Determination of 1-nitropyrene metabolites by high-performance liquid chromatography with chemiluminescence detection

Three metabolites of 1-nitropyrene, i.e., 3-hydoxy-1-nitropyrene (3-OHNP), 6-hydoxy-1-nitropyrene (6-OHNP) and 8-hydoxy-1-nitropyrene (8-OHNP), were sensitively and selectively detected by HPLC with peroxyoxalate chemiluminescence (CL) detection. In the system, the three OHNPs were reduced to their corresponding amino derivatives through a reduction column packed with Pt/Rh-coated alumina and then concentrated on a concentrator column. By rotating a switching valve, the analytes were eluted into a separator column (ODS), separated and then detected by the bis(2,4,6-trichlorphenyl)oxalate-H2O2 CL system. The detection limits (signal-to-noise ratio = 3) were in the range from 5 fmol (6-OHNP and 8-OHNP) to 12 fmol (3-OHNP) per injection. To demonstrate the proposed method, it was used to determine three compounds in the incubation mixture of 1-nitropyrene and rat S9 mix.