Optimization of GC/TOF MS analysis conditions for assessing host-gut microbiota metabolic interactions: Chinese rhubarb alters fecal aromatic amino acids and phenol metabolism

In this paper, an optimized method based on gas chromatography/time-of-flight mass spectrometry (GC-TOFMS) platform has been developed for the analysis of gut microbial-host related co-metabolites in fecal samples. The optimization was performed with proportion of chloroform (C), methanol (M) and water (W) for the extraction of specific metabolic pathways of interest. Loading Bi-plots from the PLS regression model revealed that high concentration of chloroform emphasized the extraction of short chain fatty acids and TCA intermediates, while the higher concentration of methanol emphasized indole and phenyl derivatives. Low level of organic solution emphasized some TCA intermediates but not for indole and phenyl species. The highest sum of the peak area and the distribution of metabolites corresponded to the extraction of methanol/chloroform/water of 225:75:300 (v/v/v), which was then selected for method validation and utilized in our application. Excellent linearity was obtained with 62 reference standards representing different classes of gut microbial-host related co-metabolites, with correlation coefficients (r²) higher than 0.99. Limit of detections (LODs) and limit of qualifications (LOQs) for these standards were below 0.9 nmol and 1.6 nmol, respectively. The reproducibility and repeatability of the majority of tested metabolites in fecal samples were observed with RSDs lower than 15%. Chinese rhubarb-treated rats had elevated indole and phenyl species, and decreased levels of polyamine such as putrescine, and several amino acids. Our optimized method has revealed host-microbe relationships of potential importance for intestinal microbial metabolite receptors such as pregnane X receptor (PXR) and aryl hydrocarbon receptor (AHR) activity, and for enzymes such as ornithine decarboxylase (ODC).