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A simple method for the determination of bile acids in adult human fecal samples using GC-MS is described. Bile acids are directly extracted from feces by ethanol (95%) containing 0.1 N NaOH. Extracts are purified by passage through a reversed-phase C18 silica cartridge and then analyzed by GC-MS. The present study has shown that lyophilized human feces contain mainly free bile acids, with lithocholic acid (LCA) and deoxycholic acid (DCA) as the major bile acids; however, isomers of LCA and DCA, keto-bile acids, and cholic acid are also present. Any traces of conjugated bile acids are hydrolyzed before the C18 extraction by deconjugating enzymes, which are present in feces and are activated by the addition of water during the homogenization step. Thus, the analysis of fecal bile acids can be performed without the hydrolysis step in less than 4 h in comparison to traditional techniques, which usually require at least 48 h.