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Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, eg, animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into'aqueous' and'organic'phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take∼ 48 h: 1 h for …