CHROMATOGRAPHIC DETERMINATION METHOD FOR 1-NITROPYRENE AND ITS METABOLITES IN BIOLOGICAL SAMPLES WITH FLUORESCENCE DETECTION AFTER ONLINE REDUCTION

A high performance liquid chromatographic (HPLC) method with fluorescence detection after on-line reduction for the determination of 1-nitropyrene (NP), 1-nitrosopyrene (NSP), 1-aminopyrene (AP) and N-acetylaminopyrene (AAP) has been developed. The reduction efficiency of NP and NSP on a zinc column was found to be higher than that of an electrochemical reducer. Using a HPLC system equipped with a zinc column (4.0 mm i.d. x 10 mm) and an imidazole/HClO4 (pH 6.8): acetonitrile mobile phase, detection limits (S/N = 3) of 20-30 fmol for NP, NSP and AP and 350 fmol for AAP were obtained. NP, NSP and AP were determined in the incubation mixture of NP and Salmonella typhimurium, YG1021, by this method. Time course studies showed that a large ratio of NP was metabolized in the pre-incubation step of the Ames test.