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

Fri, 2016-09-16 13:19 -- voskuhlt
TitleEvaluation of coverage, retention patterns, and selectivity of seven liquid chromatographic methods for metabolomics.
Publication TypeJournal Article
Year of Publication2016
AuthorsWernisch S, Pennathur S
JournalAnal Bioanal Chem
Volume408
Issue22
Pagination6079-91
Date Published2016 Sep
ISSN1618-2650
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. 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.

DOI10.1007/s00216-016-9716-4
Alternate JournalAnal Bioanal Chem
PubMed ID27370688
PubMed Central IDPMC4983217
Grant ListDP3 DK094292 / DK / NIDDK NIH HHS / United States
P30 DK081943 / DK / NIDDK NIH HHS / United States
P30 DK089503 / DK / NIDDK NIH HHS / United States
R24 DK082841 / DK / NIDDK NIH HHS / United States
U24 DK097153 / DK / NIDDK NIH HHS / United States
U2C ES026553 / ES / NIEHS NIH HHS / United States
UL1 TR000433 / TR / NCATS NIH HHS / United States