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

TLDR

The authors developed an integrated platform to streamline metabolomics by combining chemical analysis, identification, quantification, data reduction, and quality assurance. The platform performs two UHPLC/MS/MS2 injections—one optimized for basic species and one for acidic species—followed by MS/MS2 data search against an in-house authentic standard library containing retention times, m/z, adducts, fragments, and spectra to identify and quantify metabolites. The platform detected 339 small molecules in 24 min with 9 % median variability, enabling rapid, high‑confidence identification via a multiparameter library match and high‑throughput relative quantification of a broad spectrum of metabolites.

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/MS2) 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/MS2 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.