Metabolomic analysis of human fecal water recently aroused increasing attention with the importance of fecal metabolome in exploring the relationships between symbiotic gut microflora and human health. In this study, we developed a quantitative metabolomic method for human fecal water based on trimethylsilylation derivatization and GC/MS analysis. Methanol was found to be the best solvent for protein precipitation and extraction of fecal water metabolome. Within the optimized linear range of sampling volume (less than 50 microL), compounds showed a good linearity with a correlation coefficient higher than 0.99. The developed method showed good repeatability for both sample preparation and GC/MS analysis with the relative standard deviations lower than 10% for most compounds and less than 20% for a few other ones. The method was further validated by studying analytical variability using a set of clinical samples as well as a pooled sample. The pH value and matrix effects were the main factors affecting the accuracy of quantitative calibration curves. The increased pH value decreased the loss of short chain fatty acids during lyophilization. Spiking fecal water to a standard mixture significantly enhanced the accuracy of quantitative calibration curves, probably due to the inhibition of volatile loss during lyophilization and the increase of compound solubility in the derivatization medium. A strategy for calibration curve preparation was proposed in order to avoid the effects of pH and matrix. Totally, 133 compounds were structurally confirmed from a set of clinical samples, and 33 of them were quantified, which demonstrates the suitability of this method for a quantitative metabolomic study of human fecal water samples.
Long-chain polyunsaturated fatty acids (LC-PUFA) of the n-3 series, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, have specific activities especially in the functionality of the central nervous system. Due to the occurrence of numerous methylene-interrupted ethylenic double bonds, these fatty acids are very sensitive to air (oxygen) and temperature. Non-volatile degradation products, which include polymers, cyclic fatty acid monomers (CFAM) and geometrical isomers of EPA and DHA, were evaluated in fish oil samples obtained by deodorization under vacuum of semi-refined fish oil at 180, 220 and 250 7C. Polymers are the major degradation products generated at high deodorization temperatures, with 19.5% oligomers being formed in oil deodorized at 250 7C. A significant amount of CFAM was produced during deodorization at temperatures above or equal to 220 7C. In fact, 23.9 and 66.3 mg/g of C20 and C22 CFAM were found in samples deodorized at 220 and 250 7C, respectively. Only minor changes were observed in the EPA and DHA trans isomer content and composition after deodorization at 180 7C. At this temperature, the formation of polar compounds and CFAM was also low. However, the oil deodorized at 220 and 250 7C contained 4.2% and 7.6% geometrical isomers, respectively. Even after a deodorization at 250 7C, the majority of geometrical isomers were mono-and di-trans. These results indicate that deodorization of fish oils should be conducted at a maximal temperature of 180 7C. This temperature seems to be lower than the activation energy required for polymerization (intra and inter) and geometrical isomerization.
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