IROA metabolomics workflow for improved accuracy, identification and quantitation

A method of determining chemical identity of one or more molecules in an experimental sample by performing mass spectral analyses of the experimental sample and concurrently performing quality assurance for an associated mass spectral apparatus, which method comprises:

• a) preparing an isotopic ratio outlier analysis (IROA) standard sample comprising a known composition of metabolite compounds, which are associated with a library of mass spectral peaks reflecting each of the metabolite compounds,
• wherein the composition of metabolite compounds comprises a paired set of a first isotopically-labeled compound and a second isotopically-labeled compound, with each isotopically-labeled compound being equally present,
• wherein about 2% to about 10% by mass of a non-hydrogen/deuterium atom in the first isotopically-labeled compound is a first isotope of the non-hydrogen/deuterium atom and about 90% to about 98% by mass of the non-hydrogen/deuterium atom in the first isotopically-labeled compound is a second isotope of the non-hydrogen/deuterium atom,
• wherein about 90% to about 98% by mass of the non-hydrogen/deuterium atom in the second isotopically-labeled compound is the first isotope of the non-hydrogen/deuterium atom and about 2% to about 10% by mass of the non-hydrogen/deuterium atom in the second isotopically-labeled compound is the second isotope of the non-hydrogen/deuterium atom,
• wherein said first isotope atom and said second isotope atom are not the same,
• wherein the first isotope atom and the second isotope atom are stable to radioactive decay,
• b) preparing an experimental sample comprising one or more molecules and further comprising an Internal Standard comprising a metabolite compound of the IROA standard sample, said metabolite compound isotopically-labeled with only the heavier isotope of the first isotope and the second isotope of the non-hydrogen/deuterium atom;
• c) subjecting the IROA standard sample and the experimental sample to liquid chromatography coupled to mass spectrometry (LC/MS) to produce eluted components of the IROA standard sample and the experimental sample;
• d) separately ionizing the eluted components of the IROA standard sample and the experimental sample via mass spectrometer to produce ions of the IROA standard sample and ions of the experimental sample;
• e) implementing a windowing scheme comprising variable width and/or variable overlapping fragmentation windows;
• f) fragmenting the ions using a mass spectrometer to produce fragment ions, and, correspondingly, a pattern of mass spectral peaks for the IROA standard sample and a pattern of mass spectral peaks for the experimental sample;
• g) comparing the pattern of mass spectral peaks of the heavier isotope from the Internal Standard to the mass spectral pattern of peaks of the first isotopically-labeled compound and the second isotopically labeled compound from the IROA standard sample;
• h) comparing the pattern of mass spectral peaks of the heavier isotope from the Internal Standard to the mass spectral pattern of peaks of the one or more molecules of the experimental sample, and comparing the LC retention time of the Internal Standard to the LC retention time of the experimental sample, to identify the one or more molecules in the experimental sample;
• and
• i) determining whether the mass spectral pattern of peaks found for the IROA standard sample matches the reference library's mass spectral peaks for the IROA standard sample, in order to assess performance of the associated mass spectral apparatus, and if variances are found, correcting for those variances.