C18 solid-phase isolation and high-performance liquid chromatography/ultraviolet diode array determination of fully...

A new analytical methodology for the determination of fully methoxylated flavones (FMFs) in citrus juices is described. Isolation of the FMFs is carried out by percolation of 30 mL of clarified citrus juice (to which tetramethyl-o-kaempferol is previously added as internal standard) through a C18 Sep-Pak cartridge, washing with 3 mL of water followed by 5 mL of water/acetonitrile (3:1), and selective elution of the retained FMFs with 5 mL of water/acetonitrile (9:11). Determination of the isolated FMFs is carried out by reversed-phase high-performance liquid chromatography (HPLC) and UV diode array detection (DAD). Signals at wavelengths 320, 335, and 345 nm (bandwidth 4 nm) are simultaneously acquired, stored, plotted, and integrated. The column used is a microbore (200 x 2.1-mm) Hypersil ODS 5 microns. Elution is in gradient mode, using a ternary mobile phase (water/acetonitrile/tetrahydrofuran). Column temperature is 40 degrees C. Recovery yields are nearly 100% for all the FMFs detected and identified: isosinensetin, hexamethyl-o-gossypetin, sinensetin, tetramethyl-o-isoscutellarein, hexamethyl-o-quercetagetin, nobiletin, tetramethyl-o-scutellarein, heptamethoxyflavone, and tangeretin. Chromatographic separation of the FMFs is extremely dependent upon the minor changes of the mobile phase composition and percentages, gradient rate, and temperature. The UV spectra (230 to 400 nm) of the FMFs obtained under chromatographic conditions are given. The FMFs relative response factors at 320, 335, and 345 nm and their concentrations in hand-squeezed and commercial concentrated orange and mandarin juices are tabulated. The FMF concentration differences found among samples are discussed.