Evaluation of fresh, frozen, and lyophilized fecal samples by SPME and derivatization methods using GC×GC-TOFMS

Untargeted Metabolomic Profiling of Aqueous and Lyophilized Pooled Human Feces from Two Diet Cohorts Using Two-Dimensional Gas Chromatography Coupled with Time-of-Flight Mass Spectrometry

“…This result is thought to be attributed to the wet and slushy nature of aqueous samples, which makes it difficult to extract fatty and bulky compounds. In contrast, lyophilization makes samples more prone to release metabolites, resulting in the intensification of signals [ 26 ]. Overall, the group-type analysis also revealed similar patterns for the samples in the same storage conditions, which may be interpreted as the storage conditions having a more significant impact on the metabolic profiles than diet styles.…”

“…All 24 individual GC×GC-TOFMS chromatograms were processed using ChromaTOF ® (v.4.72; LECO, St. Joseph, MI, USA) with the same initial data processing method described by Nam et al [ 26 ] In brief, the initial method to process each chromatogram involves the exclusion of the column bleed region and setting the baseline offset to 0.9, and the expected peak widths to 12 s for the first dimension and 0.15 s for the second dimension, the peak-finding threshold of S/N to 100 searching against the peaks containing m / z 73 (trimethylsilyl fragment), with the minimum S/N ratio of 6 for the inclusion of sub-peaks. All detected peaks were searched against the NIST-MS 2017 Library mass spectral database.…”

“…These changes are due to ongoing processes such as oxidation, degradation, and bacterial or enzymatic activities [ 22 , 23 , 24 ]. Despite the awareness of the importance of sample handling for feces, there is currently no consensus on how fecal samples should be handled prior to analysis for unbiased stool metabolomics [ 25 , 26 ].…”

“…Using fresh stool samples immediately after collection (within an hour) is usually unrealistic, especially for large-scale metabolomics studies. To store samples until analysis, two sample conditions are commonly used: lyophilized feces [ 26 , 27 ] and fecal water (aqueous phase) [ 28 , 29 , 30 ]. Different sample conditions may affect the composition and metabolic profile of the sample.…”

“…For MS-based platforms, they are generally coupled with a high-performance separation instrument such as gas chromatography (GC) or liquid chromatography (LC) [ 21 , 27 , 32 , 34 ]. Amongst many analytical platforms, GC×GC-TOFMS is a well-suited tool for untargeted studies of complex metabolomics samples [ 1 , 26 , 35 , 36 , 37 ]. GC×GC employs two independent separation mechanisms with different chemistries and offers many analytical advantages compared to a conventional one-dimensional GC system.…”