Cardamonin is a chalcone that presents at high content in the seeds of Alpinia katsumadai Hayata. In recent decades, researchers have found that it is not only an edible spice, but also a remarkable herb with a wide range of pharmacological properties. However, its specific metabolic routes in vivo remain unclear while these metabolites may accumulate to exert pharmacological effects. Our study aimed to clarify the metabolic pathways of cardamonin after oral administration to rats. Here, an advanced UHPLC-Q-Exactive Orbitrap MS analytical technique was applied for efficient detection of metabolites in vivo, which especially showed benefits in obtainment of the fragment ions with relatively lower contents. We also established a novel strategy to identify metabolites based on typical fragmentation routes. The results indicated that a total of 40 metabolites could be categorized into 3 types with consideration of the particular structures and characteristic fragment ions. Then, diagnostic product ions (DPIs) of each type were summarized for further screening and identification of metabolites derived from cardamonin. Finally, methylation, demethylation, hydrogenation, hydroxylation, dehydroxylation, glucuronidation and sulfation were confirmed to be the major metabolic pathways in vivo. Our observation extended the metabolic mechanism of cardamonin and could be of great benefits to interpreting the action mechanism of cardamonin in vivo.

The screening of drug metabolites in biological matrixes and structural characterization based on product ion spectra is among the most important, but also the most challenging due to the significant interferences from endogenous species. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of prototype drug metabolites using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Although classical techniques are well-suited for achieving the partial characterization of prototype drug metabolites, there is a pressing need for a strategy to enable comprehensive drug metabolism depiction. Therefore, we present drug metabolite clusters (DMCs), different from, but complementary to, traditional approaches for mining the information regarding drugs and their metabolites on the basis of raw, processed, or identified tandem mass spectrometry (MS/MS) data. In this paper, we describe a DMC-based data-mining method for the metabolite identification of 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (HTF), a typical hydroxylated-polymethoxyflavonoid (OH-PMF), which addressed the challenge of creating a thorough metabolic profile. Consequently, eight primary metabolism clusters, sixteen secondary metabolism clusters, and five tertiary metabolism clusters were proposed and 106 metabolites (19 potential metabolites included) were detected and identified positively and tentatively. These metabolites were presumed to generate through oxidation (mono-oxidation, di-oxidation), methylation, demethylation, methoxylation, glucuronidation, sulfation, ring cleavage, and their composite reactions. In conclusion, our study expounded drug metabolites in rats and provided a reference for further research on therapeutic material basis and the mechanism of drugs.