Lc msn it tof

A liquid chromatography instrument coupled to an ion trap time-of-flight mass spectrometer (LC/MSⁿ-IT-TOF) from the Shimadzu Corporation (Kyoto, Japan) was used to identify the chemical structures of BBR and its metabolites. Liquid chromatography with tandem mass spectrometry (LC-MS/MS 8050, Shimadzu Corporation, Kyoto, Japan) and gas chromatography with mass spectrometry (GC-MS QP2010, Shimadzu Cooperation, Kyoto, Japan) were used for the analysis and quantification of BBR and its metabolites in biological samples 14 (link), 17 (link), 20 (link).

FLZ and M1 with the purity >99%, synthesized by the Department of Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China), were incubated with the gut microbiota. The method for the preparation of the gut microbiota was previously described¹¹ (link). Briefly, colonic contents from mice were pooled, and 4 g of the sample was transferred to a flask containing the anaerobic medium (Solarbio Science & Technology, Beijing, China). After thoroughly mixed and filtrated, the cultures were pre-incubated under anaerobic conditions with N₂ atmosphere at 37 °C for 30 min. Then FLZ (5 mmol/L) was incubated with the gut microbiota for 48 h with dimethyl sulfoxide as the negative control. After termination of the reaction with 3-fold of acetonitrile and centrifugation at 14,000 rpm (2K15, Sigma, St. Louis, MO, USA) for 5 min, the supernatant was dried under nitrogen flow at room temperature, and the residue was dissolved in 100 μL of acetonitrile:water (v/v) = 1:1. Then 10 μL supernatant was injected for analysis after centrifugation. A liquid chromatography instrument coupled to an ion trap time-of-flight mass spectrometer (LC/MSⁿ-IT-TOF) from Shimadzu Corporation (Kyoto, Japan) was used to identify the chemical structures of FLZ and its possible metabolites.