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Liquid chromatography (LC) separation combined with electrochemical coulometric array detection (EC) is a sensitive, reproducible, and robust technique that can detect hundreds of redox-active metabolites down to the level of femtograms on column, making it ideal for metabolomics profiling. EC detection cannot, however, structurally characterize unknown metabolites that comprise these profiles. Several aspects of LC-EC methods prevent a direct transfer to other structurally informative analytical methods, such as LC-MS and NMR. These include system limits of detection, buffer requirements, and detection mechanisms. To address these limitations, we developed a workflow based on the concentration of plasma, metabolite extraction, and offline LC-UV fractionation. Pooled human plasma was used to provide sufficient material necessary for multiple sample concentrations and platform analyses. Offline parallel LC-EC and LC-MS methods were established that correlated standard metabolites between the LC-EC profiling method and the mass spectrometer. Peak retention times (RT) from the LC-MS and LC-EC system were linearly related (r(2) = 0.99); thus, LC-MS RTs could be directly predicted from the LC-EC signals. Subsequent offline microcoil-NMR analysis of these collected fractions was used to confirm LC-MS characterizations by providing complementary, structural data. This work provides a validated workflow that is transferrable across multiple platforms and provides the unambiguous structural identifications necessary to move primary mathematically driven LC-EC biomarker discovery into biological and clinical utility.

Structural Characterization of Plasma Metabolites Detected via LC-Electrochemical Coulometric Array Using LC-UV Fractionation, MS, and NMR