Identification of nobiletin metabolites in mouse urine

Nobiletin, a major component of citrus polymethoxyflavones, has many potential significant health benefits. While the biological activities of nobiletin have been widely reported, its in vitro and in vivo metabolic fate has been rarely studied. To explore the biotransformation mechanism of nobiletin we conducted an investigation into its metabolic profile in mouse urine, by various analytical techniques. Due to sample amount limitations for isolating and characterizing an individual metabolite, two possible nobiletin metabolites were prepared in a similar multi‐step organic synthetic route: 3′‐hydroxy‐5,6,7,8,4′‐pentamethoxyflavone (3′‐demethylnobiletin) and 4′‐hydroxy‐5,6,7,8,3′‐pentamethoxyflavone (4′‐demethylnobiletin). Normal phase (silica gel) and C 18 reverse phase chromatography, as well as liquid chromatography‐mass spectrometry‐mass spectrometry, were employed in the separation of 3′‐demethylnobiletin and 4′‐demethylnobiletin, however, without success due to the structural similarities of these mono‐demethylated nobiletins. Using a chiral packed column eluted under supercritical fluid chromatography (SFC) conditions, a clear separation was achieved. Thus, by comparing the SFC profiles of metabolite mixtures with the synthesized standard compounds, the major nobiletin metabolite of mouse urine is identified as 4′‐demethylnobiletin, whereas 3′‐demethylnobiletin is a minor metabolite. In this study, the concentration of 4′‐demethylnobiletin in mouse urine is 28.9 μg/mL.