Two complementary reversed-phase separations for comprehensive coverage of the semipolar and nonpolar metabolome
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Although it is common in untargeted metabolomics to apply reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) methods that have been systematically optimized for lipids and central carbon metabolites, here we show that these established protocols provide poor coverage of semipolar metabolites because of inadequate retention. Our objective was to develop an RPLC approach that improved detection of these metabolites without sacrificing lipid coverage. We initially evaluated columns recently released by Waters under the CORTECS line by analyzing 47 small-molecule standards that evenly span the nonpolar and semipolar ranges. An RPLC method commonly used in untargeted metabolomics was considered a benchmarking reference. We found that highly nonpolar and semipolar metabolites cannot be reliably profiled with any single method because of retention and solubility limitations of the injection solvent. Instead, we optimized a multiplexed approach using the CORTECS T3 column to analyze semipolar compounds and the CORTECS C8 column to analyze lipids. Strikingly, we determined that combining these methods allowed detection of 41 of the total 47 standards, whereas our reference RPLC method detected only 10 of the 47 standards. We then applied credentialing to compare method performance at the comprehensive scale. The tandem method showed more than a fivefold increase in credentialing coverage relative to our RPLC benchmark. Our results demonstrate that comprehensive coverage of metabolites amenable to reversed-phase separation necessitates two reconstitution solvents and chromatographic methods. Thus, we suggest complementing HILIC methods with a dual T3 and C8 RPLC approach to increase coverage of semipolar metabolites and lipids for untargeted metabolomics.
KeywordsMetabolomics Untargeted profiling Mass spectrometry Global coverage Semipolar metabolome Reversed-phase
This work was supported by the National Institutes of Health (grants R35 ES028365, R21 CA191097, R01 GM05698), the Alfred P. Sloan Foundation, the Camille & Henry Dreyfus Foundation, the Edward Mallinckrodt, Jr. Foundation, and the Pew Scholars Program in the Biomedical Sciences.
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Conflict of interest
GJP is a scientific advisory board member for Cambridge Isotope Laboratories and a recipient of the Agilent Early Career Professor Award. The other authors declare that they no have competing financial interests. The authors declare that they have no nonfinancial conflicts of interest.
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