alexa Integrated, nontargeted ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems.
Immunology

Immunology

Journal of Clinical & Cellular Immunology

Author(s): Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E

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Abstract To address the challenges associated with metabolomics analyses, such as identification of chemical structures and elimination of experimental artifacts, we developed a platform that integrated the chemical analysis, including identification and relative quantification, data reduction, and quality assurance components of the process. The analytical platform incorporated two separate ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) injections; one injection was optimized for basic species, and the other was optimized for acidic species. This approach permitted the detection of 339 small molecules, a total instrument analysis time of 24 min (two injections at 12 min each), while maintaining a median process variability of 9\%. The resulting MS/MS(2) data were searched against an in-house generated authentic standard library that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as their associated MS/MS spectra for all molecules in the library. The library allowed the rapid and high-confidence identification of the experimentally detected molecules based on a multiparameter match without need for additional analyses. This integrated platform enabled the high-throughput collection and relative quantitative analysis of analytical data and identified a large number and broad spectrum of molecules with a high degree of confidence. This article was published in Anal Chem and referenced in Journal of Clinical & Cellular Immunology

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