High-performance liquid chromatographic determination of naturally occurring flavonoids in Citrus with a photodiode-array detector

Abstract 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.