Comprehensive identification and semi-quantitative profiling of 3,5,6,7,8,3′,4′-heptamethoxyflavone metabolites in rats by UHPLC-LTQ-Orbitrap mass spectrometry

3,5,6,7,8,3′,4′-Heptamethoxyflavone (HMF), one of the highest <em>O</em>-methyl numbers in the polymethoxyflavones, possesses various significant health benefits. However, the <em>in vivo</em> metabolic profile of HMF remains largely unexplored. In this study, a systematic identification and relative quantitation of HMF and its metabolites in rats were performed using UHPLC-LTQ-Orbitrap mass spectrometry combined with enzymatic hydrolysis. A total of 56 metabolites were tentatively identified, including 37 demethylated metabolites and 19 glucuronide conjugates of these demethylated products. Notably, all seven mono-demethylated metabolites of HMF were simultaneously detected and structurally discriminated for the first time in rats, along with other di-, tri-, tetra-, and penta-demethylated metabolites. Semi-quantitative analysis revealed that HMF underwent predominant biotransformation into mono-demethylated metabolites in rats, followed by progressive sequential demethylation of these metabolites to generate more di-, tri-, tetra-, and penta-demethylated metabolites. Demethylation and glucuronidation are the primary metabolic pathways of HMF <em>in vivo</em>. This study presents the first comprehensive elucidation of the <em>in vivo</em> metabolic profile of HMF, which is helpful for further understanding of its <em>in vivo</em> potential effective components and pharmacological mechanism.