ORIGINAL PAPER Hydrophilic interaction ultra performance liquid chromatography retention prediction under gradient elution Helen Gika & Georgios Theodoridis & Fulvio Mattivi & Urska Vrhovsek & Adriani Pappa-Louisi Received: 15 December 2011 / Revised: 1 April 2012 / Accepted: 2 April 2012 / Published online: 12 May 2012 # Springer-Verlag 2012 Abstract The development and application of new separa- tion mechanisms such as hydrophilic interaction chromatog- raphy (HILIC) is of high importance for the simultaneous analysis of polar molecules such as primary metabolites. However the retention mechanism in HILIC is not fully understood and as a result retention prediction tools are not at hand for this chromatographic approach. In the pres- ent report we study the utility of a simple algorithm, based on a simple linear and/or a simple logarithmic retention model, for retention prediction in HILIC gradient separation of a mixture of 23 selected compounds including (poly) amines, amino acids, saccharides, and other molecules. Uti- lizing two types of gradient elution programs with or with- out an isocratic part, retention data were collected in order to build prediction models. Starting from at least three gradient runs the prediction of analyte retention was very satisfactory for all gradient programs tested, providing useful evidence of the value of such retention time prediction methodologies. Keywords Hydrophilic interaction chromatography (HILIC) . Gradient elution . Retention prediction . Polar analytes . Metabolomics Introduction Hydrophilic interaction chromatography (HILIC) is a prom- ising alternative for the separation of analytes which owing to their hydrophilic nature are not well-retained in reversed- phase chromatography (RPLC). HILIC provides the advan- tage of strong retention together with higher compatibility and sensitivity in LC–MS systems for polar molecules, e.g., amino acids, organic acids, amines, and carbohydrates. These molecules play a central role in a number of vital biochemical processes; thus, there is great interest in the development of analytical methodologies which provide efficient analysis of these analytes. As a result HILIC has gained a wide application area ranging from biomarker discovery [1–5], to the analysis of selected groups of metabolites such as nucleosides/nucleo- tides [6–8], carbohydrates [9], amino acids [10–13], and other polar molecules. In contrast to RPLC, HILIC station- ary phases are typically polar and the mobile phase is relatively hydrophobic. Elution is often isocratic with the content of less-polar/aprotic solvents maintained close to 60 % or higher; protic solvents (typically water) act as a strong solvent. Analyte retention involves a combination of adsorption, ion exchange, and analyte partitioning from the eluent towards a water-enriched layer which surrounds the stationary phase particles; however, the mechanisms Published in the special paper collection Instrumental Methods of Analysis (IMA 2011) with guest editors Maria Ochsenkuehn- Petropoulou, Nikos Kallithrakas and Panagiotis Kefalas. Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6015-6) contains supplementary material, which is available to authorized users. H. Gika (*) Department of Chemical Engineering, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece e-mail: gkikae@auth.gr H. Gika : G. Theodoridis : F. Mattivi : U. Vrhovsek IASMA Research and Innovation Centre, Fondazione Edmund Mach, Food Quality and Nutrition Area, Via E. Mach 1, 38010 S. Michele all’Adige, Italy G. Theodoridis : A. Pappa-Louisi Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece Anal Bioanal Chem (2012) 404:701–709 DOI 10.1007/s00216-012-6015-6