Analytica Chimica Acta 468 (2002) 3–11 Natively fluorescent isoflavones exhibiting anomalous Stokes’ shifts Eva de Rijke a , Hem C. Joshi b , Huib R. Sanderse a , Freek Ariese a , Udo A.Th. Brinkman a , Cees Gooijer a,∗ a Department of Analytical Chemistry and Applied Spectroscopy, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands b Institute for Plasma Research, Bhat, Gandhinagar 382428, India Received 12 April 2002; received in revised form 1 July 2002; accepted 2 July 2002 Abstract The fluorescence behaviour of 19 flavonoids was studied. Three isoflavones (formononetin (F), ononin (FG) and daidzein (D)) were found to exhibit large Stokes’ shifts, possibly due to a change of the structure of the molecule from non-planar in the S 0 state to planar in the S 1 state. Lifetime measurements were carried out using time-correlated single photon counting spectroscopy to further characterise the mechanism. These large shifts provide a high selectivity, so that fluorescent isoflavones can be readily detected in plant samples by means of reversed-phase LC with fluorescence detection. Attention has to be paid to possibly fluorescent impurities in flavonoid standards, as was observed for daidzin (DG), which is not fluorescent itself, but has a fluorescent isomer. To distinguish between the two compounds, LC with fluorescence and MS detection was used to separate and identify the impurity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence; LC; Stokes’ shift; Lifetime measurements; Flavonoids; Isoflavones 1. Introduction Flavonoids are one of the most characteristic groups of compounds in higher plants and are present not only in flowers and leafs but also in roots [1]. They are known for their UV-B protecting properties in plants, are used as food supplements and some of them, the isoflavonoids, are known to be phytoestrogens [2]. The ultimate objective of our present research pro- gramme is to use flavonoid profiles in wetland plants as chemical indicators for stress in wetland ecosys- ∗ Corresponding author. Tel.: +31-204447540; fax: +31-204447543. E-mail address: gooijer@chem.vu.nl (C. Gooijer). tems. Obviously, this requires the use of various an- alytical methods, primarily gradient LC–UV, LC–MS and LC–MS/MS. In addition, it is of interest to find out whether fluorescence spectroscopy, known for its inherent selectivity, can be used as an alternative de- tection technique, either stand-alone or in combination with LC. To the best of our knowledge, there is no report of a systematic and comprehensive study of the native fluorescence of flavonoids, though individual mem- bers of this class received much attention. This holds especially for 3-hydroxyflavone (3HF) and some of its derivatives. 3HF has been studied since the early 1980s [3,4] and is still receiving interest from various sides [5–7]. Its fluorescence behaviour is fascinating 0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S0003-2670(02)00630-X