HPLC-MS and HPLC-NMR method development and applications for small molecule bioanalysis.

摘要

The rapidly evolving field of metabolomics or metabolic profiling focuses on the quantitative and qualitative analysis of a large or limited number of small molecules in a given biological sample. Hyphenated analytical techniques combining separation technology such as HPLC with MS or NMR are highly effective and enabling tools for metabolic profiling of small biological molecules. In this thesis the development and applications of coupled HPLC-TQMS and HPLC-microcoil/NMR techniques for investigation of drug and endogenous metabolites are presented.;First, an automated multiplex-HPLC-TQMS system capable of performing analytical separation on multiple columns following MS acquisition in multiple-reaction monitoring (MRM) mode is described. This system was utilized to perform targeted serum metabolic profiling of nucleosides in esophageal adenocarcinoma patients. A number of putative nucleoside biomarkers was discovered. Second, a novel automated HPLC-microcoil/NMR system was illustrated that performed analytical separation, pre-concentration, and NMR spectroscopy in rapid succession. The central component of the system was the online column-trapping pre-concentration technique that resulted in up to 14-fold enhancement in the NMR signal. This system was used for the separation and identification of anti-inflammatory drugs in a standard mixture, and for the identification of ibuprofen metabolites in human urine. Lastly, a "chromatogram-resolved" NMR spectroscopy technique for identification of low-abundance metabolites is described. This method, based on sample fractioning on an analytical column, allowed structural elucidation of low-concentration metabolites that were unidentifiable from NMR spectra of the whole urine.