Abstract
This study examines the information potential of comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOF MS) and variable ionization energy (i.e., Tandem Ionization™) to study changes in saliva metabolic signatures from a small group of obese individuals. The study presents a proof of concept for an effective exploitation of the complementary nature of tandem ionization data. Samples are taken from two sub-populations of severely obese (BMI > 40 kg/m2) patients, named metabolically healthy obese (MHO) and metabolically unhealthy obese (MUO). Untargeted fingerprinting, based on pattern recognition by template matching, is applied on single data streams and on fused data, obtained by combining raw signals from the two ionization energies (12 and 70 eV). Results indicate that at lower energy (i.e., 12 eV), the total signal intensity is one order of magnitude lower compared to the reference signal at 70 eV, but the ranges of variations for 2D peak responses is larger, extending the dynamic range. Fused data combine benefits from 70 eV and 12 eV resulting in more comprehensive coverage by sample fingerprints. Multivariate statistics, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) show quite good patient clustering, with total explained variance by the first two principal components (PCs) that increases from 54% at 70 eV to 59% at 12 eV and up to 71% for fused data. With PLS-DA, discriminant components are highlighted and putatively identified by comparing retention data and 70 eV spectral signatures. Within the most informative analytes, lactose is present in higher relative amount in saliva from MHO patients, whereas N-acetyl-D-glucosamine, urea, glucuronic acid γ-lactone, 2-deoxyribose, N-acetylneuraminic acid methyl ester, and 5-aminovaleric acid are more abundant in MUO patients. Visual feature fingerprinting is combined with pattern recognition algorithms to highlight metabolite variations between composite per-class images obtained by combining raw data from individuals belonging to different classes, i.e., MUO vs. MHO.Graphical abstract
Highlights
Comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometric detection (GC×GC-TOF Mass spectrometry (MS)) is a multidimensional analytical platform with great potentials for profiling and fingerprinting of complex samples such as those typically considered in metabolomics studies [1,2,3,4,5,6,7]
Here we focused our analysis on two different populations of subjects with severe obesity, according to their metabolic status
The n-alkanes mixture was prepared in cyclohexane at a concentration of 100 mg L−1; the internal standard (IS) 1,4dibromobenzene solution was prepared in toluene at a concentration of 10 g L−1 and spiked to ready-to-inject derivatized saliva samples at 50 mg L−1
Summary
Comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometric detection (GC×GC-TOF MS) is a multidimensional analytical platform with great potentials for profiling and fingerprinting of complex samples such as those typically considered in metabolomics studies (e.g., bio-fluids, tissue extracts, and exhaled volatile organic compounds [VOCs]) [1,2,3,4,5,6,7] By this technique, known and unknown analytes (i.e., targeted and untargeted compounds) can be profiled in detail (i.e., detailed profiling) thanks to the great separation power and the high sensitivity achieved by band focusing with thermal modulation [8,9,10,11]. Human obesity is a suitable clinical condition owing to its multifold association with adverse cardiometabolic events [13]
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