Comparison of supercritical fluid chromatography and liquid chromatography for the separation of urinary metabolites of nobiletin with chiral and non-chiral stationary phases.

Nobiletin (NOB), a polymethoxylated flavone found in sweet orange (Citrus sinensis) peel, is currently recognized as a promising anti-inflammatory and anti-tumor agent. It is believed that, by undergoing metabolic biotransformation in vivo, nobiletin is demethylated by hepatic P450 enzymes, yielding multiple hydroxylated metabolites. However, it has not been possible to date to separate the two demethylated nobiletin metabolites, 3'-demethyl-NOB and 4'-demethyl-NOB (regio-isomers) on reversed-phase liquid chromatography (RPLC). Additionally, both display similar mass spectrometric fragmentation, resulting in difficulties to identify the dominant metabolite. A successful separation method was developed by utilizing supercritical fluid chromatography (SFC) with chiral stationary phase. The separation was also attempted with normal-phase liquid chromatography (NPLC) in both chiral and non-chiral modes. Chromatographic separation for the two nobiletin metabolites was superior by SFC than by LC, especially using chiral stationary phase. By comparing the SFC profile of the synthesized standards, the major nobiletin metabolite in mouse urine was identified as 4'-demethyl-NOB, with the concentration of 28.9 microg/mL.