Quantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatography (HILIC)-MS/MS profiling method was developed for the efficient separation and quantification of small polar molecules, mostly primary metabolites. The method was based on an ultrahigh performance liquid chromatography (UHPLC) separation system coupled with ESI mass spectrometry on a triple quadrupole mass spectrometer, operating in both positive and negative ionisation mode using rapid polarity switching. With the developed method quantitation of 135 compounds belonging in four major classes of polar compounds (sugars, aminoacids, organic acids and amines) was achieved in a single run of 30 min. The method was applied to grape extracts from different varieties and provided information on primary metabolite content. Multivariate statistical analysis was applied using the concentrations found, with the aim of investigating the differences in metabolite profiles. Classification of grapes according to their skin colour was carried out using principle component analysis based on the concentration variation of a number of the metabolites studied.

► A new HILIC–MS/MS method for the determination of 124 polar metabolites. ► ESI (positive/negative) ionisation of aminoacids, amines, organic acids, saccharides. ► Utilisation of new sub-3 μm stationary phase material (HILIC Amide). ► Provides quantitative results that complement untargeted metabolomics studies. ► Applicability in different fields: biochemistry, food science, clinical chemistry.