Quantitation of polymethoxylated flavones in orange juice by high-performance liquid chromatography.

A quantitative high-performance liquid chromatographic (HPLC) procedure for the determination of the five major polymethoxylated flavones (PMFs) in orange juice has been developed. It employs a unique ternary solvent system with coupled UV-fluorescence detection. The dual detectors were employed to determine the presence of interfering substances and served as a cross check on quantitation. Stop flow UV and fluorescence scanning was used to identify peaks and determine the presence of impurities. Although all five citrus PMFs fluoresce, some HPLC fluorescence peaks were too small to be of much practical use. All five citrus PMFs could be quantitated satisfactorily with the fixed wavelength UV (313 nm) detector. The HPLC procedure has been used to evaluate each step in the preparation. The optimum extracting solvent was selected and one time consuming step was eliminated, as it was found to be unnecessary. HPLC values for nobiletin and sinensetin are in good agreement with the thin-layer chromatographic (TLC) values in the literature. HPLC values for the other three flavones were considerably lower than those reported in the literature. The HPLC procedure is considerably faster than the TLC procedure with equal or superior precision and accuracy.