Roger Kautz, Michael Pollastri, Carolyn Lee-Parsons, G. Satyrayana Reddy, Bruce Kristal
Date of Award
Doctor of Philosophy
Department or Academic Unit
Department of Chemistry and Chemical Biology
chemistry, analytical chemistry, LC-MS-NMR, mass spectrometry, metabolomics, nuclear magnetic resonance, vitamin D
Analytical Chemistry | Chemistry
This thesis centers on the applications of mass spectrometry based techniques and nuclear magnetic resonance (NMR) to the structural elucidation of low molecular weight analytes in complex mixtures. In the first chapter, a detailed overview of the challenges associated with the integration of LC-MS and NMR and the approaches that have been taken towards overcoming their mutually incompatible features in order to create an efficient analytical platform is discussed.
Chapter 2 introduces an integrated LC-MS-NMR platform developed here at Northeastern that provides high sensitivity for both MS and NMR. The platform uses a post-column nanoSplitter for online LC-MS, and offline microdroplet NMR. The nanoSplitter provides nanoelectrospray from 4 mm columns whilst collecting most of the flow for offline NMR. Nanoelectrospray reduces signal suppression and increases sensitivity when compared to conventional electrospray. Microfluidic sample loading, whereby the analyte is carried as a droplet in an immiscible oil, increases sample loading efficiency into a microcoil NMR probe, for conducting NMR in the most mass sensitive way. Because MS and NMR have different sample and time requirements, NMR is conducted offline, which also allows for evaluation of MS data in prioritizing goals for NMR analysis. The utility of this platform is shown in the analysis of plant cell cultures. Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. The utility of our LC-MS-NMR approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced to produce benzophenanthridine alkaloids. Preliminary HPLC-UV analysis provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the optimal yield of alkaloid products, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level.
In Chapter 3, a robust and highly sensitive analytical method that combines the strengths of mass spectrometry, and NMR is developed. The end-goal of the work presented is to utilize this analytical method in the structural elucidation of metabolites affected by diet. These metabolites had previously been identified as markers of interest in a metabolomics study (the study of all low molecular weight analytes in a biological system) using liquid-chromatography-electrochemical detection (LC-EC). The structural characterization of these metabolites would aid in identifying metabolic pathways altered by changes in diet and their association to disease risk. Although highly sensitive (LODs at femtomole level), LC-EC provides little structural information thus the need for MS and NMR. The validity of the developed analytical method is demonstrated in the structural elucidation of metabolites in human plasma. For MS, a high-resolution mass spectrometer operated with full-scan MS alternating between positive and negative ion mode, and with high-energy collisional dissociation (HCD) all ion fragmentation in each mode for MS/MS. Additional structural information of volatile and semi-volatile metabolites is also gained from GC-MS analysis. Microcoil NMR, with limits of detection at 2 nmoles, is used as a follow-up to MS analysis. By integrating data from mass spectrometry, microcoil NMR and LC-EC, nine metabolites from human plasma are unambiguously identified.
In Chapter 4, the superseding role of GC-MS for structural elucidation of Vitamin D compounds is demonstrated. The hormonally active form of Vitamin D, 1α,25(OH)2D3 (Vitamin D3), has therapeutic effects in various diseases including cancer. The major limitation to using Vitamin D3 as a therapeutic agent is its potent calcemic activity, which leads to hypercalcemia. Hypercalcemia leads to the formation of renal stones, soft tissue calcification and can be lethal. A second limitation of the use of Vitamin D3 as a therapeutic agent is its short half-life caused by its rapid metabolism and inactivation by CYP 24A1 hydroxylase. In this chapter, a combination of GC-MS and HPLC were utilized to investigate the metabolism and stability of two vitamin D analogs which were designed to resist metabolic inactivation by CYP24A1 and with reduced calcemic effect. The metabolism of the C-3 epimer, a natural metabolite of 1α,25(OH)2D3 by CYP24A1, is also investigated . 1α, 25(OH)2-3epi-D3 differs from Vitamin D3 in the configuration of the hydroxyl group at C-3 and has been shown to have the same therapeutic effects as Vitamin D3 but without the calcemic effects.
In Chapter 5, some preliminary results demonstrating a metabolite extraction scheme utilizing chelating agents for improved metabolite recovery in metabolomics are presented. This chapter also includes some recommendations for future research based on the studies presented in this dissertation.
Rose Muthoni Gathungu
Gathungu, Rose Muthoni, "The integration of mass spectrometry and NMR for structural characterization of trace-level analytes in complex mixtures" (2012). Chemistry Dissertations. Paper 43. http://hdl.handle.net/2047/d20002669
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