ELSEVIER Journal of Chromatography A, 761 (1997) 315-321 JOURNALOF CHROMATOGRAPHY A Short communication Quantitative analysis of flavonoids by reversed-phase high- performance liquid chromatography • a ~ C Alan Crozier ' , Einar Jensen b, Michael E.J. Lean , Morag S. McDonald a "Plant Molecular Science Group, Bower Building, Division of Biochemistry and Molecular Biology, Institute ¢~fBiomedical and Life Sciences, University of Glasgow, Glasgow G 12 8QQ, UK bDepartment of Pharmacology, University of Troms¢, Science Park. N-9037 Troms¢, Norway CDepartment of Human Nutrition, Universi~ of Glasgow, Queen Elizabeth Building, Royal Infirmary, Glasgow G31 2ER. UK Received 27 August 1996; accepted 1 October 1996 Abstract Large variations were found in the effectiveness of five reversed-phase high-performance liquid chromatography columns used for the analysis of free and conjugated flavonoids. The best results were obtained with C~x Nova-Pak, C,s Symmetry and C~s Genesis columns but substantial band broadening and peak asymmetry were observed when free flavonoids were chromatographed on ODS-Hypersil and LiChrospher RP-I 8 supports. The Symmetry and Genesis columns provided gradient elution separations of rutin, quercetin-3-glucoside, quercitrin, myricetin, luteolin, quercetin, apigenin, kaempferol and isorhamnetin. This procedure was used for the quantitative analysis of endogenous flavones and flavonols in acid hydrolysed extracts from onions and celery. Keywords: Plant extracts; Flavonoids I. Introduction Flavonoids are a large family of secondary plant metabolites, comprising anthocyanins, flavonols, flavones, catechins and flavonones, and many are present in plant tissues in relatively high concen- trations as sugar conjugates [1[. There is increasing interest in flavonoids because it is becoming apparent that they are an important group of compounds in both plants and animals. In plants their production is induced by and they provide protection against UV-B radiation (see Ref. [2]). Flavonoids are also involved *Corresponding author. in the regulation of pollen tube growth in the stigma [3,4] and they act as regulatory signals in the transcription of nodulation genes in Rhizobium cells as the first step towards legume root nodule forma- tion and symbiotic nitrogen formation [5,6]. Flavonols and flavones are of particular importance in the human diet as there is evidence that they act as antioxidants [7,8], and epidemiological studies have indicated that their consumption is associated with a reduced risk of cancer [9-12] and cardiovascular disease [13-15]. The main dietary sources of the flavonols, primarily as conjugates of quercetin (I) and kaempferol (II), and the corresponding flavones, luteolin (III) and apigenin (IV), are vegetables, fruit and beverages (Scheme 1) [16,171. Among the more ubiquitous flavonoids over 50 0021-9673/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S0021-9673(96)00826-6