C18 Solid-Phase Isolation and High-Performance Liquid Chromatography/Ultraviolet Diode Array Determination of Fully Methoxylated Flavones in Citrus Juices

Abstract

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.

Separation and quantitation of orange juices using liquid chromatography of polymethoxylated flavones

“…Figure 3 shows a high content in SIN and NOB, a relative low content in HEX, SCU and HEP and a low content in TAN. These results are in agreement with precedent studies on PMF orange juice contents [9][10][11][12].…”

“…PMF Content in orange juice depends of pressing level and the kind extraction process used. Sendra et al [9] have shown that the PMF contents are 10 to 40 fold lower in handly pressing juices (mean of 0.33 mg L -1 ) versus 2.8 to 10.8 mg L -1 in industrial orange juices.…”

“…The PMF contents were expressed in percentage of total peaks taken in account [9] and in mg L -1 . Concentration in mg L -1 for SIN, SCU and TAN were determined by external calibration method using response factor of standards.…”

“…The composition study of PMF in essential oils and in orange and mandarin juices has been largely investigated using thin layer chromatography [6], and five PMF have been identified: SIN, NOB, HEP, tetramethylscutellarein (SCU) and TAN in oranges juices and essential oils. Then, separation techniques using gas chromatography [7] and liquid chromatography (isocratic and gradient elution) have been developed [8][9][10], allowing a rapid and precise determination. Nevertheless, due to the low content in PMF found in mandarin and orange juices compared to the PMF content in essential oils of this citrus (from 200 to 500 fold lower in juices), many extraction techniques have been developed.…”

“…One of them consist in a liquid/liquid extraction using organic solvents such as benzene or chloroform [10][11][12]. Another technique used for PMF extraction, the concentration on C 18 cartridges after juice filtration on celite, have significantly simplified the sample preparation [9]. In this paper, we have used a rapid method of separation and quantitation of the six main PMF occurring in orange juices [SIN, hexamethoxyflavone (HEX), NOB, SCU, HEP and TAN] using an isocratic liquid chromatographic method with a C 18 cartridge extraction system, to separate and quantify PMF.…”