Introduction
Metabolomics aims to characterize and quantify the complete small molecule complement, or metabolome, of a biological system. The metabolome consists of a diverse mixture of small molecules, including amino acids, sugars and phosphosugars, and biogenic amines and lipids. Untargeted metabolomics is exceptionally challenging due to the requirement to both identify and quantify hundreds of different compounds with limited a priori knowledge of the metabolites. It is, therefore, advantageous to use a detection system that is not only capable of sensitive detection of specific molecules in an untargeted way, but can also provide accurate mass information for confident confirmation and structural elucidation of unknowns.
Gas chromatography-mass spectrometry (GC-MS) is routinely used for metabolomics applications due to its inherent advantages, especially its chromatographic resolution, reproducibility, peak capacity, and convenient spectral libraries. GC provides excellent chromatographic separation capability for biomarker discovery using untargeted metabolomics, but has previously been hampered by the lack of high-end mass spectrometry support providing the dynamic range, accurate mass, and scan rate sufficient to analyze very complex samples, such as mammalian muscle tissue. The polar nature of the majority of central metabolites means that derivatization must be performed to allow effective volatilization and ensure good chromatography. High sample throughput and advanced automation is required for metabolomic analysis, especially for clinical metabolomics. This work demonstrates the application of a complete untargeted metabolomics workflow using a novel Thermo Scientific™ Orbitrap™ MS-based GC to detect biomarkers for time of death in a rat model. Estimation of postmortem interval (PMI) is one of the most critical, yet difficult, tasks in forensic investigation, particularly after the cadaver has equilibrated to the ambient environmental temperature. Current methods to determine PMI are inaccurate and primarily based on visual inspection of the body. A laboratory-based method, using a robust biomarker for PMI, would assist forensic investigation. This GC-MS configuration using an Orbitrap-based detector enables ultra-high mass resolution, sub-ppm mass accuracy, a large dynamic range, and a scan rate commensurate with the efficient quantitative analysis of highly complex metabolomic samples. The high resolution, mass accuracy, and scan speed is critical for consistent data deconvolution to permit the detection of species from overlapping TIC peaks, allowing for an untargeted metabolomics pipeline. Accurate mass electron ionization (EI) fragment patterns are also suitable for matching against the widely available NIST and Wiley libraries for tentative compound identification, while providing accurate mass for more in-depth characterization.
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