Quantitative Metabolomics Using Isotope Residue Outlier Analysis (Iroa (R)) With Internal Standards

Various research strategies involving biomarker discovery and mechanistic studies in system biology depend on reproducible and reliable quantification of all metabolites from tissue(s) of interest. Contemporary analytical methods rely on mass spectrometry-based targeted and/or untargeted metabolomics platforms. The robustness of these analyses depends on the cleanliness of the samples, accuracy of the database, resolution of the instrument, and, the most variable of the list, the personal preferences of the researcher and the instrument operator. In this chapter, we introduce a simple method to prepare murine liver samples and carry it through the Isotope Ratio Outlier Analysis (IROA (R)) pipeline. This pipeline encompasses sample preparation, LC-MS-based peak acquisition, proprietary software-based library creation, normalization, and quantification of metabolites. IROA (R) offers a unique platform to create and normalize a local library and account for run-to-run variability over years of acquisition using the internal standards (IROA (R)-IS) and long-term reference standards (IROA (R)-LTRS).