1260 infinity binary lc system

For BA profiling, 20 μL of serum was mixed with 60 μL of acetonitrile (1% NH₄OH), including lithocholic acid-D₅ as the internal standard, and supernatant was separated by centrifugation. The liver tissue was weighed and homogenized in 6 volume of aqueous methanol (water/methanol 1:1 v/v). Next, 100 μL of each homogenate was added to 300 μL of acetonitrile (1% NH₄OH v/v), including lithocholic acid-D₅ as the internal standard. After centrifugation, the supernatant was transferred to a new Eppendorf vial for subsequent centrifugation. Each supernatant was transferred to a sample vial for analysis.
Next, 5 µL of the supernatant was injected into a liquid chromatography–tandem mass spectrometry (LC-MS/MS) for analysis (6490 QQQ MS; Agilent Technologies, Santa Clara, CA). BA separation was achieved using a 1260 Infinity Binary LC System (Agilent Technologies) equipped with a 100× 2.1 mm (Waters BEH C18) column. LC-MS/MS was operated in the negative mode with electrospray ionization. Mass chromatograms and spectra were acquired using MassHunter Workstation data Acquisition software (Agilent Technologies). Analysis of BAs was processed using Quantitative Analysis software (Agilent Technologies). Details are provided in theSupplemental Experimental Procedures.

Enzyme assays based on isoliquiritigenin as a substrate were performed at 25 °C for 10 min in a 200 μl reaction containing 50 mM Tris–HCl (pH 7.5), 50 μM substrate, and 10 μg of purified recombinant proteins; control reactions contained a protein extract of E. coli BL21 (DE3) carrying an empty pET-32a plasmid. The reactions were extracted twice in 200 μl of ethyl acetate. Because naringenin chalcone is unstable in buffer solution, the reaction was performed at room temperature, and the substrate was added last. After mixing for 1 min, an equal volume of ethyl acetate was added immediately, and the reactions were extracted twice in 200 μl of ethyl acetate. After removal of the solvent under vacuum, the residue was dissolved in 100 μl of methanol. The samples were separated through a reverse-phase C18 column (XDB-C18, 5 μm; Agilent, Santa Clara, CA, USA) using HPLC (1260 Infinity Binary LC system, Agilent) equipped with a multiwavelength diode array detector using the previously reported procedure (Cheng et al., 2018 (link)). Enzyme kinetic assays were performed following the procedure reported previously (Cheng et al., 2018 (link)).