Chemicals and reagents

All solvents and reagents except those described below were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Analytical reagent-grade and LC/MS-grade solvents were used for the sample preparation and the LC/MS mobile phase, respectively. Dimethylazodicarboxylate, N,N′-diisopropylcarbodiimide, dimethylaminopyridine, and lithium iodide were purchased from FUJIFILM Wako Pure Chemical Corporation (Miyazaki, Japan). A Bond Elut C18 (500 mg/6 ml) solid phase extraction cartridge was obtained from Agilent (Tokyo, Japan).

Abbreviations and trivial names of BAs used in this study are shown in the supplemental Table S1. Standard products such as Pam, Ste, Ole, and linoleic acid (Lin) were purchased from FUJIFILM Wako Pure Chemical Corporation. d₃-Pam and ¹³C₁₈-Ste were purchased from Sigma Chemicals (St. Louis, MO). CA, CDCA, UDCA, DCA, LCA, and hyocholic acid were also purchased from Sigma Chemicals. [2,2,3,4,4-d₅]-CA (d₅-CA, internal standard (IS) for trihydroxy BAs), [2,2,4,4-d₄]-DCA (d₄-DCA, IS for dihydroxy BAs), [2,2,4,4-d₄]-LCA (d₄-LCA, IS for monohydroxy BAs), [2,2,4,4-d₄]-glycocholic acid (GCA) (d₄-GCA, IS for glycine-conjugated trihydroxy BAs), [2,2,4,4-d₄]-taurocholic acid (TCA) (d₄-TCA, IS for taurine-conjugated trihydroxy BAs), [2,2,4,4-d₄]-glycochenodeoxycholic acid (GCDCA) (d₄-GCDCA, IS for glycine-conjugated di- and mono-hydroxy BAs), and [2,2,4,4-d₄]-taurochenodeoxycholic acid (TCDCA) (d₄-TCDCA, IS for taurine-conjugated di- and mono-hydroxy BAs) were obtained from CDN Isotopes (Quebec, Canada). AlloCA, isoCA, 3-oxo-CA, 7-oxo-DCA, 12-oxo-CDCA, UCA, isoUCA, alloCDCA, isoCDCA, 3-oxo-CDCA, 3-oxo-alloCDCA, 7-oxo-LCA, UDCA, isoUDCA, 3-oxo-UDCA, alloDCA, isoDCA, isoalloDCA, 3-oxo-DCA, 3-oxo-alloDCA, 12β-OH-LCA, 12β-OH-isoLCA, 12-oxo-LCA, 12-oxo-isoLCA, alloLCA, isoLCA, isoalloLCA, 3-oxo-LCA, 3-oxo-alloLCA, hyodeoxycholic acid, murideoxycholic acid, CA-Δ⁴-3-one, DCA-Δ^4,6-3-one, CDCA-Δ⁴-3-one, LCA-Δ^4,6-3-one, DCA-Δ⁴-3-one, LCA-Δ⁴-3-one, nor-CA, GCA-Δ⁴-3-one, TCA-Δ⁴-3-one, GCDCA-Δ⁴-3-one, and TCDCA-Δ⁴-3-one were gifts from Professor Takashi Iida (Nihon University, Tokyo Japan). CA-3S, CDCA-3S, UDCA-3S, DCA-3S, lithocholic acid 3-sulfate (LCA-3S), GCA-3S, GCDCA-3S, GUDCA-3S, GDCA-3S, GLCA-3S, TCA-3S, TCDCA-3S, TUDCA-3S, TDCA-3S, TLCA-3S, [2,2,3,4,4-d₅]-GCDCA-3S (d₅-GCDCA-3S, IS for glycine-conjugated BA 3-sulfates), [2,2,3,4,4-d₅]-TCDCA-3S (d₅-TCDCA-3S, IS for taurine-conjugated BA 3-sulfates), and [2,2,3,4,4-d₅]- CDCA-3S (d₅-CDCA-3S, IS for BA 3-sulfates) were synthesized using previously reported methods (31).