Structural Elucidation of Genistein Metabolites in Rats Based on UHPLC-LTQ-Orbitrap Mass Spectrometry

For further study of bioactive components of Genistein in vivo, a method of ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was established to comprehensively profile its metabolism in rats. Genistein was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) solution. Rats in drug group were given a dose of 200 mg/kg body weight orally. 0.5% CMC-Na aqueous solution (2 mL) was administrated to rats in control group. The obtained biological samples were pretreated using solid phase extraction method to exclude protein and solid residue precipitation. Samples were separated on a Waters Acquity BEH C18 column (2.1 mm×100 mm×1.7 μm) with 0.1% formic acid aqueous solution-acetonitrile solution as the mobile phases in gradient elution, and then analyzed by LTQ-Orbitrap mass spectrometry equipped with an ESI ion source in negative mode. According to the obtained accurate mass measurements and mass fragmentation patterns, a total of 31 metabolites (including Genistein) were tentatively or unambiguously identified. The results demonstrated that Genistein underwent multiple in vivo metabolic reactions, including dehydration, methylation, glucuronide conjugation, sulfate conjugation and their composite reactions. The results not only provided novel and useful data to comprehensively understand the metabolic mechanism and material basis of Genistein for further researches of safety, toxicity and efficacy, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug related constituents in vivo.