Jana Rousova

Date of Award

January 2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Alena Kubatova

Abstract

One of the challenges in characterization of complex environmental matrices and renewable materials is the determination low molecular weight polar compounds (LMWPCs). Depending on the matrix, the targeted LMWPCs include acids, aldehydes, sugars, sugar alcohols or aminoacids. To investigate the role and impact of these species, a development of accurate and precise analytical protocols is essential. One of the challenges in analysis of LMWPCs is their volatility and thus potential losses during sample preparation. The various matrices including renewable materials may contain >100 compounds that are either non-targeted (i.e., their identity is not known prior to the analysis) or targeted (specific compounds within the matrix). Gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS, respectively) are the preferred methods of analysis because they adequately address the simultaneous identification of numerous non-targeted compounds and quantification of targeted compounds. Analysis of low molecular weight polar compounds in four different matrices and materials, each representing a unique challenge, are presented in this dissertation including characterization of biologically produced succinic acid (Chapter II), investigation of the composition of a methanolic extract of Pulicaria jaubertii and its fractions (Chapter III), serum of rats after exposure to resveratrol (Chapter IV) and atmospheric particulate matter (PM, Chapter V).

Biologically produced succinic acid needs to be free of undesirable compounds, such as short-chain carboxylic acids, which cause odor, and sugars and sugar alcohols, which can partake in a Maillard reaction in the presence of nitrogen containing compounds. We have adapted derivatization followed by GC-MS to identify and quantify more than 120 impurities in several succinic acid samples. This study focused on petroleum based succinic acid as well as bio-based samples that use a modified E. coli strain for fermentation. To enable an accurate quantification of both the target product and common impurities, we evaluated the acetonitrile extraction efficiency as an alternative to direct derivatization, and then compared several derivatization agents for trimethylsilylation. A prior ACN extraction was shown to be essential to detect impurities in trace concentrations. N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) was most efficient for derivatization of saccharides and low molecular weight monocarboxylic acids. However, the presence of pyridine was shown to be necessary for derivatization of saccharides and polyalcohols with BSTFA, whereas low molecular weight acids had to be quantified without pyridine. Fourteen representative bioproduced succinic acid samples differing in production stage and cultivation method were characterized. The screening of initial-process (1st stage of synthesis) samples showed that monocarboxylic acids were the most abundant and suggested the occurrence of saccharides. Thus, we have developed a method allowing for quantification of carboxylic acids and saccharides with limits of detection between 0.02–0.3 ng. In the initial-process bacterial samples and petrochemical sample, formic, acetic, lactic, oxalic, benzoic, citric and malic acids as well as glycerol, butanediol and glucose were found in a range of 0.02–1160 Ö§/g. In final-process samples, formic and acetic acid, and glucose were found in concentrations lower than 0.001%, thereby demonstrating the effectiveness of the process as well as the applicability of the method for quality control measure of the process.

Pulicaria jaubertii is a Middle Eastern medicinal plant with anti-obesity potential. To characterize its biologically active compounds, extraction with methanol was performed. This extract was further fractionated to hexane, dichloromethane and water/methanol. Analysis by GC-MS and LC coupled with high resolution mass spectrometry (HRMS) demonstrated the presence of catechin-like moieties in the dichloromethane and methanolic fractions and suggested that these components were partially responsible for the bioactivity of these fractions. Our data indicate that fractions derived from PJ exhibit anti-adipogenic properties in part owing to the presence of catechin-like compounds.

Trans-resveratrol (3,5,4’-trihydroxy-trans-stilbene) (RES) is a polyphenol found in many foods, such as peanuts, berries and red wine. In this study we developed a sensitive method using LC coupled to electrospray ionization (ESI) with high resolution time of flight (TOF) MS for the determination of RES. This method enabled an investigation of a relationship between tumor growth in rats and concentration of RES and its primary metabolites, trans-resveratrol-3-O-sulfate (R3S) and trans-resveratrol-3-O-β-D-glucuronide (R3G), in rat serum after RES exposure (5 or 25 mg/kg/day). RES levels in rat serum were near the limit of detection, showing concentrations of 4ѱ and 12Ñ´ ng/mL for low and high-dose exposure, respectively. Compared to RES, higher concentrations were found for its metabolites (R3G:4.8Ñ°.3 and 6.8Ñ°.3 Ö§/mL; R3S:0.27Ñ°.09 and 0.34Ñ°.04 Ö§/mL, respectively). Using LC-ESI-HRMS, for the first time, we measured the matrix-affected limits of detection (LODs) in plasma (3.7, 82.4, and 4.7 ng/mL for RES, R3G, and R3S, respectively), which were comparable to those reported in previous work using LC tandem mass spectrometry, but with a benefit of obtaining a full mass spectral profile. The additional novelty of our study is in synthesis and application of deuterated recovery standards enabling accurate and precise quantification. In order to develop a robust method, the ESI conditions were optimized using a multilevel full factorial design of experiments.

Understanding the occurrence of polar organic species in PM is essential because they are significant constituents of atmospheric carbonaceous PM and are also suggested to serve as cloud condensation nuclei. In this study, we propose a new analytical method allowing for the simultaneous methylation of the majority of carboxylic acids and derivatization of aldehydes (in contrast to sequential trimethylsilylation) enabling an easier interpretation of acids’ mass spectra without any interference from hydroxy groups. The sequential trimethylsilylation is still employed though targeting only the remaining aromatic acids and compounds with hydroxy groups. The developed quantitative method for simultaneous determination of aldehydes and acids using PFBHA؈Cl in methanol results in oximes and methyl esters, respectively; with the limits of detection between 0.04–1 μg/mL. The method has been successfully applied to a broad range of species with various functionalities (ca. 95 compounds), including long chain monocarboxylic acids, dicarboxylic acids, aromatic acids, ketoacids, hydroxyacids and aldehydes. The developed protocol was applied to wood smoke and urban air standard reference material 1648b PM. The aldehydes were observed in concentrations 10–3000 Ö§/g in wood smoke PM and 10–900 Ö§/g in urban air PM, while the observed acids were in concentrations 20–1800 Ö§/g in wood smoke PM and 15–1200 Ö§/g in urban air PM. The most prominent aldehydes were syringaldehyde and vanillin in wood smoke PM and glyoxal in urban air PM. The most abundant acids in both PM samples were short-chain dicarboxylic acids (≤C10), while wood smoke PM had a high abundance of hydroxyacids (vanillic and malic acids), as well as ketoacids (glutaric and oxalacetic), urban air PM also featured a high abundance of long-chain monocarboxylic acids (≥C16).

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