HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF NATURALLY-OCCURRING FLAVONOIDS IN CITRUS WITH A PHOTODIODE-ARRAY DETECTOR

High-performance liquid chromatography (HPLC) coupled with ultraviolet-visible spectrophotometry using a photodiode-array detector was used as a routine method for the simultaneous separation and determination of 25 naturally occurring Citrus flavonoids. The separation system consisted of a C-18 reversed-phase column, a gradient system of 0.01 M phosphoric acid (A) and methanol (B), and a photodiode-array detector. Each of the 25 flavonoids was eluted from the column with a gradient system composed of three periods: (1) 0-55 min, 70-55% (v/v) A in B, (2) 55-95 min, 55-0% A in B, and (3) 95-100 min, isocratic, 100% B, and quantified by spectrophotometric detection at 285 nm. Identifications of specific flavonoids were made by comparing their retention times (t(R)) and UV spectra with those of standards. The relative standard deviations of t(R) values were 0.029-0.321%. The recoveries of pure eriocitrin, naringin, hesperidin and tangeretin added to tissues prepared from Unshiu (Citrus unshiu Marc.) and Hirado-buntan (Citrus grandis Osbeck f. Hirado) and subsequent extraction were 97.47-103.13% from the mesocarp and 96.87-104.93% from the juice with standard deviations of 2.32-5.72% and 2.18-5.96%, respectively.