Untargeted Serum Metabolic Profiling by Comprehensive Two-Dimensional Gas Chromatography-High-Resolution Time-of-Flight Mass Spectrometry.

While many laboratories take appropriate care, there are still cases where the performances of untargeted profiling methods suffer from a lack of design, control, and articulation of the various steps involved. This is particularly harmful to modern comprehensive analytical instrumentations that otherwise provide an unprecedented coverage of complex matrices. In this work, we present a global analytical workflow based on comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry. It was optimized for sample preparation and chromatographic separation and validated on in-house quality control (QC) and NIST SRM 1950 samples. It also includes a QC procedure, a multiapproach data (pre)processing workflow, and an original bias control procedure. Compounds of interest were identified using mass, retention, and biological information. As a proof of concept, 35 serum samples representing three subgroups of Crohn's disease (with high, low, and quiescent endoscopic activity) were analyzed along with 33 healthy controls. This led to the selection of 33 unique candidate biomarkers able to classify the Crohn's disease and healthy samples with an orthogonal partial least-squares discriminant analysis Q(2) of 0.48 and a receiver-operating-characteristic area under the curve of 0.85 (100% sensitivity and 82% specificity in cross validation). Fifteen of these 33 candidates were reliably annotated (Metabolomics Standards Initiative level 2).