1. PhD ABSTRACT
In this work the concurrent use of nuclear magnetic resonance (NMR) spectroscopy
and mass spectrometry, both off-line and on-line has been shown to provide an
efficient and accurate means of characterising novel drug metabolites in biological
fluids. In particular the use of 1H NMR spectroscopy as a generic and universal
detector for the analysis of HPLC fractions derived from preparative fractionation of
biological fluids has been demonstrated. This has been facilitated by the fact that the
majority of drug candidates are organic molecules containing protons, and their
detection by 1H NMR spectroscopy is independent of polarity, size, volatility and the
presence or absence of a chromophore, thereby making this an extremely efficient
approach. The coupling of NMR spectroscopy, mass spectrometry and HPLC
together to create a completely integrated system (LC/NMR/MS) has also been
demonstrated, and has subsequently been applied to several drug metabolism
problems. The serial and parallel modes of operation have been both investigated and
optimised. The development of mass-directed NMR spectroscopy in stop-flow mode
for the direct characterisation of urine metabolites of novel drug candidates, with out
any sample pre-treatment, has also been reported. This work presents the application
of these techniques to drug metabolite characterisation using both clinical and pre-
clinical samples. Several noteworthy metabolite structures are presented, including a
novel N-O-glucuronide and NIH-shift metabolites, which are reported here
systemically in human sera for the first time. The advantages of these combined
systematic approaches for metabolite identification are described. Where necessary,
improvements to the techniques used have been proposed, which will be the subject
of future work.