2.4. Bile Acids Measurement
SEPARATION
Fasting plasma samples were obtained in the morning from each subject at four time points (before and after AD and HD) to standardize the conditions and reduce the influence of circadian rhythms. These samples were used to analyze bile acids.Specifically, samples were analyzed using a LC–MS/MS system, employing a method adapted from Daniel N et al. [All analytes were quantified using tandem mass spectrometry with an API6500 instrument equipped with an electrospray ionization source (Applied Biosystems, Concord, ON, Canada). Chromatographic separation employed a Nexera ultra-high-pressure liquid chromatography (UHPLC) instrument (Shimadzu Scientific Instruments, Columbia, MD, USA) and a 150 × 2.1 mm Poroshell 120 EC-C18; 2.7 μm particles column (Agilent, Santa Clara, CA, USA). The chromatographic system utilized a 5 mM gradient of water, ammonium acetate at pH 7.7, and acetonitrile at a flow rate of 300 μL/min. The source temperature was set at 500 °C.