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Evaluation of coverage, retention patterns, and selectivity of seven liquid chromatographic methods for metabolomics

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Abstract

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.

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

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Abbreviations

A:

Adenine/adenosine

Arg:

Arginine

C:

Cytosine/cytidine

ESI:

Electrospray ionization

G:

Guanine/guanosine

HILIC:

Hydrophilic interaction liquid chromatography

His:

Histidine

HMDB:

Human Metabolome Database

HPE:

Human plasma extract

Ile:

Isoleucine

LC-MS:

Liquid chromatography–mass spectrometry

Leu:

Leucine

LSER:

Linear solvation energy relationships

Lys:

Lysine

MM:

Mixed mode (stationary phase)

MP:

Mobile phase

m/z :

Mass-to-charge ratio

Phe:

Phenylalanine

QSSR:

Quantitative structure–selectivity relationships

Q-TOF:

Quadrupole time-of-flight (mass spectrometer)

RP:

Reversed phase

RSD:

Relative standard deviation

SP:

Stationary phase

T:

Thymine/thymidine

Val:

Valine

WAX:

Weak anion exchanger

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Acknowledgments

The authors would like to thank Prof. Wolfgang Lindner (University of Vienna) for the generous loan of HILIC columns used in this study. Research reported in this publication was supported in part by grants from the National Institutes of Health (DK094292, DK089503, DK082841, DK081943, U2C-ES-026553, DK097153, and UL1TR000433).

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Authors and Affiliations

  1. Division of Nephrology, Department of Internal Medicine, University of Michigan, 5309 Brehm Center, 1000 Wall St., Ann Arbor, MI, 48105, USA

    Stefanie Wernisch & Subramaniam Pennathur

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  1. Stefanie Wernisch
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  2. Subramaniam Pennathur
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Correspondence to Subramaniam Pennathur.

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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.

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The authors declare that no conflict of interest exists.

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Wernisch, S., Pennathur, S. Evaluation of coverage, retention patterns, and selectivity of seven liquid chromatographic methods for metabolomics. Anal Bioanal Chem 408, 6079–6091 (2016). https://doi.org/10.1007/s00216-016-9716-4

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  • DOI: https://doi.org/10.1007/s00216-016-9716-4

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