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. 2016 Sep;408(22):6079-91.
doi: 10.1007/s00216-016-9716-4. Epub 2016 Jul 1.

Evaluation of Coverage, Retention Patterns, and Selectivity of Seven Liquid Chromatographic Methods for Metabolomics

Free PMC article

Evaluation of Coverage, Retention Patterns, and Selectivity of Seven Liquid Chromatographic Methods for Metabolomics

Stefanie Wernisch et al. Anal Bioanal Chem. .
Free PMC article


Liquid chromatography-mass spectrometry-based metabolomics studies require highly selective and efficient chromatographic techniques. Typically employed reversed-phase (RP) methods fail to target polar metabolites, but the introduction of hydrophilic interaction liquid chromatography (HILIC) is slow due to perceived issues of reproducibility and ruggedness and a limited understanding of the complex retention mechanisms. In this study, we present a comparison of the chromatographic performance of a traditional RP-C18 column with zwitterionic, amide-, alkyl diol-, and aminoalkyl-based HILIC and mixed-mode columns. Our metabolite library represents one of the largest analyte sets available and consists of 764 authentic metabolite standards, including amino acids, nucleotides, sugars, and other metabolites, representing all major biological pathways and commonly observed exogenous metabolites (drugs). The coverage, retention patterns, and selectivity of the individual methods are highly diverse even between conceptually related HILIC methods. Furthermore, we show that HILIC sorbents having highly orthogonal selectivity and specificity enhance the coverage of major metabolite groups in (semi-) targeted applications compared to RP. Finally, we discuss issues encountered in the analysis of biological samples based on the results obtained with human plasma extracts. Our results demonstrate that fast and highly reproducible separations on zwitterionic columns are feasible, but knowledge of analyte properties is essential to avoid chromatographic bias and exclusion of key analytes in metabolomics studies. Graphical Abstract The chromatographic parameters of 764 authentic metabolite standards provide the basis for a comparison of coverage, selectivity and orthogonality of 7 reversed-phase (RP), mixed-mode (MM) and hydrophilic interaction liquid chromatography (HILIC) methods.

Keywords: High-performance liquid chromatography; Hydrophilic interaction liquid chromatography; Mass spectrometry; Metabolomics; Reversed phase; Selectivity.

Conflict of interest statement

Compliance with Ethical Standards

No experiments requiring Institutional review board (IRB) approval were carried out. The biological samples in this study were obtained from commercial sources and therefore IRB exempt.

The authors declare that no conflict of interest exists.


Fig. 1
Fig. 1. Properties of metabolites in the analyte set
a: Sub-groups of metabolites. Percentage based on total library (764 compounds). b: Octanol-water partition coefficients logD of 726 ionizable compounds at pH=3. (Missing values: Data not available.) c: Acid dissociation constants pKa for 652 acidic and 381 basic moieties. Note: Some metabolites contain >1 ionizable group.
Fig. 2
Fig. 2. Library coverage of RP column A and mixed-mode and HILIC columns B–G
a: Chromatographic peak-shape for all 764 compounds. b: Well-retained compounds with retention factors k ≥ 1.
Fig. 3
Fig. 3. Coverage comparison of RP-C18, mixed-mode and HILIC methods
Numbers inside circles: Metabolites exclusively detected with each method. Center: compounds detected with both. Percentages relative to detectable compounds in library (714). a: Metabolites with good peakshapes. b: Well-retained compounds with good peakshapes and retention factors k ≥ 1. c: Overlapping selectivity ranges for zwitterionic HILIC methods (B vs. E), zwitterionic and neutral SPs (E and B vs. F), SPs with terminal amino groups (E vs. G), neutral HILIC (D vs. F) and for a mixed-mode column run in RP and HILIC modes (C vs. D).
Fig. 4
Fig. 4. Coverage of metabolite groups
Shaded bars: retention factor>1, good peak-shape, colored bars: number of metabolites not captured by RP-C18 method A. For method A, “exclusive” compounds are not detected by B–F. b. Complementarity of phosphorylcholine-based column E and other HILIC columns for amino acids subgroup.
Fig. 5
Fig. 5. Shifting peaks during HILIC column conditioning with human plasma extract
During the first runs, reproducibility of the total ion chromatogram (TIC) is poor. After completed conditioning, retention times are stable with an RSD of <2%. Chromatograms recorded with method B.
Fig. 6
Fig. 6. Extracted ion chromatograms of proteinogenic amino acids obtained with methods B (left panel) and E (right panel)
Hydrophobic amino acids elute earlier and highest retention times are observed for basic amino acids lysine (Lys) and arginine (Arg).

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