Alternate reversed-phase and hydrophilic interaction liquid chromatography coupled with mass spectrometry for broad coverage in metabolomics analysis.

Abstract Because of the greatly different physicochemical properties of metabolites, comprehensive metabolite profiling analysis has always been a challenging task. Reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) have been used to the analysis of nonpolar metabolites and polar metabolites, respectively. In this work, an alternate HILIC/RPLC-mass spectrometry (MS) approach was developed for the comprehensive and high-throughput analysis of polar and nonpolar metabolites. HILIC and RPLC are respectively performed on two ultra-high performance LC (UHPLC) systems, and coupled to one mass spectrometer to acquire the data. When HILIC gradient elution is running RPLC is in a washing and equilibration state, and vice versa. As a result, the total analysis time was reduced by about one third to 25.4 min. Two hundred and eight representative standards including at least twelve types of commonly met metabolites, SRM 1950 plasma, serum, urine and liver tissue samples were used to test the established alternate HILIC/RPLC-MS method. The results demonstrated that the method possessed high metabolite coverage. The developed method was validated to have good linearity and repeatability. As an example of application, 61 significantly changed metabolites in the colon cancer tissues were defined by this established method.