Usefulness of cyclodextrins for detection in molecular fluorescence. Application to xenobiotics and drugs

Dossier Luminescence spectroscopy ■ Usefulness of cyclodextrins for detection in molecular fluorescence. Application to xenobiotics and drugs P. Prognon, A. Kasselouri, M. C. Desroches and G. Mahuzier Faculté de Pharmacie de Châtenay-Malabry, Laboratoire de Chimie Analytique, 5, rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France cyclodextrins derivative controls the entire or partial inclusion of the guest molecule. Moreover, the hydrophobicity of the organic compounds is also of crucial importance with regards to the fit of the molecule in the internal cavity of CDs. It is assumed that the driving forces of such interactions are: hydrogen bonding, van der Waals forces or hydrophobic interaction [1,2]. Native CDs give rise to numerous hemisynthetic derivatives by grafting the outer hydroxyls of the molecules by various functional groups either hydrophobic or hydrophilic [3] in order, for instance, to decrease or increase their water solubility. This inclusion capability, common at various degrees, to all CDs derivatives, makes them good candidates for molecular recognition and thus, explain their extensive chromatographic and electrophoretic use especially for chiral separation [4]. On the other hand, the inclusion capability of CDs are of great interest in stabilizing, solubilizing substances [5]. Selective precipitation or extraction can be, also, achieved using CDs [5]. Without claiming to be exhaustive, the aim of this short report is to underline the potential interest of CDs in an other field of analytical chemistry: fluorescence spectrometry to improve the sensitivity and selectivity of the detection of included compounds. Cyclodextrins (CDs) present the unique ability to entrap molecules. In analytical chemistry, they have been mainly used for separation purpose. The present review deals with their potential interest towards the detection by molecular fluorescence of the included compounds mainly xenobiotics and drugs. As some of the included molecules exhibit native fluorescence due to their aromaticity, the addition to their aqueous solution of CDs entails, in some cases, a large enhancement of their emission of fluorescence. This feature appears of particular interest from an analytical point of view, either for direct detection of the guest molecule or as a detection mode after separations techniques (i.e.: chromatography or electrophoresis). Decrease in rotation motion of entrapped molecule and (or) decrease in solvent relaxation appear as the main causes of the enhancing emission effect. Some example picked up from pharmaceutical and bioanalytical literature are presented in order to demonstrate the interest of CDs in the field of the fluorescence techniques. Preliminary considerations The first observation of fluorescence enhancement upon inclusion in βCD aqueous solutions of the fluorophore: 1 anilino-8-naphtalene sulfonate (ANS) was due to Cramer in 1967 [6]. This prompted many authors to use fluorescence techniques as a spectroscopic tool to study the mechanisms of the inclusion process in solution. As an example, Harada and coworkers [7] as early as 1976 showed that the fluorescence of 2-p-toluidinyl naphtalene sulfonate (TNS) was increased by 25 times with βCD and up to 25 and 30 times with polymers containing cyclodextrins. The authors already stated that the fluorescence increase observed with TNS was mainly due to the restriction of intramolecular rotation in the rigid environment of the cavity and/or to the exclusion of solvent relaxation. The direct consequence is a decrease in the vibrationnal desactivation. Thus, the CDs acts as Introduction Cyclodextrins (CDs) are cyclic oligomers of α 1-4 linked Dglucose and can form inclusion complexes with a variety of organic compounds in aqueous solution. The α, β, γ, cyclodextrins have six, seven and eight glucose units respectively. They are called native cyclodextrins as they are issued from bacillus macerans fermentation. The size of the internal cavity of native cyclodextrins increase from circa 5 Å for α CD to 8 Å for γ CD. Consequently, the geometry of the guest compound is the first factor that rules the inclusion process. In other words, the internal diameter of the 664 ANALUSIS, 2000, 28, N° 8 © EDP Sciences, Wiley-VCH 2000 Article available at http://analusis.edpsciences.org or http://dx.doi.org/10.1051/analusis:2000280664 Dossier Luminescence spectroscopy spectroscopic shield, protecting the fluorescing singlet state or the phosphoring triplet state (see the article of Muñoz de la Peña et al. in this issue) from the external quenchers. Actually, the CDs cavity looks like an organic solvent surrounding the entrapped molecules, which often yields to higher quantum efficiency. Modifying the microenvironment of the caged analyte can, also, be interpreted as increasing the water solubility by complexation of a hydrophobic molecule. These two metallic ions were fluorometrically determined by means of authraquinonic ligands, i.e.: 1,4–dihydroxyanthraquinone and 1-amino-4 hydroxyantraquinone, respectively. Neither of these ligands was water soluble enough to prepare convenient working solutions of the analytical reagents. In contrast, in the presence of 10–2 M βCD, 10–4 M aqueous solutions of both ligands could be prepared and thus permitted trace determination of the metallic ions of interest. This resulted from a partial inclusion of the reagent in the β-CD cavity. As analytical consequence, a significative increase in sensitivity was obtained, i.e.: for beryllium the limit of detection (LOD), as impurity in organic material, was lowered to 3 ng/ml in the presence of β-CD instead of 17 ng/ml in the absence of CDs. In that kind of pioneering work, the authors focused their efforts on the quantitative aspects of the inclusion process (i.e.: Job’s plot for stoichiometry determination, affinity constant calculation). These early studies were confirmed by others [8] for instance in an inclusion study using benzene derivative probes that showed that the fluorescence enhancing was ascribed to the increase of the radiation rate constant (kr) of the excited state of the entrapped molecules. Similarly, the water insoluble and non fluorescent pigment retinal exhibited, after addition of β-CD, a strong emission due to the solubilizing effect after encapsulation in the interior of the β-CD cavity [20]. Once more, it should be pointed out that these elegant spectroscopic [9-10] studies were fundamentally conducted with the aim of getting a better insight in the host-guest interaction but without any analytical application purpose. The fluorescence probe acted in such a system only as a “molecular spy” to elucidate the supramolecular interactions involved between various cyclodextrins derivatives and guest organic species. Though, out of scope of our analytical purpose, it is worth noting that this probing approach still remains, of great interest, since it allows rapid, and simple a (...truncated)