2.4. Detection of bile acids by LC–MS/MS

In view of not having precesion experiemental equipments, we decided to hire a medical analysis company. We picked Shanghai Baiqu Biomedical Technology Co.,Ltd, and sent the materials to them. They detects serous bile acid content based on LC–MS/MS platform. Take 100 μL serum sample in EP tube, add 400 μL extraction solution methanol:acetonitrile (volume ratio 1:1, containing 1% formic acid and 62.5 nmol/L internal standard), vortex for 30 s, mix well, sonicate in an ice water bath for 5 min, let stand at 20°C for 1 h, centrifuge at 4°C and 13,400 g for 15 min, and take 75 μL supernatant to injection vial for LC–MS/MS analysis. Mobile phase conditions: Vanquish (Thermo Fisher Scientific) ultrahigh‐performance liquid chromatography using Waters ACQUITY UPLC BEH C18 (150 × 2.1 mm, 1.7 mm) μm. Waters liquid chromatography column is used for the chromatographic separation among the target compound. Phase A of liquid chromatography is 1 mmol/L ammonium acetate and 0.1% acetic acid aqueous solution, and phase B is acetonitrile. The temperature of the column incubator is 50°C, the sample tray is set at 4°C, and the injection volume is 1 μL. Mass spectrometry conditions: Q Active HFX high‐resolution mass spectrometer, using parallel reaction monitoring (PRM) mode for mass spectrometry analysis. The ion source parameters are as follows: spray voltage = +3500/−3100 V, sheath gas (N2) flow rate = 40, aux gas (N2) flow rate = 15, sweep gas (N2) flow rate = 0, aux gas (N2) temperature = 350°C, and capillary temperature = 320°C. The original UPLCMS/MS data are processed using MultiQuant software (version 3.0.3) for peak detection, calibration, and standardization. Statistical analysis was conducted using SPSS 22.0 (IBM) statistical software, and p < .05 was considered statistically significant. The original data were processed, and the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS‐DA) of the serum bile acid metabolic profile were performed using SIMCA (V15.0.2, Umetrics). After verifying that the original model was suitable for subsequent analysis, the VIP value was calculated using R language, the difference of bile acid was screened out, and the p value was determined by the two‐tailed t‐test (VIP > 1.0 and p < .05).