| Summary: | Global metabolomic analysis of urine offers great potential for detection of early warning markers of disease. Current methods focus on rapid sample preparation and high throughput analyses at the expense of the detection of low abundance metabolites. The aim of this study was to develop sensitive analytical methods for metabolomic profiling. Methods were developed to use nanoflow ultra high performance liquid chromatography-nanospray ionization-mass spectrometry (nUHPLC-nESI-TOFMS), normally used for proteomics, for metabolomic analyses of urine samples. Compared with a conventional UHPLC-ESI-TOFMS, the use of a nanoflow-nanospray platform increased the sensitivity to a standard mixture of metabolites by 2-2000 fold. Highly repeatable results for retention time and metabolome peak area were achieved, where the coefficients of variation were <0.2% and <30% respectively for the majority of peaks present in the urine metabolome. To further increase sensitivity and enable small injection volumes, a sample preparation method was developed using polymeric anion and cation exchange mixed mode solid phase extraction with pre-concentration. Combined with the nano platform, this enabled the detection of low abundance signalling molecules (estrogens, eicosanoids and unconjugated androgens) not usually detected with conventional methods. A pre-analysis normalisation technique based on osmolality concentrations was used to reduce sample variability due to differing urine concentrations. These methods were used to investigate the metabolomic consequences of HIV infection and patient response to combined antiretroviral therapy (cART). No significant differences in metabolomic profiles between HIV positive and negative patients were observed. However, disruption of bile acid profiles and decreased concentrations of selected carnitines, steroid conjugates, polypeptides and nucleosides were detected in patients on cART therapy indicating disrupted lipid and protein metabolism but improved immunological function associated with antiretroviral medication. These finding highlight the importance of these newly developed SPE sample preparation and nUHPLC-nESI-TOFMS analysis methods for global profiling of the urinary metabolome.
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