has shown to be a promising technique in analysis of pesticide residues in fatty foodstuffs.

In water analysis, large sample volumes are needed due to low MRLs. The solid phase extraction (SPE) technique allows a concentration of large sample volumes and simplifies the tedious laboratory work with traditional separation funnels. A new approach was to use non-polar solvents for the sample extraction from the earlier used polymeric column. Both these techniques provide low solvent consumption, short extraction times and ability to automate the manual steps.

An LC-MS/MS multi residue method was finally developed for pesticide residues in fruit and vegetables. The technique is robust and sensitive and allows a simultaneous determination of 57 pesticides and metabolites in one single analysis and without any clean-up steps. The sensitivity was improved to achieve the maximum residue limits needed by EU. Several multi step methods, which involve more costly analysis, has been replaced by this technique.

Sample preparation is often a bottleneck in systems for chemical analysis. The aim of this work was to investigate and develop new techniques to address some of the shortcomings of current sample preparation methods. The goal has been to provide full automation, on-line coupling to detection systems, short sample preparation times and high-throughput.

A new technique for sample preparation that can be connected on-line to liquid chromatography (LC) and gas chromatography (GC) has been developed. Microextraction in packed syringe (MEPS) is a new solid-phase extraction (SPE) technique that is miniaturized and can be fully automated. In MEPS approximately 1 mg of sorbent material is inserted into a gas tight syringe (100-250 μL) as a plug. Sample preparation takes place on the packed bed. Evaluation of the technique was done by the determination of local anaesthetics in human plasma samples using MEPS on-line with LC and tandem mass spectrometry (MS-MS). MEPS connected to an autosampler was fully automated and clean-up of the samples took one minute. In addition, in the case of plasma samples the same plug of sorbent could be used for about 100 extractions before it was discarded.

A further aim of this work was to increase sample preparation throughput. To do that disposable pipette tips were packed with a plug of porous polymer monoliths as sample adsorbent and were then used in connection with 96-well plates and LC-MS-MS. When roscovitine in human plasma and water samples was used as model substance, a 96-plate was handled in two minutes.

Metabolomics is a rapidly growing field in “omics” research where metabolites are analyzed in biological systems. Over the past decade, mass spectrometry (MS) based metabolomics has been used for its superior analytical performance to reveal how these biological systems respond to genetic and environmental changes. MS is both sensitive and selective and is capable for providing comprehensive information for metabolic profiling by combining separation methods such as liquid chromatography (LC-MS) or gas chromatography (GC-MS). However, in untargeted metabolomics identification of small molecules is the bottleneck. In the research described here, I have combined both in silico and experimental methods for compound dereplication of natural products using MS-based metabolomics.

Chapter 1 addresses the advancement of fragmentation and mass spectral trees used for unknown metabolite identification. Tools used for metabolite identification from the past 10 years are discussed, including algorithms, software, mass spectral libraries, and databases that implement fragmentation and mass spectral trees. Due to the inherent complexity of natural products in plants and microbes, unknown compound identification is increasingly difficult and limiting. Resolving this problem requires better computational tools and informative data such as those acquired by multi-stage mass spectrometry (MSⁿ). MSⁿ yields more fragmentation data and allows for more complex structural elucidation as needed for compounds with positional isomers. The limitation with using tandem mass spectrometry (MS/MS) only is that many ions are shared between positional isomers and full structural information is not available to elucidate an unknown metabolite. Fragmentation and mass spectral trees both describe the fragmentation processes of a metabolite and aid in fragmentation rule generation and substructure identification. The major difference between fragmentation and mass spectral trees is that fragmentation trees use elemental compositions to describe the fragmentation process and mass spectral trees or ion trees use precursor and product ion spectra from MSⁿ mass spectral acquisition. As a result, there has been a large increase in efforts to develop MS^{n > 2} data and tools for both structure elucidation and spectral annotations with the use of fragmentation and mass spectral trees in recent years.

Chapter 2 describes research and development of iTree, a MSⁿ mass spectral tree library of plant natural products and its aid in compound identification of natural products. In metabolomics, mass spectral library searching is a standard method for compound identification, correctly known as compound dereplication. Mass spectral libraries are either freely or commercially available and can contain both experimental and in silico MS/MS reference spectra. The coverage of MS^{n > 2} reference spectra is much smaller in many of these MS/MS libraries and databases. To date the largest MS^{n > 2} libraries are HighChem and mzCloud, which also support mass spectral trees. The chemical coverage of such libraries and databases are very low in comparison to the number of known compounds. iTree was developed to expand the coverage of fragmentation spectra for natural products. iTree contains more than 2,000 natural products and more than 9,000 ion tree spectra annotated with in silico generated substructures from both Mass Frontier 7.0 and CFM-ID. iTree is freely available through MassBank of North America (MoNA), an open-access mass spectral database. As a result of the high number of natural products, and specifically flavonoid aglycones, previously published fragmentation rules were studied and validated. A new rule for flavanonols was proposed as a loss of –CCO to occur specifically for this class. In addition, iTree was used to profile secondary metabolites in the roots and nodules of the host plant Datisca glomerata. More than 100 natural products were identified by combining LC-MSⁿ, high resolution LC-MS/MS, and ion tree analysis using iTree. Overall, iTree has shown to provide a method to facilitate metabolite identification for plant natural products.

Although MS^{n > 2} data is more useful for complex structural elucidation, the predominant data used in untargeted metabolomics is MS/MS. For this reason, in silico tools that focus on the interpretation of MS and MS/MS spectra alone must be evaluated. In Chapters 3 through 5, I discuss how the Critical Assessment of Small Molecule Identification (CASMI) has allowed for such an evaluation by presenting unknown challenge data sets to the metabolomics community to evaluate the tools and methods they currently use for unknown compound identification. The results submitted by each user are compared and discussed to provide greater insight into how in silico tools can be further improved to aid in the advancement and accuracy of unknown compound identification methods.

Chapter 3 focuses specifically on the performance of MS-FINDER, a software that uses MS and MS/MS spectra for structural elucidation of unknown compounds, presented in the CASMI 2016 Category 1. (Abstract shortened by ProQuest.)

The work in this dissertation presents microfluidic methods developed for the study of biological dynamics. The requirements for the methods development was to create approaches with the ability to perform dynamic cell stimulation, measurement, and sample preparation. The methods presented herein were initially developed for the study of pancreatic islet biology but are expected to be translatable to other applications. In another study, a method to interface transmission electron microscopy (TEM) with microfluidics methods was developed.

The primary biological topic of interest investigated was the mechanisms of inter-islet synchronization. To test this, a microfluidic device fabricated from poly(dimethylsiloxane) (PDMS) was used to culture and stimulate pancreatic islets. Intracellular calcium ([Ca²⁺]i) imaging was performed with a fluorescent indicator, Fura-2-acetoxymethyl ester (Fura-2 AM). Under constant glucose (11 mM), islets demonstrated asynchronous and heterogeneous [Ca²⁺]i oscillations that drifted in period. However, when exposed to a glucose wave (11+/– 1 mM, 5 min period) islets were entrained to a common and consistent [Ca²⁺]i oscillation mode. The effect of islet entrainment on cellular function was investigated by measuring gene expression levels with microarray profiling. Calcium-dependent genes were found to be differentially expressed. Furthermore, it was speculated that islet entrained produced a beneficial effect on cell function and upkeep.

While [Ca²⁺]i imaging is an acceptable proxy measurement for insulin, it is not a viable reporter for other islet peptides and direct measurement is desired. Electrophoretic affinity assays can be performed on a microfluidic device in a serial manner to measure peptide release from an on-chip cell culture in near real-time. Successful analysis of electrophoretic affinity assays depends strongly on the preservation of the affinity complex during separations. Elevated separation temperatures due to Joule heating promotes complex dissociation leading to a reduction in sensitivity. To address this limitation, a method to cool a glass microfluidic chip for performing an affinity assay for insulin was achieved by a Peltier cooler localized over the separation channel. The Peltier cooler allowed for rapid stabilization of temperatures, with 21 °C the lowest temperature that was possible to use without producing detrimental thermal gradients throughout the device. Kinetic capillary electrophoresis analysis was utilized as a diagnostic of the affinity assay and indicated that optimal conditions were at the highest attainable separation voltage, 6 kV, and the lowest separation temperature, 21 °C, leading to 3.4% dissociation of the complex peak during the separation. These optimum conditions were used to generate a calibration curve and produced 1 nM limits of detection (LOD), representing a 10-fold improvement over non-thermostated conditions.

To date, most approaches for measurement of rapid changes in insulin levels rely on separations, making the assays difficult to translate to non-specialist laboratories. To enable rapid measurements of secretion dynamics from a single islet in a manner that will be more suitable for transfer to non-specialized laboratories, a microfluidic online fluorescence anisotropy immunoassay was developed. A single islet was housed inside a microfluidic chamber and stimulated with varying glucose levels from a gravity-based perfusion system. The total effluent of the islet chamber containing the islet secretions was mixed with gravity-driven solutions of insulin antibody and cyanine-5 (Cy5) labeled insulin. After mixing was complete, a linearly polarized 635 nm laser was used to excite the immunoassay mixture and the emission was split into parallel and perpendicular components for determination of anisotropy. Key factors for reproducible anisotropy measurements, including temperature homogeneity and flow rate stability were optimized, which resulted in a 4 nM LOD for insulin with < 1% RSD of anisotropy values. The capability of this system for measuring insulin secretion from single islets was shown by stimulating an islet with varying glucose levels. As the entire analysis is performed optically, this system should be readily transferable to other laboratories.

To increase the number of analytes that can be simultaneously monitored by a fluorescence anisotropy immunoassay, frequency encoding was introduced. As a demonstration of the method, simultaneous competitive immunoassays for insulin and glucagon were performed by measuring the ratio of bound and free Cy5-insulin and fluorescein isothiocyanate (FITC)-glucagon in the presence of their respective antibodies. A vertically polarized 635 nm laser was pulsed at 73 Hz and used to excite Cy5-insulin, while a vertically polarized 488 nm laser pulsed at 137 Hz excited FITC-glucagon. The total emission was split into parallel and perpendicular polarizations and collected onto separate photomultiplier tubes. The signals from each channel were demodulated using a fast Fourier transform, resolving the contributions from each fluorophore. Anisotropy calculations were carried out using the magnitude of the peaks in the frequency domain. The method produced the expected shape of the calibration curves with LOD of 0.6 and 5 nM for insulin and glucagon, respectively. (Abstract shortened by ProQuest.)

Single cell metabolomics provides new insights into understanding cellular heterogeneity of small molecules, and individual cell response to environmental perturbations. With high sensitivity and specificity, mass spectrometry (MS) has become an important tool for analyzing metabolites, lipids, and peptides in individual cells. Facing significant challenges, single cell and subcellular analysis by MS requires technical advances to answer fundamental biological questions, for example the phenotypic variations of genetically identical cells. The work presented in this dissertation describes my efforts to develop and apply capillary microsampling MS with ion mobility separation (IMS) for the analysis of single cells and subcellular compartments.

Chapter 1 introduces MS based analytical techniques for single cell and subcellular analysis. Recent advances of sampling and ionization methods for MS analysis of volume-limited samples are reviewed with emphasis on ambient ionization techniques, cell micromanipulation methods, and rapid gas phase separations.

In Chapter 2, the application of capillary microsampling electrospray ionization (ESI)-IMS-MS for metabolic and lipidomic analysis of single Arabidopsis thaliana epidermal cells is presented. Distinct metabolite compositions and metabolic pathways are identified among basal and pavement cells, and trichomes. These three specialized epidermal cells serve different functions in the plant leaf, and our single cell MS data reveals the corresponding metabolic pathways.

In Chapter 3, it describes the utilization of capillary microsampling ESI-IMS-MS for the analysis of metabolites and lipids in single human hepatocellular carcinoma cells. Cellular physiological states and their heterogeneity in response to xenobiotics treatment, and lipid turnover rates are explored. Here, IMS helps to enhance molecular coverage, facilitate metabolite and lipid identification, resolve isobaric ions, and minimize background interference. Comparing cells affected by metabolic modulators to unaffected counterparts reveals dramatic reduction in the availability of energy in the former.

In Chapter 4, the combination of fluorescence microscopy with capillary microsampling ESI-IMS-MS for selective analysis of identified cell subpopulations at a single cell level is demonstrated. Molecular differences and heterogeneity corresponding to cells in distinct mitotic stages are explored. Pairwise correlations between relative metabolite levels among individual mitotic cells are also studied.

In Chapter 5, the subcellular distributions of neuropeptides in individual identified neurons are explored by capillary microsampling ESI-IMS-MS. Distinct peptide distributions between the cytoplasm and nucleus are revealed. Mass spectra provide direct evidence for high abundance of these peptides in the nucleus despite the scarcity of immunostaining results supporting their presence there. A new neuropeptide is discovered and sequenced by MS in a single cell.

In Chapter 6, the current state of single cell and subcellular metabolomics is discussed. Major challenges include the low-throughput of current sampling techniques, low molecular coverage of metabolites, lipids and peptides, and external perturbations introduced by the sampling and ionization processes. In addition to exploring new solutions to these challenges, future advances will lead to the development of systems biology at the single cell level, to nano- and micro-fabricated tools to study perturbations in a lab-in-a-cell framework, and to coupling with optical manipulations and microfluidic techniques to investigate subcellular heterogeneity.

Human biomonitoring is an analytical challenge to find environmental organic chemicals of varying polarity, persistency, and potential toxicity in a suitable, ideally non-invasive matrix at ppb levels that are significantly above method blanks. Compared with more traditional matrices of adipose tissue, serum, and urine, toenail clippings samples are non-invasive, compact, can be shipped without refrigeration, stored indefinitely at room temperature, and processed without concerns for biohazards. With both hydrophilic and hydrophobic layers, toenails contain 1-2% lipid, which is several times higher than serum. Toenails grow slowly and are trimmed every 2-3 months, which offers the potential to integrate both chronic and pulsed episodic exposures. Using toenail samples (65 to 340 mg) donated from four individuals and an indoor house cat, the hypothesis that toenails are a suitable biomonitoring matrix was tested by analyzing for persistent pesticides, over 50 PCB congeners, moderately persistent PBDEs, and transient compounds of triclosan and bisphenol A by using GC/High Resolution MS (GC/HRMS) analysis and for unsuspected compounds using GC/full scan MS. Although not fully digested and dissolved, toenails averaged 1.22% lipid (sd 0.20%, n=10). Lipid was separated and determined using a new small single-use 2-g S-X3 gel permeation chromatography flash column with high purity nitrogen. Multiple toenail samples from one individual were collected for over a year for replicate analysis, p,p’- DDE averaged 0.82 ng/g-nail, sd 0.28, n=5 and 65.2 ng/g-lipid, sd 15.3, n=5 on lipid-adjusted basis. Trans-nonachlor averaged 3.08 ng/g-nail, sd 1.03, n=5; mean 254 ng/g-lipid, sd 97, n=5. PBDE 28 averaged 0.29 ng/g-nail, sd 0.10, n=5; mean 24.8 ng/g-lipid, sd 13.3, n=5. PBDE 85 averaged 0.25 ng/g-nail, sd 0.06, n=5; mean 20.8 ng/g-lipid, sd 6.2, n=5. PBDE 153 averaged 1.82 ng/g-nail, sd 0.51, n=6; mean 150 ng/g-lipid, sd 49.3, n=6. Most effectively biomonitored in toenails were normally transient triclosan (mean 58.3 ng/g-nail, sd 6.6, n=2), chlordanes, DDT, PBDEs, and PCBs including congeners with 2,5- or 2,3,6-chlorine substitution (PCBs 52, 49, 44, 70, 95, 101, 87, and 110), which are suspect neurotoxins, but are rarely found in extant serum biomonitoring data. Toenail soap wash samples indicated little (< 4%) or no exogenous contamination, except for the musks galaxolide and tonalide in most samples, which ranged up to 30%, likely from topical application. The one cat toenail sample had elevated concentrations of PBDEs and especially chlordanes. Unsuspected tentatively identified compounds included a UV Filter compound, octocrylene, a hydroxyl-methyl benzothiazole, and several compounds used in flavors or fragrances.

Mass spectrometry (MS) is an efficient analytical tool due to a number of advantages for providing high sensitivity, high-throughput analysis and less sample consumption. Since 1897, MS has continued to evolve and significant developments have occurred in the area of ionization techniques. Since 2010, inlet ionization techniques have shown the potential to compete in the areas of sensitivity and sample conservation with electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI). Matrix assisted inlet ionization (MAI) produces multiply charged ions within a mass spectrometer inlet without the need of high voltage, a laser or nebulizing gas. For MAI, sample preparation can be critical affecting the sensitivity, reproducibility and carry over. This dissertation presents new sample preparation techniques which have shown substantial improvement in sensitivity and reproducibility. The limit of detection can be achieved to less than 50 amol with the new sample preparation technique. Furthermore, this new sample introduction process enhance matrix assisted ionization (MAI) and solvent assisted ionization (SAI)’s ability to couple with Thin-layer chromatography.

Electrokinetic chromatography is a variation of capillary electrophoresis that allows for the separation of nonionic analytes by selective interaction with an ionic pseudostationary phase dissolved in the background electrolyte. The utility of electrokinetic chromatography to characterize pseudostationary phases and pseudostationary phase–solute interactions has been recognized since its introduction. The objective of this dissertation was to use electrokinetic chromatography and copolymer stabilized lipid bilayer nanodiscs as a pseudostationary phase to characterize small molecule-lipid bilayer interactions.

Styrene-maleic acid copolymers were used to stabilize cylindrical sections of lipid bilayer in solution, forming nanodiscs. The nanodiscs are formed based on strong hydrophobic interactions between the styrene moiety, on the copolymer, and the alkyl tails of the lipids. Using the nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was characterized. Linear solvation energy relationship analysis was employed to characterize the changes in solvent environment of the nanodiscs of varied copolymer to lipid ratio, copolymer chemistry and molecular weight, and lipid composition. Increases in the lipid to copolymer ratio resulted in smaller, more cohesive nanodiscs with greater electrophoretic mobility. Nanodisc structures with copolymers of different chemistry and molecular weight were compared and showed changes in solvent characteristics and selectivity. Seven phospholipid and sphingomyelin nanodiscs of different lipid composition were characterized. Changes in lipid head group structure had a significant effect on bilayer?solute interactions. In most cases, changes in alkyl tail structure had no discernible effect on solvation environment.

The nanodisc pseudostationary phase was also used to study sphingomyelin stereochemistry. Various studies have produced conflicting results regarding whether interactions with lipid bilayers are or can be stereoselective. Using sphingomyelin nanodiscs stereoselective interactions between a pair of atropisomers, R-(+)/(S)-(–) 1,1'-Bi-2-naphthol, were demonstrated.

Finally the dissociation constants between sphingomyelin nanodiscs and solvochromatic analytes were measured and then validated using steady state fluorescence. Using nanodisc affinity capillary electrophoresis, dissociation constants were derived on the same order of magnitude as the dissociation constants derived using the fluorescent technique. Future directions of this project will be to study peptide and protein interactions with lipid bilayers of interest.