DETERMINATION OF QUERCETIN IN HUMAN PLASMA USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C-18 Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% viv acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and mu Bondapak C-18 column (150 x 3.9 mm I.D., 10 mu m particle size). The detection limit (A(375nm)) for quercetin in plasma was 0.1 mu g/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.