Lc qtof ms

The characterization of bioactive compounds present in EECS was carried out by LC-QTOF-MS (Agilent Technologies, Santa Clara, CA, USA, Model no: 6545 QTOF).

The characterization of bioactive phytochemicals present in the Fr. 3 was investigated using LC-QTOF-MS (Agilent Technologies, Santa Clara, CA, United States, Model no: 6545 QTOF) (Sasidharan et al., 2022c ).

The AFB1 degradation ability was confirmed using an HPLC system with an ultraviolet (UV) diode array detector at 360 nm; the specific detection conditions are listed in Table 1. LC-Q-TOF/MS (Agilent) was applied to analyze the AFB1 degradation products. All samples were filtered through a sterile 0.22 μM pore filter (Agilent) before testing.

Enzyme reactions were performed in 100 µl of buffer (50 mM Tris-HCl pH 8.0) containing 50 µg purified protein, 1 mM UDP-Glc (electronic supplementary material, figure S1, Yuanye Biotech, Shanghai, China) and 100 µM glycyrrhetinic acid (Sigma-Aldrich, Shanghai, China). After 6 h of incubation at 37°C, the reactions were stopped by the addition of 100 µl methanol. The conversion rates were calculated by HPLC peak area (A_product/A_substrate + A_product × 100%). The supernatants obtained by centrifugation (13 500 r.p.m. for 5 min) were analysed by Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA) and LC-QTOF-MS (Agilent Technologies, Santa Clara, CA, USA), and internal standards (GA-3-O-monoglucose and GA-30-O-monoglucose) were supplied by Prof. Ye Min's laboratory (State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University).

Phenolic and flavonoid compounds in BsE fraction were identified by liquid chromatograph–quadrupole time-of-flight mass spectrometer (LC-QTOF MS, Agilent Technology, Santa Clara, CA, USA). The chromatographic separation was run on a Zorbax Eclipse Plus C18 column (150 × 2.1 mm, particle size 1.8 µm) at 25 °C. The injection volume was 2 µL, and the flow rate was kept at 0.2 mL/min. Elution was carried out by using two mobile phases: eluent A (0.1% formic acid in water) and eluent B (acetonitrile). The gradient program was as follows: 5% B (3 min), 23% B (22 min), 35% B (10 min), and 5% B (10 min).
MS was operated with Dual AJS source, and the instrument parameters were as follows: gas temperature, 325 °C; gas flow, 13 L/min; nebulizer, 35 psi; sheath gas temperature, 275 °C; sheath gas flow, 12 L/min; VCap, 4000 V; nozzle, 2000 V; fragmentor, 175 V; skimmer, 65 V; and octopole RF peak, 750. The scan range of the ion trap was 100–1500 m/z for MS and 50–1500 m/z for MS/MS. The mass spectra were recorded in both negative and positive ion modes. The reference mass ions purine at 112.9856 (for negative mode) and 121.0508 (for positive mode), as well as the HP921 at 1033.9881 (for negative mode) and 922.0098 (for negative mode), were used for continuously correcting any mass drift. All data were analyzed by LC/MS Data Acquisition and Qualitative Analysis Workflows software.

Proteomic profiling in all the study groups was performed via shotgun proteomic analysis after the depletion of the 12 most abundant proteins using immuno-affinity kits from a 10 µL plasma sample. Then, the proteins were reduced and alkylated, and trypsin-based digestion was carried out to generate peptides using LC-qTOF-MS (Agilent, California, USA) analysis (Figure S2.4). The detailed protocol is given in Supplementary Materials File S2 (Section S1.1.3).