Liquid chromatography-mass spectrometry platforms hyphenated with coulometric array and microscale nuclear magnetic resonance detection

This manuscript centers on the hyphenation of analytical detection technologies, specifically, the coupling of liquid chromatography-mass spectrometry (LC-MS) to the complementary analytical methods of electrochemical array (EC-array) detection and nuclear magnetic resonance (NMR). Chapter 1 provides a detailed overview of the specific detection methods used throughout this dissertation.;Chapter 2 focuses on the combination, in parallel, of LC-MS and EC-array detection methods into a streamlined platform and its application to drug metabolism studies. The platform's performance was evaluated by demonstrating retention of chromatographic integrity between the two detectors where retention times and peak widths at half height between the EC-array and MS were reproducible with relative standard deviations (RSD) < 10 %. Additionally, through a comparison of EC-array and MS relative limits of detection the system's compatibility for parallel metabolite analysis is clearly established, detecting down to 600 pg injected on column with merely femtogram levels being delivered to the MS.;Chapter 3 applies the LC-EC-array-MS platform to a more detailed metabolic assessment of the oral drug SPB as a histone deacetylation (HDAC) inhibitor in a safety and tolerability study of SPB in HD patients. An iterative process was developed with LC-EC-array and parallel LC-EC-array-MS detection for characterizing these metabolites. 10 metabolites were identified in treated subjects including indole species in urine that are not directly related to structural modifications of SPB, but were only found in SPB treated HD patients. The application of the process was directed at understanding metabolic pathways that differ among HD individuals when being treated with SPB and when not treated. These previously unreported metabolites resulting from SPB therapy may have both implications both on the disease processes in HD and a secondary effect of the therapeutic intervention in combination with HDAC processes. Both of these aspects will all be discussed.;In Chapter 4, two innovations in microscale analysis, nanoSplitter LC-MS (Chapter 2) and microdroplet NMR were combined for the identification of unknown compounds found at low concentrations in complex sample matrices as frequently encountered in metabolomics or natural products discovery. Microdroplet NMR is a droplet microfluidic NMR loading method providing several-fold higher sample efficiency than conventional flow-injection methods. Performing NMR offline from LC-UV-MS accommodated the disparity between MS and NMR in their sample mass and time requirements, as well as allowing NMR spectra to be requested retrospectively, after review of the LC-MS data. Interpretable 1D NMR spectra were obtained from analytes at the 200 ng level, in 1-hour-per-well automated NMR data acquisitions.;In Chapter 5, the synthesis, isolation and analytical characterization of DNA-adducts, using the microscale LC-MS-NMR platform, is described. These adducts include both N-(deoxyguanosin-8-yl)-aminobiphenyl (C8-dG-ABP) and N-(deoxyguanosin-8-yl)-aminobiphenyl-d9 (C8-dG-ABP-d 9), as well as the identification of various isomeric compounds associated with the two adducts. This characterization was achieved using the LC-MS-NMR platform described in Chapter 4 of this thesis, but, with manual microdroplet injections into the microcoil NMR as opposed to using the automated sample handler. This change was made in order to more effectively recover the analytes for post-NMR use and allow interactive NMR acquisition, as well as provide more efficient sample injections for trace analysis compounds.;Chapter 6 offers recommendations for future research based on the studies presented in this dissertation. (Abstract shortened by UMI.)...