2.9. Assessment of Bile Acids in Intestines

A UPLC−MS/MS method was used to assess sixteen bile acids, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), α-muricholic acid (α-MCA), β-muricholic acid (β-MCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), lithocholic acid (LCA), taurocholic acid (TCA), glycocholic acid (GCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), tauro-α-muricholic acid/tauro-β-muricholic acid (Tα-MCA/Tβ-MCA), tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), taurohyodeoxycholic acid (THDCA) and taurolithocholic acid (TLCA), in cecal and colonic contents. After thawing at 4 °C for 30 min, the sample (~50 mg) was placed in a 1.5-mL centrifuge tube and weighed accurately. A total of 200 μL of water was added to a test tube, and the mixture was ultrasonicated for 5 min. Then, 1 mL of methanol was added. The sample was vortexed for 5 min followed by ultrasonic treatment for 1 h and centrifugation (4 °C, 12,000 rpm) for 5 min. The supernatant (100 μL) and isotope standard solutions (10 μL, 2 μg/mL CA-d4 and LCA-d4) were loaded into a 1.5-mL centrifuge tube and vortexed for 2 min. The mixture was then transferred to vials for LC−MS/MS testing. A Waters ACQUITY UPLC I-CLASS chromatograph (Waters, Milford, USA) equipped with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm, Waters, USA) was used for separation. The column temperature was 50 °C. The mobile phase consisted of water/acetonitrile (10:1, v/v, containing 1 mmol/L ammonium acetate, phase A) and acetonitrile/isopropanol (1:1, v/v, phase B) with a flow rate of 0.26 mL/min. Analyte isolation was performed by elution gradient, and the injection volume was 5.0 μL. The MS data were collected using a QTRAP 6500+ LOW MASS system (AB Sciex, Framingham, MA, USA) in negative ion mode. The analysis was performed based on the following parameters: ion source voltage 2.0 kV, ion source temperature 150 °C, desolvation temperature 600 °C, desolvation gas flow 1000 L/h, cone voltage 21 V, cone gas flow 10 L/h.

Total bile acid concentrations were calculated as the sum of the concentrations of all sixteen bile acids. The primary BAs included CA, TCA, GCA, CDCA, α-MCA, β-MCA, and Tα-MCA/Tβ-MCA. The secondary BAs included DCA, GDCA, LCA, HDCA, UDCA, TLCA, THDCA, GUDCA, and TUDCA. 12α-Hydroxylated bile acids (12α-OH BAs) included CA, DCA, TCA, GCA, GDCA, and TDCA. Non-12α-hydroxylated bile acids (non-12α-OH BAs) included CDCA, α-MCA, β-MCA, LCA, HDCA, UDCA, Tα-MCA/Tβ-MCA, TLCA, THDCA, GUDCA, and TUDCA. The concentrations of primary BAs, secondary BAs, 12α-OH BAs, and non-12α-OH BAs were calculated based on the sum of the concentrations of each type of bile acid mentioned above.