A Comprehensive LC–MS Metabolomics Assay for Quantitative Analysis of Serum and Plasma

Background/Objectives: Targeted metabolomics is often criticized for the limited metabolite coverage that it offers. Indeed, most targeted assays developed or used by researchers measure fewer than 200 metabolites. In an effort to both expand the coverage and improve the accuracy of metabolite quantification in targeted metabolomics, we decided to develop a comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that could quantitatively measure more than 700 metabolites in serum or plasma. Methods: The developed assay makes use of chemical derivatization followed by reverse phase LC-MS/MS and/or direct flow injection MS (DFI-MS) in both positive and negative ionization modes to separate metabolites. Multiple reaction monitoring (MRM), in combination with isotopic standards and multi-point calibration curves, is used to detect and absolutely quantify the targeted metabolites. The assay has been adapted to a 96-well plate format to enable automated, high-throughput sample analysis. Results: The assay (called MEGA) is able to detect and quantify 721 metabolites in serum/plasma, covering 20 metabolite classes and many commonly used clinical biomarkers. The limits of detection were determined to range from 1.4 nM to 10 mM, recovery rates were from 80% to 120%, and quantitative precision was within 20%. LC-MS/MS metabolite concentrations of the NIST^® SRM^®1950 plasma standard were found to be within 15% of NMR quantified levels. The MEGA assay was further validated in a large dietary intervention study. Conclusions: The MEGA assay should make comprehensive quantitative metabolomics much more affordable, accessible, automatable, and applicable to large-scale clinical studies.