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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 (RP) 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 profiling. An aminopropyl-based HILIC/MS method was optimized on the basis of mobile-phase additives and pH, followed by evaluation of reproducibility. When applied to the untargeted study of perturbed bacterial metabolomes, the …