Elimination of bilirubin interference in fluorimetric determination of fluorescein by phase-resolved fluorescence spectrometry

Phase-resolved fluorescence spectrometry is used to eliminate bilirubin interference in the fluorimetric quantitation of fluorescein, despite extensive excitation and emission spectral overlap of the two species. In this method, it is the difference in fluorescence lifetimes of the two species that provides the selectivity parameter. Results are given for a nulling approach and for a multiple phase-angle approach. In the nulling approach, intensity is measured at a detector phase angle at which the intensity for bilirubin is zero. In the multiple phase-angle approach, intensities at several phase angles are used to generate a matrix which is solved for the concentrations of fluorescein and bilirubin. Although a steady-state method yielded low results for fluorescein concentration in the presence of bilirubin concentration about 3 μM, the phase-resolved methods yielded correct values for fluorescein concentration with bilirubin concentration up to 10 μM.