Metabolomics involves qualitative and/or quantitative analysis of hundreds of metabolites in a complex sample. As most of the metabolites are polar in nature (for example, amino acids, nucleotides, carboxylic acids), their chromatographic separation and analysis present a difficult challenge. Recently, hydrophilic interaction chromatography (HILIC) has become a useful tool for analysis of such polar molecules. In this chapter, we present a simple HILIC LC-MS/MS method for targeted multiple reaction monitoring (MRM)-based relative quantitation of hundreds of polar metabolites in a complex sample. The method uses an aminopropyl column with acetonitrile as weak solvent and 20 mM ammonium acetate buffer (pH 9.4) as strong solvent. The method does not use any ion pairing reagent making it suitable for analysis in both positive and negative ionization mode using a simple LC-MS/MS instrument setup. The method has been thoroughly tested, validated, and applied to a variety of samples such as bacteria, yeast, plants, human body fluids, and cell cultures.
Key wordsMetabolomics metabolite profiling LC-MS/MS chromatography MRM HILIC