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While the liver produces primary bile acids from cholesterol, gut bacteria transform these into diverse secondary forms which act as powerful signaling molecules. Since bile acid changes are increasingly linked to health and disease, their accurate measurement in the gut and circulation is essential. Bile acid analytical evaluations, however, are challenging as many co-elute in liquid chromatography (LC), share identical mass spectrometry (MS) precursor masses, and produce similar tandem mass spectrometry (MS\/MS) spectra. As a result, conventional LC\u2212MS\/MS workflows struggle to differentiate bile acids and have motivated the addition of orthogonal separations such as ion mobility spectrometry (IMS). Here, we assess optimal bile acid extraction parameters for stool, serum, and plasma; compare LC conditions; and assess electrospray ionization performance across polarities. Additionally, we introduce a publicly available multidimensional reference library containing LC retention times, IMS collision cross section values, and accurate precursor masses for 280 unique bile acids (264 endogenous and 16 deuterium-labeled species) including unconjugated, host-conjugated, and microbially conjugated bile acids. This multidimensional library empowers bile acid identification in complex samples and enables a more comprehensive exploration of their biological roles and disease associations.<\/jats:p>","DOI":"10.1093\/sumbio\/qvag039","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T12:03:32Z","timestamp":1789473812000},"source":"Crossref","is-referenced-by-count":0,"title":["Unlocking the bile acid universe: advanced workflows and a multidimensional library of 280 unique species"],"prefix":"10.1093","volume":"3","author":[{"given":"Guozhi","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, NC 27514 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily C","family":"Vincent","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of North Carolina at Chapel Hill , Chapel Hill, NC 27514 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Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, Departments of Pharmacology and Pediatrics, University of California at San Diego , La Jolla, CA 92093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ipsita","family":"Mohanty","sequence":"additional","affiliation":[{"name":"Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, Departments of Pharmacology and Pediatrics, University of California at San Diego , La Jolla, CA 92093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victoria","family":"Deleray","sequence":"additional","affiliation":[{"name":"Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, Departments of Pharmacology and Pediatrics, University of California at San Diego , La Jolla, CA 92093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Zhang","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health , Bethesda, MD 20894 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul A","family":"Thiessen","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health , Bethesda, MD 20894 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evan E","family":"Bolton","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health , Bethesda, MD 20894 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6868-8145","authenticated-orcid":false,"given":"Emma L","family":"Schymanski","sequence":"additional","affiliation":[{"name":"Luxembourg Centre for Systems Biomedicine, University of Luxembourg , 6 Avenue du Swing, 4367 Belvaux ,","place":["Luxembourg"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pieter C","family":"Dorrestein","sequence":"additional","affiliation":[{"name":"Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, Departments of Pharmacology and Pediatrics, University of California at San Diego , La Jolla, CA 92093 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Casey M","family":"Theriot","sequence":"additional","affiliation":[{"name":"Department of Population Health and Pathobiology, North Carolina State University , Raleigh, NC 27607 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