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 due to inadequate retention. Our objective was to develop a 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. A 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 due to solubility and retention limitations of the injection solvent. Instead, we optimized a multiplexed approach using the CORTECS T3 column to analyze semipolar compounds and the CORTECS C8 to analyze lipids. Strikingly, we determined that combining these methods enabled detection of 41 of the total 47 standards whereas our reference RPLC method only detected 10 of 47. We then applied credentialing to compare method performance at the comprehensive scale. The tandem method showed over a 5-fold 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.
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