Application of novel SPE-NMR protocols and flow-chemistry synthesis for comprehensive annotation of the human urinary metabolome, metabolite identific

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Metabolite structure elucidation is considered a bottleneck in metabonomics/metabolomics - metabolites found within tissues and biofluids (the most common being urine, as it is collected non-invasively and easily available) can be identified and proven to be biomarkers characteristic of specific phenotypes, such as disease states. However, the human urinary metabolome remains only partially mapped. There is hence a need to expand the annotation of the human urine metabolome in order to improve our capacity for characterization of population phenotypes and discovery of biomarkers related to diseases and diet. Currently, the two most powerful analytical techniques used for metabolite annotation and identification are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, as they provide orthogonal qualitative data, on top of absolute and relative quantification in a precise and high-throughput manner. NMR is a powerful technique in the characterisation of metabolites - the quantitative, non-destructive, and rapid processing of samples, as well as the inherent reproducibility of the data produced, can give experimenters an additional dimension of information about biofluids, which are orthogonal to the datasets produced by mass spectrometry.Solid Phase Extraction (SPE) can be thought of as liquid-solid chromatography, in which a packed column is used to separate out compounds within a complex mixture based on interactions such as polarity and hydrophobicity. This allows for the compounds to be extracted, enriched, and purified for MS/NMR analysis. Silica- and polymer-based solid phase extraction utilises columns with modifications (such as C18 octadecyl chains) attached to a silica or polymer 'backbone'. Different modifications with different chemistries allow for a different retention profile.Samples pre-treated with SPE methods can be utilized for isolation of molecules and subsequent structure elucidation using various 1D and 2D NMR techniques and MS. The different chemistries of SPE cartridges (such as cation and anion exchange) can be utilised in order to modulate and simplify NMR spectra of biofluids. This approach is similar to one previously reported by Zhang et al, in which negatively or positively charged silica nanoparticles were added to a biofluid sample, causing weakening and suppression of the NMR signals of positively or negatively charged metabolites, respectively. The approach helped to remove overlap in 13C-1H HSQC spectra by removal of crosspeaks, aiding characterisation of the sample. The aim of the project is to utilise cartridges of differing chemistries, in order to produce protocols for the selective fractionation of human urine based on metabolite class. Variations of SPE experiments will be done by varying solvent systems, urine concentrations, and cartridge chemistries. Protocols which can be transferred onto an automated SPE system can then be developed. LC-MS and direct infusion (DIMS) will also be used to aid metabolite identification. A key aspect of the identification process involves the comparison of putatively annotated compounds to authentic standards. However, many standards (and their spectra) are not available due to lack of demand, high tailored-synthetic costs, or simply because the standard in question was not previously required. The use of organic flow chemistry has been popularised by the group of Prof. Steven Ley. Flow kits can be set up in a modular fashion, enabling microfluidic experiments to be run for the synthesis of natural products. This approach has several advantages over 'traditional' synthesis: the modular nature of the equipment allows for setups to be changed as necessary for any given experiment, the entire rig can be automated for better ease of use and control over a system, and the reactions in question run at high efficiencies, creating less waste - hence being a keystone in the future of sustainable chemistry.