Simultaneous quantitative analysis of metabolites using ion-pair liquid chromatography-electrospray ionization mass spectrometry

We have developed an analytical method, consisting of ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IP-LC-ESI-MS), for the simultaneous quantitative analysis of several key classes of polar metabolites, like nucleotides, coenzyme A esters, sugar nucleotides, and sugar bisphosphates. The use of the ion-pair agent hexylamine and optimization of the pH of the mobile phases were critical parameters in obtaining good retention and peak shapes of many of the above-mentioned polar and acidic metabolites that are impossible to analyze using standard reversed-phase LC/MS. Optimum conditions were found when using a gradient from 5 mM hexylamine in water (pH 6.3) to 90% methanol/10% 10 mM ammonium acetate (pH 8.5). The IP-LC-ESI-MS method was extensively validated by determining the linearity (R2 > 0.995), sensitivity (limit of detection 0.1-1 ng), repeatability, and reproducibility (relative standard deviation