Separation and detection of a benzo[a]pyrene metabolite with capillary electrophoresis in the presence of DNA using laser-induced fluorescence

A novel approach was developed for the separation and detection of a benzo[a]pyrene B[a]P–DNA metabolite (tetrol I-1) in the presence of DNA using CE and laser induced fluorescence. Tetrol I-1 in the presence of DNA is intercalated and undergoes fluorescence quenching. Thus, an equilibrium is established between the intercalated tetrol I-1 and the uncomplexed tetrol I-1. It is only the uncomplexed tetrol that is fluorescent in the presence of DNA. The tetrol I-1 fluorescence intensities, at two concentrations of tetrol I-1 (0.005 and 0.01mg ml−1), were observed while varying the DNA concentration. Stern–Volmer plots were constructed of the fluorescence intensity of the uncomplexed tetrol I-1 versus DNA concentration. From the slopes of the Stern–Volmer plots quenching constants were determined. The quenching constants are essentially the same as an association constant for tetrol I-1 with DNA. The average value obtained for the association constant for the two concentrations of tetrol I-1 was 0.22±0.02 mg ml−1. It was thus demonstrated that uncomplexed tetrol I-1 can be separated from DNA by CE and an association constant for tetrol I-1 bound to DNA can be obtained from the fluorescence quenching data.