Although the objective of any ‘omic’ science is broad measurement of its constituents,such coverage has been challenging in metabolomics because the metabolome is comprised of a chemically diverse set of small molecules with variable physical properties.While extensive studies have been performed to identify metabolite isolation and separation methods,these strategies introduce bias toward lipophilic or water-soluble metabolites depending on whether reversed-phase liquid chromatography (RPLC) or hydrophilic interaction liquid chromatography (HILIC) is used,respectively.Here we extend our consideration of metabolome isolation and separation procedures to integrate RPLC/MS and HILIC/MS for generic profiling of central carbon metabolism (CCM) in E.coli cells.An optimal combination of HILIC and RPLC columns were selected by comparing the performance of several available columns to achieve the best separation efficiency and reproducibility.High-resolution MS and MS/MS capacity of our workflow enables accurate metabolite identifications.Therefore,we aim to establish a robust,sensitive and generic LC-MS/MS platform to acquire the maximum amount of biological information related to central carbon metabolism by employing a dual separation scheme combining RPLC-ESI and HILIC-ESI MS analyses.Furthermore,we developed a new LC-MS-based scheme for specific analysis of cofactors and energy currency molecules involved in E.coli CCM.These metabolites of special biological significance are very difficult to capture in large-scale metabolomic surveys.Targeted approaches for their extraction,enrichment and LC-MS analysis were developed to achieve the most accurate identification and quantitation of this CCM metabolite subset.