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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
Authors:Nathaniel W. Snyder  Maya Khezam  Clementina A. Mesaros  Andrew Worth  Ian A. Blair
Affiliation:Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania
Abstract:Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.
Keywords:Biochemistry   Issue 75   Chemistry   Molecular Biology   Cellular Biology   Physiology   Medicine   Pharmacology   Genetics   Genomics   Mass Spectrometry   MS   Metabolism   Metabolomics   untargeted   extraction   lipids   accurate mass   liquid chromatography   ultraperformance liquid chromatography   UPLC   high resolution mass spectrometry   HRMS   spectrometry
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