The ability to effectively analyze particulate matter (PM2.5) in air is becoming increasingly pertinent. Allen Goldstein of the University of California in Berkeley is studying the semi-volatile fraction of organic compounds in PM2.5 through the use of the thermal desorption aerosol gas chromatograph (TAG) system. However, as conventional GC does not provide adequate separation power, the development of comprehensive two-dimensional gas chromatography (GC × GC) was required. GC × GC works more effectively by utilizing a modulator that periodically traps and focuses analytes from a primary column onto a secondary column. This allows for the primary and secondary columns to separate the analytes based on two different properties. This report focuses on the continuing study and enhancement of a modulator designed by Ognjen Panić during his Masters project. Improving and testing the robustness of this dual stage modulator was originally the focus of this project. However, this study led to the development of a single stage modulator. In addition to that, the effect of modulator characteristics such as length of the restriction, total length of the modulator and wall thickness on the modulator performance were studied. A robustness test of the single stage modulator was also completed. Experiments conducted tested the characteristics of the new modulator to ensure it performed effectively and would satisfy the requirements of the TAG system. A study comparing the sensitivity of conventional gas chromatography and GC × GC was also preformed. The sensitivity of GC × GC was on average an order of magnitude better than that of 1D GC.

No description available.

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This dissertation illustrates an efficient purification method for citrus limonoids and flavonoids, while examining their effects on cytochrome P450 inhibition and apoptotic induction on human neuroblastoma (SH-SY5Y) and colonic adenocarcinoma (Caco-2) cells. The first study developed a bulk purification method for limonoids, from seeds and molasses of citrus fruits, using a combination of chromatographic techniques. This also resulted in an efficient purification method for naringin and hesperidin from citrus byproducts. The second study investigated the inhibitory effects of purified limonoids and flavonoids on the enzymatic activities of different isoforms of human cytochrome P450. O-Dealkylase and hydroxylase activities of CYP1A2, CYP1B1, CYP3A4 and CYP19, using specific substrates such as ethoxyresorufin (ethoxyresorufin O-dealkylase, EROD), methoxyresorufin (methoxyresorufin O-dealkylase, MROD), and dibenzylfluorescein (DBF), were found to be significantly (P < 0.001) reduced at micromolar levels. A kinetic analysis showed competitive and non-competitive modes of inhibition by limonoids, on CYP19 hydroxylase activity. The results corroborate the active role of limonoids in the redox cycling mechanisms. The third study examined the antioxidant and apoptotic inducing ability of limonoid glucosides on human neuroblastoma cells. Four limonoid glucosides, LG (17beta-D glucopyranoside limonin), OG (obacunone 17beta-D glucopyranoside), NAG (nomilinic acid 17beta-D glucopyranoside), and DNAG (deacetylnomilinic acid 17beta-D glucopyranoside), have shown superoxide scavenging at millimolar levels. Micromolar amounts of LG and OG induced rapid necrosis of SH-SY5Y cells. Cytotoxicity was correlated (P = 0.046) to a concentration and timedependent increase in caspase 3/7 activity. Analyses of DNA content during the S phase of the cell cycle indicated reductions of 86.6% for LG and 82.3% for OG as compared to untreated. The results validate the antineoplastic distinctiveness of limonoid glucosides. In the fourth study, cytotoxic effects of limonoid aglycones and glucosides were assessed on human SH-SY5Y neuroblastoma and colon carcinoma (CaCo-2) cell lines and compared with the non-cancerous Chinese hamster ovary (CHO) cells. Significant (P < 0.001) cytotoxic effects were observed only on cancerous cells, over 24 to 36 h. The study revealed a marked increase in the DNA content of aneuploidic cells, which results in cell cycle arrest. The results confirm that glycosides are the most active apoptotic inducing form.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 145-148).

The Nitrogen Phosphorus Detector is a sensitive, selective device used in gas chromatography. It responds selectively towards nitrogen and phosphorus containing organic compounds with detection limits in the picogram range. The detector is of great importance for the measurement of trace levels of drugs, pesticides and herbicides in biological matrices and the environment. There is, however, some dispute in the literature regarding the detector's response mechanism. The detector is based on a hydrogen-air diffusion flame. Two electrodes polarise the flame with a potential difference of about 200 V and the current through the flame is measured using an electrometer amplifier. The selectivity of the system relies on the presence of an alkali metal source, usually rubidium. In the presence of nitrogen- and phosphorus-containing organics, C~ and PO· anions are formed, yielding a current which is the measured response. It has been suggested that this selective response arises from a charge transfer reaction between the rubidium excited states and ~ or PO· and P02• radicals. Using an AlGaAs diode laser, the rubidium excited state population can be modulated and the influence on detector current monitored. Rubidium resonance-enhanced ionisation, laser-induced fluorescence and emission spectroscopy have all been used to further probe the response mechanism of the detector. Results have demonstrated that during response the C~ radical concentration increases. In addition the diode laser can modulate the excited state rubidium concentration altering it by a factor of 2. However despite more that doubling the Rubidium excited state concentration no increase in detector response is observed. From these observations it has been concluded that the above mentioned charge transfer reaction plays little if any role in detector response.

published_or_final_version / Chemistry / Master / Master of Philosophy

This thesis begins with an examination of the feasibility of using the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method, and its variants, for the co-extraction of nonpolar and polar pesticides, including highly polar quaternary ammonium salts (QA) and organophosphates (OP). QuEChERS was performed on a mixture of nine different chemical classes of pesticides, with triphenyl phosphate (TPP) as the internal standard, on aqueous Chinese lettuce samples, at 0.1 and 1.0 μg/mL levels. QuEChERS provided superior sample recovery and precision (RSD) for nonpolar pesticides than it did for its polar variants, including most of the polar pesticides and several carbamates, as judged from quintuplicate analyses. A modification of the extraction buffer and cleanup reagent significantly enhanced the recovery of highly polar QA and carbamates at sub–microgram-per-milliliter levels, while maintaining the performance of the other nonpolar pesticides. Thus, QuEChERS is a versatile and convenient methodology for multi-residue analyses, although unambiguous confirmation and quantification of some polar pesticides, namely chlormequat, mepiquat, and glyphosate, required separate analyses using tedious and time-consuming single-residue methods. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) provides the potential advantages of low detection limits, simple sample preparation procedures, and high selectivities for multi-residue analyses of organic toxics in food. In this study, a novel methodology based on LC-MS/MS was developed for the simultaneous analyses of both polar and nonpolar pesticides; an online-coupled hydrophilic-interaction chromatography/reversed-phase (HILIC-RP) separation platform, featuring a HILIC column for online sample enrichment and a downstream high-flow injector for solvent mixing and reconditioning, was constructed to allow efficient, concomitant analyses of both hydrophilic and hydrophobic pesticides from a single sample injection event. The performance of the HILIC-RP system, verified using a standard pesticide mixture, was satisfactory. 6 out the 7 polar pesticides were successfully retained on the HILIC column and 11 out of the 15 nonpolar pesticides were transferred to and separated by the RP column; the remaining compounds were determined as flow-through prior to the HILIC separation. No sensitivity loss of the polar pesticides was observed when using this hyphenated system. The calibration curves for 20 out of the 22 standard pesticides exhibited excellent linear responses (R2 > 0.995) over a typical working concentration range. The performance of the HILIC-RP platform was comparable with those of individual one-dimensional HILIC and RP analyses, thereby potentially eliminating redundant quantification procedures and allowing highthroughput multi-residue analyses of toxic organic compounds. / published_or_final_version / Chemistry / Master / Master of Philosophy

abstract / Chemistry / Doctoral / Doctor of Philosophy

Cancer is one of the leading causes of morbidity and mortality in developed and developing nations. It is estimated that 86% of new cases and 64% of death due to cancer are from Africa and 13.1 million deaths are estimated to occur worldwide by the year 2030. Cancer death rates have not subsided despite recent advances in cancer drug development and treatment. Present cancer drug regimens are limited due to unpredictable efficiency, severe side effects ,resistance and high cost. Plants provide a vast array of natural compounds such as terpenoids, phenolics and alkaloids with antiproliferative pro-apoptotic and antioxidant effects. Plants are principal sources of compounds for drug discovery and development of several clinically proven useful anticancer drugs. The present study focused on the isolation of compounds from the Holarrhena floribunda (H. Floribunda) leaves for their potential anticancer activities. Standard methods were employed to assess the antiproliferative potential, apoptosis, cell cycle analysis and reactive oxygen species of the methanolic leaf extract (MLE) of H. floribunda. The standard methods of isolation such as column chromatography, thin layer Chromatography, high performance liquid chromatography (hplc) and nuclear magnetic Resonance (nmr) were used to isolate and purify bioactive compounds from the leaves. To Elucidate the mechanism of cytotoxicity of the isolated compounds, apoptosis effect was studied by flow cytometry analysis using the Apopercentagetm dye, Annexin-V/PI stain, induction of cspase-3 using the Caspase-3/7 glo assay kit and Parp-1 deactivation using Western blot analysis. The mode of action was further assessed by evaluating reactive oxygen species (ros), mitochondrial toxicity, light and fluorescent microscopic morphological evaluations of F-actin and topoisomerase-I relaxation assay. In addition, potential cancer prevention of the plant was also evaluated by assessing the antioxidant activity of the flavonoids compounds isolated from the mle. The results of the present study show that the MLE of H. floribunda inhibited the proliferation of the cancer cell lines (HeLa, HT-29 and MCF-7) in a dose- and timedependent manner. The anti-proliferative activity of the methanolic extract is selective towards cancer cells more than normal KMST-6 fibroblast cells used in the study. The extract showed cytotoxicity, cell cycle arrest, induced generation of ROS and apoptosis. The methanolic extract of the leaves led to the isolation of two pure steroidal alkaloid and four flavonoid compounds. The two steroidal alkaloids - holamine and funtumine were found to be selectively cytotoxic to human cancer cells more than the normal fibroblasts. The cytotoxicity of the two steroidal alkaloids was mediated through the induction of apoptosis. The apoptosis induction was found to be triggered by the activation of caspase-3, deactivation of PARP-1, increased ROS, cell cycle arrest at G0/G1 and G2/GM phases, mitochondria toxicity, F-actin disorganization and topoisomerase-I inhibition. However, four flavonoids (kaempferol-3-O-rutinoside, quercetin-3-galactoside/glucoside, quercetin-3-O-glucoside and kaempferol-3-O-glucoside) isolated were subjected to antioxidant activity assay using oxygen radical absorbance capacity (ORAC), ferric reducing/antioxidant power (FRAP), trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation inhibition. Two flavonoids with a quercetin nucleus were found to be active in the entire antioxidant spectrum, except pro-oxidant activity while the remaining two compounds with a kaempferol nucleus were selective in their activity. Structure-activity relationship analysis shows that the activity of the flavonoids depends on the ortho dihydroxyl group on the B-ring of the flavonoids. The present study on H. floribunda has shown that the leaves contain bioactive compounds with potential as anticancer agents and cancer protective and preventive activity. More work is still needed to elucidate other possible anticancer mechanisms that might be relevant to the cytotoxic effects of the two isolated alkaloids. / Philosophiae Doctor - PhD