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Different hydrophilic interaction liquid chromatography (HILIC) stationary phases have been evaluated using different chemometric methods with the aim of their application in metabolomics studies. Experimental factors, such as the type of HILIC stationary phase (i.e. amide, amine, zwitterionic and diol) and the mobile phase conditions (organic co-solvent, pH and ionic strength) were assessed using a full factorial experimental design. A test sample mixture of metabolites with diverse physicochemical properties (amino acids, nucleotides, nucleosides, and sugars among others) was analyzed by liquid chromatography with a diode array detector (LC-DAD) using five different HILIC columns. Application of multivariate curve resolution alternating least squares (MCR-ALS) method, allowed the full chromatographic peak resolution of all mixture constituents. This approach was particularly helpful in the case of methanol samples where the quality of the chromatographic separation (resolution) was lower in consequence of the co-solvent perturbation on the water layer formation at the surface of the stationary phase. Then, Berridge chromatographic response function (CRF), based on peak resolution, retention times and number of peaks, was used for the investigation of the best HILIC column configuration for future metabolomics studies. The best chromatographic configuration resulted in being the amide and zwitterionic HILIC stationary phases in combination with acetonitrile as organic co-solvent of the mobile phase. © 2017 The Royal Society of Chemistry.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement No. 320737. Research funding from MINECO Spain Grant No. CTQ2015-66254-C2-1-P is also acknowledged. The authors are grateful to Ms. Marta Fontal from IDAEA-CSIC (Barcelona, Spain) for technical assistance in the use of the LC-DAD system.Peer reviewe

Chemometric evaluation of hydrophilic interaction liquid chromatography stationary phases: resolving complex mixtures of metabolites