Untargeted Serum Metabolic Profiling by GC×GC-HRTOF-MS

Whilst 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 (GC×GC) coupled to high resolution time-of-flight mass spectrometry (HR-TOF-MS). It was optimized for sample preparation and chromatographic separation, and validated on in-house QC samples and NIST SRM 1950 samples. It also includes a QC procedure, a multi-approaches data (pre)processing workflow and an original bias control procedure. Compounds of interest were identified using mass, retention and biological informations. As a proof of concept, 35 serum samples representing 3 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 31 unique candidate biomarkers able to classify Crohn’s disease and healthy samples with OPLS-DA Q2 0.48 and ROC AUC 0.85 (100% sensitivity and 82% specificity in cross validation). 15 of these 33 candidates were reliably annotated (MSI level 2).