High-performance liquid chromatographic analysis of polyhydroxyflavones using solid-phase borate-complex extraction.

Abstract

A high-performance liquid chromatographic method using a solid-phase borate-complex extraction pretreatment was studied for the selective quantitation of polyhydroxyflavones in vegetables, red wine and human blood plasma. Homogenate, extract and intact samples were applied to phenylboric acid cartridges to retain polyhydroxyflavones on the solid-phase by forming the borate-complex, followed by elution of the retained analytes with an acidic solvent. Reversed-phase chromatography with diode array detection allowed the simultaneous separation of rutin, myricetin, fisetin, morin, quercetin and kaempferol without significant interference from other components, indicating high selectivity of the solid-phase borate-complex extraction. The absolute recoveries of quercetin, fisetin and rutin were superior to those of kaempferol, myricetin and morin, suggesting a difference in the complex formation efficiency between 1,2- and 1,3-diol structures. When using fisetin as an internal standard, polyhydroxyflavones were quantified in the concentration range 0.10-30.0 microg/ml. In replicate spiking experiments with standards, the mean relative recoveries ranged between 75.7 and 104.6%, and the intra-and inter-assay C.V.s ranged between 0.8 and 10.2% for onion, wine and plasma samples. The proposed method will be applicable to nutritional and pharmacokinetic experiments of polyhydroxyflavones.