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

Abstract

Liquid chromatography-mass spectrometry (LC-MS)-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). 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 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.

Challenges and emergent solutions for LC‐MS/MS based untargeted metabolomics in diseases

“…101 In another comprehensive study, the chromatographic performance of RPLC, zwitterionic, amide-, alkyl diol-, and aminoalkyl-based HILIC and RP/ HILIC MMC on 714 metabolite standards and human plasma was compared. 124 Only 37% of the metabolite standards displayed good peak shape and good retention (retention factor k > 1) on RPLC, and as expected, a combination of MMC or HILIC with RPLC could expand the coverage up to 52-66% of the standards. Because the combination of different chromatography methods could increase metabolome coverage, MDLC approaches, which have been commonly adopted in proteomics, are also of interest in metabolomics.…”

“…Although ANP performed better in the analysis of polar metabolite standards compared to RPLC (80% of the standards eluted in the solvent front in RPLC), zwitterionic HILIC showed the best chromatographic performance and the greatest coverage for the metabolite standards and polar metabolites in urine . In another comprehensive study, the chromatographic performance of RPLC, zwitterionic, amide‐, alkyl diol‐, and aminoalkyl‐based HILIC and RP/HILIC MMC on 714 metabolite standards and human plasma was compared . Only 37% of the metabolite standards displayed good peak shape and good retention (retention factor k > 1) on RPLC, and as expected, a combination of MMC or HILIC with RPLC could expand the coverage up to 52‐66% of the standards.…”

Comparison of fully wettable RPLC stationary phases for LC‐MS‐based cellular metabolomics

“…Several previous studies have focused on metabolome coverage, peak capacity, and overall performance of different columns suitable for LC‐based metabolomics applications . For example, in the study of Wernisch et al., the comparison focused on investigation of metabolome coverage, retention time profile, peak shape, and selectivity from different hydrophilic interaction liquid chromatography (HILIC) columns . Walles et al.…”

“…RPLC remains the most popular mode of HPLC for practical broad scale metabolome coverage as the retention characteristics of these materials are well-suited to a range of moderately polar and nonpolar Several previous studies have focused on metabolome coverage, peak capacity, and overall performance of different columns suitable for LC-based metabolomics applications [10]. For example, in the study of Wernisch et al, the comparison focused on investigation of metabolome coverage, retention time profile, peak shape, and selectivity from different hydrophilic interaction liquid chromatography (HILIC) columns [11]. Walles et al also compared separation efficiency and precision of two metabolites: norverapamil and o-demethylverapamil in human-liver microsomal incubates analysis on two sub-2 m particle columns, with results indicating that there was no substantial improvement when changing from 1.8 m to 1.7 m particle size [12].…”

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…Recently, a comparison of the chromatographic performance of a traditional ODS column with various HILIC and mixed-mode columns showed that ODS and zwitterionic HILIC columns are the best combination for wide metabolite coverage. 51) e poor quality control and incompatibilities among the data acquisition procedures seriously hamper meta-analysis of biomarkers using metabolome datasets.…”