Two complementary reversed-phase separations for comprehensive coverage of the semipolar and nonpolar metabolome

Abstract

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. Graphical abstract Analysis of semipolar and nonpolar metabolites necessitates two reversed-phase chromatography (RPLC) methods, which extend metabolome coverage more than fivefold for untargeted profiling. HILIC hydrophilic interaction liquid chromatography.

Keywords: Global coverage; Mass spectrometry; Metabolomics; Reversed-phase; Semipolar metabolome; Untargeted profiling.

Fig. 1

Comparison of 47 small-molecule standards between columns. The T3 demonstrated the best performance for semipolar standards. The C8 had the best performance for nonpolar standards. Combined, the two CORTECS methods had the best overall performance with 41 of the 47 standards being reliably detected. Red indicates that a standard was undetected, eluted in the void volume, or had an unreliable peak shape. Yellow indicates peak tailing or fronting, band broadening, or asymmetry (see Fig. 2 for examples).

Fig. 2

Optimizing mobile-phase buffers and RPLC. A: Adding 5 mM phosphate buffer to the mobile phase increases the signal intensities of semipolar metabolites. The ratio between signal intensity with and without phosphate buffer shows an increase in signal for all standards but histamine. B–C: EIC and TIC of serotonin shows retention on T3 but not C8. D: Mass spectra from T3 and C8 show less serotonin signal suppression on T3 relative to C8. E: EIC of phosphatidylcholine (24:0/24:0) shows optimal peak shape for C8, peak tailing for T3 and C18, and peak degradation for C18+. F: EIC for diacylglycerol (18:1/18:1) and diacylglycerol (18:0/18:2). The structural isomers start to be resolved on the T3 and C18, but not the C8.

Fig. 3

Comparison of peak intensities and widths between columns. A: Standards detected in XBridge C18, CORTECS T3, and CORTECS C8 experiments have similar intensities. See ESM (Table S1) for full chemical names of each standard. B: Ratio of standard peak widths detected on the CORTECS C8 and T3 methods with respect to low and high log P values.

Fig. 4

Metabolites detected from E. coli samples. A: Ten semipolar metabolites were identified from analysis with the T3 method, but were mostly undetected and poorly resolved with other methods. B: Ten complex lipids were identified from analysis with the C8 method with optimal peak shape. The lipids were undetected with the XBridge C18 and CORTECS T3 methods. See Fig. 1 for more detailed descriptions of colors.