On the applicability of comprehensive two-dimensional gas chromatography combined with a fast-scanning quadrupole mass spectrometer for untargeted large-scale metabolomics.

Comprehensive two-dimensional gas chromatography mass spectrometry (GC×GC-MS) offers excellent chromatographic separation performance and superior sensitivity. As such, it is eminently suitable for the analysis of complex biological samples. The applicability of a GC×GC instrument equipped with a fast-scanning qMS detector for large-scale untargeted metabolome analyses was investigated. We optimized the dimensions of an apolar×medium-polar column combination in order to meet detector requirements and to compromise between separation performance and analysis time. The final method enabled a sufficient separation (R≥1.2 or higher) of approx. 90% of all analytes detected in urine within less than 1h. Using the qMS at maximum scan speed (20,000u/s) and choosing a scan range of m/z 60-550, a data acquisition frequency of 33Hz and usually at least 10-13 data points per (2)D peak above the baseline were achieved. Peak area as well as peak height could thus be determined precisely (mean RSD 2.5%). Spectral skewing was limited regarding the data points covering the upper peak half. As a consequence, peak apex spectra could be used for the alignment of analytes in different samples. The linear dynamic range was 1-2.5 orders of magnitude, depending on the analyte. In addition, the slow transition into saturation beyond the linear dynamic range made it possible to exploit an extended "working range" for relative quantification. Long-term stability of the system was demonstrated by the analysis of more than 300 human urine study samples for which detailed repeatability and intermediate precision data are provided. In summary, the GC×GC-qMS system proved to be applicable for untargeted large scale metabolome analyses.