Detailed study of fluorescent properties of cyclodextrin complexes for sensitive automated determination of...

A simple and rapid method for determining diclofenac in a sequential injection system was developed for the selective and sensitive on-line determination of the analyte in sample from a metabolic study. The fluorescence properties of complexes of 13 types of cyclodextrins and their derivatives, including salts, were studied off-line, while two of the complexes were then used in the flow conditions. Optimisation was carried out with (2-hydroxypropyl)-γ-cyclodextrin, which is fluorescent and the intensity of its complex with diclofenac was found to be lower. In the flow system, we tested different types of mixing of zones and stop-flow periods. Simple mixing using flow reversal and a 1-min stop-flow in the holding coil were sufficient to rapidly determine diclofenac, even at low concentrations with a limit of quantitation of 1 × 10^-6 mol/L. The relationship between fluorescence intensity and diclofenac concentration showed a different pattern in the two intervals: decreasing in the range of 1-5x10^-7 mol/l and linearly increasing in the range of 1-5x10^-6 mol/l, which was used for analysis. Real samples from a metabolic activity study based on the activation of xenobiotics recognition receptors were analysed, and the results were compared with off-line liquid chromatography/mass spectrometry measurements. The differences corresponded to the complexity of the matrix and were not significant. The trend of diclofenac metabolism was obvious. The developed method is prepared for the fully automated, real-time monitoring of the metabolic study with more frequent sampling than just once daily and the prediction of sample collection for the mass spectrometric determination of diclofenac metabolites.