Metworks version 1.3

A Thermo Xcalibur 2.1 workstation (Thermo Scientific) was adopted for acquiring and processing HR-ESI-MS¹ and MSⁿ data. To obtain as many product ions of daidzein metabolites as possible, the peaks detected with intensity over 10,000 for negative ion mode and 50,000 for positive ion mode were selected for further structural characterization. The chemical formula for all parent ions were calculated from accurate mass using a formula predictor with the parameters set as followings: C [6–35], H [5–50], O [0–15], S [0–5], N [0–5], and ring double bond (RDB) equivalent value [0–15]. Meanwhile, MetWorks (Version 1.3) and Mass Frontier (Version 8.0) software (Thermo Scientific, Waltham, MA, USA) were utilized for mass fragmentation behaviors analysis, structural elucidation, and chromatographic peaks extraction.

A Thermo Xcalibur 2.1 workstation (Thermo Scientific, Bremen, Germany) was utilized to acquire and process HR-ESI-MS¹ and MSⁿ data. Based on the established screening templates of metabolites, by searching the metabolites with specific molecular weight to compare the high-resolution extracted ion chromatograms of the Control Group and the Drug Group, the metabolites related to phillyrin were selected. To obtain as many phillyrin metabolite ions as possible, the peaks detected with intensity over 10,000 for negative ion mode and 50,000 for positive ion mode were selected for further structural characterization. Based on the exact mass of the metabolite and the set elemental composition, the formula predictor could accurately calculate the chemical formula of all parent ions. The type and number of atoms were set as follows: C [0–40], H [0–60], O [0–25], S [0–2], N [0–5], and ring double bond (RDB) equivalent value [0–15]. At the same time, MetWorks (Version 1.3) and Mass Frontier (Version 8.0) software (Thermo Scientific, Waltham, MA, USA) were used as auxiliary tools for mass fragmentation behaviors analysis and structural identification.