To verify the reliability of the retention time-based annotation, two standards (Gly-LCA and Gly-UDCA) were purchased, and three new AA-BAs standards (Gln-CA, Val-CA, and Ser-CA) were synthesized. These identified AA-BAs according to the predicted retention time were successfully confirmed by comparing their extracted ion chromatograms of fecal samples with those of pure standards (Figure ). The above results indicated that AA-BAs could be identified based on the predicted retention time with high confidence.