The development of enabling mass spectrometry platforms for the quantification of diverse lipid species in human urine is of paramount importance for understanding metabolic homeostasis in normal and pathophysiological conditions. Urine represents a non-invasive biofluid that can capture distinct differences in an individual's physiological status. However, currently there is a lack of quantitative workflows to engage in high throughput lipidomic analysis. This study describes the development of a MS/MS(ALL) shotgun lipidomic workflow and a micro liquid chromatography-high resolution tandem mass spectrometry (LC-MS/MS) workflow for urine structural and mediator lipid analysis, respectively. This workflow was deployed to understand biofluid sample handling and collection, extraction efficiency, and natural human variation over time. Utilization of 0.5 mL of urine for structural lipidomic analysis resulted in reproducible quantification of more than 600 lipid molecular species from over 20 lipid classes. Analysis of 1 mL of urine routinely quantified in excess of 55 mediator lipid metabolites comprised of octadecanoids, eicosanoids, and docosanoids generated by lipoxygenase, cyclooxygenase, and cytochrome P450 activities. In summary, the high-throughput functional lipidomics workflow described in this study demonstrates an impressive robustness and reproducibility that can be utilized for population health and precision medicine applications.
Keywords: Lipid analysis; Lipid metabolism; Mass spectrometry; Molecular species; Phospholipid analysis; Second messengers.