Optimization of sample extraction and injection-related parameters in HILIC performance for polar metabolite analysis. Application to the study of a model of pulmonary hypertension.

ABSTRACT

HILIC is a separation technique increasingly used for the study of polar metabolites. However, HILIC suffers of a drawback related to the solvents used for these analyses acetonitrile as sample solvent leads to the best chromatographic peaks, but it is not capable to extract/dissolve the most polar compounds. In this work we evaluated the use of several strategies for the extraction of polar compounds from plasma samples and, although methanol was the ideal solvent for analyte extraction, distorted peaks were obtained in the chromatography. Different strategies were tested included changing the solvent after extraction or modifying the injection volume and type. Finally, the best solution was using the lowest injection volume possible and to employ a simple sandwich injection including acetonitrile during the injection of the sample. This remarkably improves the peak shape when methanol is used in the sample. We evaluated and applied two HILIC-MS methods, in positive and negative ionization modes, on plasma samples from pigs with pulmonary hypertension produced by aorto-pulmonary shunting to identify metabolic signatures underlying damage to the right heart. Our analyses revealed altered relevant metabolic pathways, suggestive of oxidative stress and reduced energy demands.