Improving the quality of biomarker candidates in untargeted metabolomics via peak table-based alignment of comprehensive two-dimensional gas chromatography–mass spectrometry data

• Chromatographic factors affecting peak alignment in untargeted metabolomics by GC × GC/TOF MS.
• The impact of mass spectrum matching on peak alignment across 63 chromatograms was analyzed.
• Peak resolution has a large impact on alignment, especially for very high- or low-intensity peaks.
• A workflow is proposed for the selection of viable biomarkers in untargeted metabolomics projects.

The potential of high-resolution analytical technologies like GC × GC/TOF MS in untargeted metabolomics and biomarker discovery has been limited by the development of fully automated software that can efficiently align and extract information from multiple chromatographic data sets. In this work we report the first investigation on a peak-by-peak basis of the chromatographic factors that impact GC × GC data alignment. A representative set of 16 compounds of different chromatographic characteristics were followed through the alignment of 63 GC × GC chromatograms. We found that varying the mass spectral match parameter had a significant influence on the alignment for poorly-resolved peaks, especially those at the extremes of the detector linear range, and no influence on well-chromatographed peaks. Therefore, optimized chromatography is required for proper GC × GC data alignment. Based on these observations, a workflow is presented for the conservative selection of biomarker candidates from untargeted metabolomics analyses.