A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites

•We developed a novel IP-LC–MS protocol for the identification and quantification of polar metabolites.•The IP method resolves biologically interesting molecules with good linearity and repeatability.•We demonstrate the versatility of method by determining the metabolomic signature of 3 biological systems. We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC–MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values