Journal of Chromatography B: Biomedical Sciences and Applications
Short communicationHigh-performance liquid chromatographic determination of quercetin and isorhamnetin in rat tissues using β-glucuronidase and acid hydrolysis
Introduction
Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one; 3,3′,4′,5,7-pentahydroxy flavone) is a polyphenolic product of the phenylpropanoid biosynthetic pathway in plants and is therefore an integral part of the mammalian diet both as an aglycone and as β-glycosidic conjugates [1]. In vitro models indicate that this flavonoid has numerous biological roles, including antioxidant function, vasodilatory and platelet anti-aggregation effects and the maintenance of capillary integrity [2]. Early studies with rats indicated that quercetin had little nutritional relevance as the aglycone and the β-glycosides appeared to be destroyed by intestinal microorganisms in the small intestine and lower bowel [3], [4]. Recently, however, quercetin and its methylated derivative, isorhamnetin, have been detected in plasma and urine of rats maintained on quercetin-rich diets [5]. This suggests that a significant proportion of the aglycone may be absorbed and further metabolised by tissues. Consequently, to further elaborate on the bioavailability of quercetin, we have developed methodology for extraction using β-glucuronidase incubation and acid hydrolysis with subsequent HPLC detection to quantify tissue concentrations of quercetin and isorhamnetin in rats supplemented with the aglycone for 2 weeks.
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Animals and diets
Ten individually-housed weanling male rats of the Rowett Hooded Lister strain were offered, ad libitum, for 10 weeks a standard torula-yeast based semisynthetic diet [6]. Five rats were then offered the diet containing quercetin at a concentrations of 5 g/kg for a further 2 weeks and the remainder were maintained on the original ration. Diets were stored at −40°C before use.
Rats were anaesthetised with ether and blood removed by cardiac puncture into heparinised evacuated tubes (Becton
Results and discussion
The chromatographic peaks of quercetin and its methylated derivative, isorhamnetin, were well separated (retention times 13.07±0.02 and 28.54±0.79 min, respectively) (Fig. 1). There was some indication of the coelution of endogenous components with quercetin, although the peaks were comparatively small. Moreover, a peak with a retention time of 24.82±0.20 min may be kaempferol which differs from quercetin only by the absence of an hydroxyl group on position 5 of the B-ring. Although the spiking
Acknowledgements
We are grateful for financial support from the Scottish Executive for Rural Affairs Department (SERAD).
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