Fluorescence Detection of Flavonols in HPLC by Postcolumn Chelation with Aluminum

Abstract

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 Al(3+), 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 Al(3+). 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.

Absorption and antioxidant effects of quercetin from onions, in man

“…The quercetin concentration in plasma peaked 2 h after ingestion at 248.4 AE 103.9 ngaml (Figure 1), corresponding to a plasma quercetin concentration of 0.73 AE 0.30 mM, and then decreased, returning to baseline levels 24 h after ingestion. These results are comparable to those of Hollman et al (1996) in a similar study where two subjects consumed 215 g of fried onions.…”

“…However, more recently, using more accurate analysis techniques, Hollman et al (1995) demonstrated that 24% of orally administered quercetin aglycone was absorbed. In a further study they found the quercetin glucosides present in onions were shown to be absorbed even more ef®ciently than the aglycone (Hollman et al, 1996).…”

“…This assay was carried out according to a technique developed by Hollman et al (1996). In a 4 ml vial, 1.0 ml of methanol containing 2 gal tert-butyl hydroxyquinone (TBHQ), 0.4 ml of 10 M HCl and 0.6 ml of plasma were mixed.…”

“…Nieder et al (1991) reported that¯avonol glycosides from Ginkgo biloba could be absorbed, but no details were given about quantities ingested and how ef®ciently they were absorbed. More recently a sensitive technique for measuring¯avonoids in plasma has been developed (Hollman et al, 1996). Plasma quercetin levels were found to reach maximum plasma concentrations in two subjects about 2.7 h after the ingestion of a fried onion breakfast (Hollman et al, 1996).…”

“…More recently a sensitive technique for measuring¯avonoids in plasma has been developed (Hollman et al, 1996). Plasma quercetin levels were found to reach maximum plasma concentrations in two subjects about 2.7 h after the ingestion of a fried onion breakfast (Hollman et al, 1996). These ®ndings suggest that signi®cant¯avo-noid absorption may occur following onion ingestion and point to a possible role for quercetin in protecting LDL against oxidative modi®cation.…”