Metabolite identification and annotation procedures are necessary for the discovery of
biomarkers indicative of diet, lifestyle, and disease. NMR spectroscopy remains a powerful tool
in metabolic profiling — however, despite its reproducibility, ease of use, and quantitative data
generation, its relatively limited sensitivity remains a bottleneck; low-concentration compounds
become indistinguishable from noise, and the signals of more concentrated compounds regularly
obscure those of less concentrated compounds. The aim of the project was to develop SPE-NMR
protocols utilising different cartridge chemistries, using both natural and artificial urine mixtures,
to produce unique retention profiles useful for metabolic profiling. These methods can then
be incorporated as part of an analyst’s ‘toolbox’ for metabolite identification and structural
elucidation.
Different cartridge sorbents and conditions were utilised in order to produce unique retention
profiles for different compound classes. Each elution demonstrated differing retention profiles
for each method — replicates of the same method, however, had little difference between
spectra, guaranteeing the reproducibility of the spectra. We found that application of the
developed SPE methods to biofluids (such as urine) can be used to selectively retain metabolites
based on compound taxonomy or other key functional groups, reducing peak overlap through
concentration and fractionation of unknowns, and hence promising greater control over the
metabolite annotation/identification process. Several NMR peaks demonstrating the presence of
3-hydroxyhippurate, unreported in literature or databases, were revealed through the use of the
developed methods on human urine — a range of analytical methods were subsequently utilised
in order to annotate the revealed peaks. Finally, standard operating procedures were written
and validated for an automated SPE system, such that the SPE methods can be more generally
used by researchers; this automated procedure was used to attempt to selectively retain specific
glucuronide metabolites found in human urine, with retention being demonstrated where the
moiety contains aromatic or otherwise significantly hydrophobic functional groups.