Large scale targeted metabolomics assay for quantitative plasma profiling Using the SCIEX Triple Quad or systems

Untargeted serum metabolomics studies have detected, identified and documented over 75% of endogenous compounds. 1 However, only a handful of robust and sensitive targeted methods are in existence to quantify large panels of endogenous metabolites in a single assay. Methods for the quantification of plasma metabolites have great utility for many applications, including disease research, food omics and pharmaceutical development, among others. However, the wide structural diversity of metabolites, and hence the broad chromatographic and ionization properties of these compounds, have posed an immense challenge in developing high-throughput, large scale targeted LC-MS methods for semi-quantitative purposes. Here, a large, high-throughput metabolite method consisting of 336 serum metabolites was developed and tested using SRM 1950 plasma. The chromatographic separations were performed on a Kinetex F5 (Phenomenex, CA) column using standard reversed-phase mobile phases for reliable separations of a majority of serum analytes. Development of the MRM method was done both from standards and from testing in matrix, as well as leveraging the CAS numbers, accurate mass information, compound nomenclature and groups from Human Metabolite Data Base (HMDB). Both polarity switching and the Scheduled MRM algorithm were used to ensure the robust analysis of this large panel of analytes in a single injection. The assay was tested on both the SCIEX Triple Quad 7500 system and the QTRAP 6500+ system. 3