Development of a Quantitative Metabolomic Approach to Study Clinical Human Fecal Water Metabolome Based on Trimethylsilylation Derivatization and GC/MS Analysis

摘要

Metabolomic analysis of human fecal water recentlynaroused increasing attention with the importance of fecalnmetabolome in exploring the relationships between symbioticngut microflora and human health. In this study, wendeveloped a quantitative metabolomic method for humannfecal water based on trimethylsilylation derivatization andnGC/MS analysis. Methanol was found to be the bestnsolvent for protein precipitation and extraction of fecalnwater metabolome. Within the optimized linear range ofnsampling volume (less than 50 μL), compounds showedna good linearity with a correlation coefficient higher thann0.99. The developed method showed good repeatabilitynfor both sample preparation and GC/MS analysis with thenrelative standard deviations lower than 10% for mostncompounds and less than 20% for a few other ones. Thenmethod was further validated by studying analytical variabilitynusing a set of clinical samples as well as a poolednsample. The pH value and matrix effects were the mainnfactors affecting the accuracy of quantitative calibrationncurves. The increased pH value decreased the loss ofnshort chain fatty acids during lyophilization. Spiking fecalnwater to a standard mixture significantly enhanced thenaccuracy of quantitative calibration curves, probably duento the inhibition of volatile loss during lyophilization andnthe increase of compound solubility in the derivatizationnmedium. A strategy for calibration curve preparation wasnproposed in order to avoid the effects of pH and matrix.nTotally, 133 compounds were structurally confirmed fromna set of clinical samples, and 33 of them were quantified,nwhich demonstrates the suitability of this method for anquantitative metabolomic study of human fecal waternsamples.