| Summary: | 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. An investigation of an eight compound mixture, representative of the diversity typically encountered in physiological systems, both in neat solution and a serum matrix with limited sample cleanup demonstrates the system's ability to handle biological samples without concern for biological matrix effects. Finally, by using the nanoelectrospray
LC-EC-array-MS system, its unique abilities in preliminary metabolomics analyses were highlighted through successful identification of unknown sodium phenyl butyrate (SPB) drug metabolites in Huntington's disease (HD) patient plasma. 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. Using a method employing gradient LC with
EC-array, UV and Fluorescence (F) (LCECA/UV/F), treated patient plasma and urine gave individual-specific patterns of ca. 20 SPB metabolites which may relate to the selection of subjects for extended trials of SPB. The structural identification of these metabolites was of critical importance, since characterization will aid in the understanding of mechanisms of drug action and possible side effects. 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. The system also showed excellent intra- and inter-detector reproducibility with retention time RSD values less than 2%, and sample recovery on the order of 93%. When applied to a cyanobacterial extract showing antibacterial activity, the platform recognized several previously-known metabolites, down to the 1% level, in a single 30 μ,g injection, and prioritized one unknown for further study. 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-d9), 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. Both the LC-MS fraction collection and manual injection microdroplet NMR analyses were evaluated for sample recovery and injection efficiency, using a dG standard, prior to adduct analysis. Each adduct was analyzed using LC-MS-micrcoilNMR and subsequently
recovered for future use in in vitro and in vivo studies correlating DNA adduct isomer persistence to biological endpoints such as apoptosis, gene transcription, mutagenesis and cancer. Chapter 6 offers recommendations for future research based on the studies presented in this dissertation.
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