Fluorescence detection of flavonols in HPLC by postcolumn chelation with aluminum

Flavonols are dietary antioxidants which may prevent coronary heart disease. To be able to study absorption of flavonols in humans, we developed a postcolumn derivatization with aluminum for HPLC with fluorescence detection. Variables governing postcolumn chelation, such as water content, buffer, organic modifier of the eluent, concentration of Al³⁺, presence of acetic acid in the postcolumn reagent, and temperature, were studied and optimized. Of the flavonoids, only flavonols that contain a free 3-hydroxyl and 4-keto oxygen binding site form fluorescent complexes with Al³⁺. The method has a detection limit of 0.15 ng/mL for quercetin, 0.05 ng/mL for kaempferol, 0.45 ng/mL for myricetin, and 0.05 mg/mL for isorhamnetin, thus improving detectability of quercetin 300-fold as compared to that possible with UV detection. The reproducibility relative standard deviation of the method is 1.4%. This extremely sensitive method enables, for the first time, determination of flavonols in body fluids after consumption of a normal diet.