A protocol for the preparation of tissue extracts for the targeted analysis of ca. 150 polar metabolites, including those involved in central carbon metabolism is described, using a reversed-phase ion pair U(H)PLC-MS method. Data collection enabled by multiple-reaction monitoring provides highly specific, sensitive acquisition of metabolic intermediates with a wide range of physicochemical properties and pathway coverage. Technical aspects are discussed for method transfer along with the basic principles of sample sequence setup, data analysis, and validation. General comments are given to help the assessment of data quality and system performance.
Keywords: Ion pair chromatography; Mass spectrometry; Metabolites; Metabonomics; Targeted analysis.