Isotopic Ratio Outlier Analysis (IROA) for Quantitative Analysis.

There is always a tension within the omics sciences between trying to measure biological molecules rapidly and measuring accurately. Metabolomics as an omics science tries to measure the small biochemicals rapidly, in a single pass, but the current state of the art cannot provide the reproducibility or accuracy needed for clinical use or even daily reproducibility for larger experiments. The IROA TruQuant measurement system uses a daily "Long-Term Reference Standard (LTRS)" and a chemically identical Internal Standard (IS) to provide validated chemical identity, daily QA/QC on instrument and sample preparation, and accurate reproducible quantitation that is comparable across days, instruments, and even, for most compounds, chromatographic methods. The LTRS is, as the name implies, a Long-Term Reference Standard that is always the same and should therefore provide very similar results on a large but finite collection of compounds. All of the compounds in the LTRS are isotopically signed with formula indicating IROA patterns so they cannot be mistaken for one another. Because of the precise IROA patterns, a software-driven analysis of the compounds seen daily can determine the performance of the instrument in terms of sensitivity, in-source fragmentation, and chromatographic and injection stability and provide completely reproducible quantitation.

Quantitative Metabolomics Using Isotope Residue Outlier Analysis (IROA®) 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®) pipeline. This pipeline encompasses sample preparation, LC-MS-based peak acquisition, proprietary software-based library creation, normalization, and quantification of metabolites. IROA® 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®-IS) and long-term reference standards (IROA®-LTRS).

Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis™, an isotopic-labeling technique for accurate biochemical profiling.

Metabolomics or biochemical profiling is a fast emerging science; however, there are still many associated bottlenecks to overcome before measurements will be considered robust. Advances in MS resolution and sensitivity, ultra pressure LC-MS, ESI, and isotopic approaches such as flux analysis and stable-isotope dilution, have made it easier to quantitate biochemicals. The digitization of mass spectrometers has simplified informatic aspects. However, issues of analytical variability, ion suppression and metabolite identification still plague metabolomics investigators. These hurdles need to be overcome for accurate metabolite quantitation not only for in vitro systems, but for complex matrices such as biofluids and tissues, before it is possible to routinely identify biomarkers that are associated with the early prediction and diagnosis of diseases. In this report, we describe a novel isotopic-labeling method that uses the creation of distinct biochemical signatures to eliminate current bottlenecks and enable accurate metabolic profiling.