Determination of(+)-anti- benzoapyrene diol-epoxide-DNA adducts metabolited from benzoapyrene in bivalves by high-performance liquid chromatography with fluorescence detection

Objective To develop a simple and highly sensitive high-performance liquid chromatography/fluorescence method for the determination of benzoapyrene(BaP)-tetrols released after acid hydrolysis of(+)-anti- benzoapyrene diol-epoxide(BPDE)-DNA adducts metabolited from benzoapyrene in bivalves.Methods The tissue DNA in bivalves were extracted by reagent kit and the(+)-anti-BPDE-DNA adducts were hydrolyzed in 0.1 mol/L HCl at 90℃ for 4 hours.The acid-hydrolysis products(BaP-tetrols)of DNA adducts were extracted by ethylacetate and measured by high-performance liquid chromatography/fluorescence detection.Samples were separated on CenturySIL C18-BDS column(150 mm×4.6 mm,5 μm)and the elution was isocratic using a 55% methanol:45% water(v/v)mobile phase at a flow rate of 1.0 mL/min;fluorescence detection was conducted at 265 nm(excitation)and 395 nm(emission),with the injection volume of 20 μL.Results The operating linear range was at least 0.5-100 ng/mL(r²=0.9960)and the detection limits(S/N=3)was 0.3 ng mL^-1.Coefficients of variation for within-run and between-run assays were 2.8%-4.2% and 3.2%-5.8%,respectively.The content of(+)-anti-BPDE-DNA adducts were 13.44-152.7 μg/kg and the relative standard deviations(RSDs)were 3.0%-6.5%.The average recoveries were 86.9%-91.6% and the RSDs were 3.1%-7.3%,respectively.Conclusion The method was proved to be sensitive,accurate,rapid and could be applied to the determination of(+)-anti-BPDE-DNA adducts in bivalves.