Serially coupling hydrophobic interaction and reversed-phase chromatography with simultaneous gradients provides greater coverage of the metabolome

Madalina Oppermann,Stefan Weidt,Karl E. V. Burgess,Richard J. Burchmore,Matthew J. Dalby,Ken Cook,Jennifer Haggarty

doi:10.1007/s11306-014-0770-7

Abstract

The serial coupling of a reversed-phase liquid chromatography (RPLC) column to a hydrophilic interaction liquid chromatography (HILIC) column has been developed in recent years for the detection of polar and nonpolar metabolites. TCA intermediates, bile acid standards and numerous polar and non-polar metabolites extracted from beer were analysed using a combined RPLC/HILIC method. Non-polar metabolites were retained by the RPLC column. Polar metabolites not retained by the RPLC column were retained and separated by the HILIC column. The results from this study validate this simple yet powerful metabolomics approach.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-014-0770-7) contains supplementary material, which is available to authorized users.

Highlights

The field of metabolomics has the difficult task of attempting to characterise and quantifying the vast array of metabolic compounds present in biological systems
The serial coupling of a reversed-phase liquid chromatography (RPLC) column to a hydrophilic interaction liquid chromatography (HILIC) column has been developed in recent years for the detection of polar and nonpolar metabolites
Some of the most important compound classes in metabolomics fall into these groups: carbohydrates, organic acids, amino acids and nucleotides chromatograph poorly on reversed phase (RP) HPLC, but retain and separate well with some hydrophilic interaction liquid chromatography (HILIC) columns, —the ZIC-pHILIC zwitterionic surfactant column

Summary

Introduction

The field of metabolomics has the difficult task of attempting to characterise and quantifying the vast array of metabolic compounds present in biological systems. Diametrically opposed organic concentration at the start of each separation has led to the development of on-line RPLC/ HILIC valve switching systems (Wang et al 2008; Thomas et al 2010; Wang and Lehmann 2008) These methods, successful, are not suited for routine use in most laboratories as dedicated interfaces are required for handling mobile phase incompatibilities. This paper describes a metabolomics method in which a sample can be injected onto both RP and HILIC columns in tandem for the analysis of polar and non-polar compounds in a single injection This method allows for the independent optimisation of both columns and removes the need for separate sample preparations or the addition of ion pairing reagents. 10 lL of each sample was injected onto the column in quadruplicate

Beer samples

Identification

Chemicals and reagents

Standards

Chromatographic conditions

Evaluation of reproducibility

Beer sample